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The efficacy of an experimental Pasteurella hemolytica vaccine as measured by sero-conversion in Awassi lambs in Jordan
Small Ruminant Research 45 (2002) 201–208
The efficacy of an experimental Pasteurella hemolytica vaccine as measured by sero-conversion in Awassi lambs in Jordan Y.H.M. Al-Tarazi∗ Department of Veterinary Basic Sciences, Faculty of Veterinary Medicine, Jordan University of Science and Technology, P.O. Box 3030, Irbid 22110, Jordan
Abstract An inactivated iron regulating protein (IRP) vaccine containing nine Pasteurella haemolytica serotypes was used. Two groups of Awassi lambs were used during the experiment. One group of 10 lambs was vaccinated and the second unvaccinated group of 10 lambs considered as negative control. Blood samples were collected on day 0, 2 and 4 weeks (post-vaccination) and 4 weeks after a revaccination. Antibody titers against capsular antigen of P. haemolytica serotypes A1, A2, A6, A7 and T3 was evaluated for both groups of lambs by indirect haemagglutination test (IHT). Antibody titers against P. haemolytica A2 outer membrane IRPs and the 35 kDa proteins was evaluated by enzyme linked immunosorbent assay (ELISA) and by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Results showed that the vaccine used induced sero-conversion against the capsular antigen, major outer membrane proteins (OMPs) and IRPs of P. haemolytica in the vaccinated lambs. Results also showed that one inoculation of the vaccine was insufficient to induce high antibody titers. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Pasteurella haemolytica; Vaccine; Lambs; ELISA; Jordan
1. Introduction Vaccines against pasteurellosis in sheep have been available for many years, but their efficacy has been equivocal (Donachie, 1994). Vaccine research on pneumonic pasteurellosis in sheep has primarily focused on Pasteurella haemolytica serotype A2 (Donachie et al., 1986). In the early 1980s, vaccine research showed that specific pathogen free (SPF) lambs could be protected against pneumonic pasteurellosis caused by serotypes A1, A6 and A9 using a vaccine containing a sodium salicylate extract (SSE) of these serotypes (Gilmour et al., 1983). It showed a high degree of protection which was ∗ Tel.: +962-2-7201000x22009; fax: +962-2-709-5123. E-mail address: [email protected] (Y.H.M. Al-Tarazi).
serotype-specific and antibodies were raised against bacterial cell surface antigens such as outer membrane proteins (OMPs), lipopolysaccharide and the capsule (Donachie et al., 1984). A similar vaccine comprising serotype A2 did not show protection exceeding 50% (Gilmour et al., 1983). Research towards an effective P. haemolytica A2 vaccine, arose from studies using SPF lambs which were experimentally infected by aerosol with the A2 serotype. These lambs recovered following antibiotic treatment and were subsequently immune to challenge with the Parainfleunza Type 3 (PI3) virus and P. haemolytica serotype A2 (Donachie et al., 1986). The sera of these lambs were found to contain antibodies against different virulence factors of P. haemolytica, including antibodies against the leukotoxin, the capsule and the lipopolysaccharide.
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Other research work has been carried on the iron regulating protein (IRP) antigens, which are present in the outer membrane of P. haemolytica A2 recovered from the pleural fluid of SPF lambs experimentally infected with A2. They can also be detected in P. haemolytica strains grown on iron depleted media. They are absent in the OMPs of P. haemolytica grown in nutrient broth (Donachie and Gilmour, 1988). Vaccines prepared from IRPs and SSE of P. haemolytica significantly enhance protection against A2 challenge, confirming its role in inducing immunity (Gilmour et al., 1991). In the current investigation, a newly developed vaccine available from a commercial company was used because anecdotal evidence suggested that pneumonic pasteurellosis in sheep and goats in Jordan was not prevented by available vaccines.
2. Materials and methods 2.1. Iron regulating protein vaccine This was an experimental, IRP vaccine, containing killed P. haemolytica of serotypes A1, A2, T3, T4, A6, A7, A9, T10 and T15. The viable counts for each serotype used in the vaccine was 5 × 108 CFU/ml. They had been grown in iron deprived media. The vaccine contained aluminium hydroxide as an adjuvant. 2.2. Animals Two groups of Awassi lambs were used, one group of 10 animals were vaccinated with the experimental vaccine and 10 unvaccinated animals served as a control group. The 3-month-old lambs were obtained from ewes which had not been vaccinated against P. haemolytica and were chosen at random. The animals were kept under a closed rearing system in separate pens at the Tafila fattening station in Jordan. 2.3. Vaccination schedules
2.4. Blood sampling This was done on day 0, 2 and 4 weeks before second vaccination and 4 weeks after the booster from all lambs included in the study. 2.5. Antibody measurements The antibody titers were measured for P. haemolytica serotypes A1, A2, T3, A6, A7 and A9 for both groups of lambs by the indirect haemagglutination test (IHT), which was performed in microtiter plates by a method modified by Fraser et al. (1983). The antibody titers against P. haemolytica serotype A1 outer membrane IRPs and the 35 kDa protein were measured by an enzyme linked immunosorbent assay (ELISA) as described by Donachie and Jones (1982). The microtiter plates were coated with 100 mg per well outer membrane IRPs and with 100 mg per well 35 kDa protein. The positive control used was anti-P. haemolytica serotype A1 anti-serum from a convalescent sheep. The positive control sera were used at dilutions of 1:10 to 1:50,000 each dilution in two wells. The optical density (OD) was read at 492 nm. The mean of the OD of two readings for each dilution was calculated and allocated “∗” or “+” rating. The scheme started from the highest reading which was designated Table 1 The optical density (OD) of the positive control P. haemolytica anti-A1 sheep serum tested against outer membrane iron regulating proteins by ELISA Serum dilutions
Mean of OD (from duplicate readings)
Interpretation
1:10 1:50 1:100 1:500 1:1000 1:5000 1:10000 1:50000
Over 2.303 2.022 1.083 0.704 0.270 0.178 0.117
∗a ++++b +++c ++d +e ±f Negative Negative
a
Two injections of 2 ml each of the vaccine were given subcutaneously, on day 0 and 4 weeks later to one group, while the other group remained unvaccinated as controls.
Highly positive. Very strong. c Positive. d Intermediate. e Weak positive. f Unclear. b
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Table 2 The optical density (OD) readings of the positive control P. haemolytica anti-A1 sheep serum tested against 35 kDa protein by ELISA
3. Results
Serum dilution
Mean of OD (from duplicate readings)
Interpretation
1:10 1:50 1:100 1:500 1:1000 1:5000 1:10000 1:50000
Over 2.235 1.674 0.669 0.412 0.209 0.077 0.017
∗a +++++b ++++c +++d ++e +f ±g Negative
No antibody titers were detected in the sera of the IRP vaccine group on day 0 nor in the sera from day 0 and 2, 4 and 8 weeks of the unvaccinated group for all P. haemolytica serotypes tested. In the vaccinated group, a titre 1:8 was detected against serotypes A1, A2, A7 and A9 2 weeks post-vaccination (Table 3). Other serum samples either did not show sero-conversion or gave a value of 1:4. The arithmetic mean for the antibody titre for all sera tested against different serotypes was <8. Four weeks after vaccination more animals showed sero-conversion for all serotypes tested, with higher means than those found after 2 weeks (Table 4). Eight weeks after the first vaccination and 4 weeks following re-vaccination, 80% of the sera tested for serotypes A1, A6, A7, and 70, 60 and 60% for serotypes T3, A2 and A9 showed a titres <1:32, respectively. The mean of the antibody titre for all serotypes tested ranged from 8.8 to 13.6 (Table 5).
a
Highly positive. Very strong positive. c Very strong. d Positive. e Intermediate. f Weak positive. g Unclear. b
as ∗ (highly positive) through +++++ (very strong positive) to negative as illustrated in Tables 1 and 2. The negative control was sera from a SPF lamb derived from a non-vaccinated ewe and used at dilutions of 1:50 in eight wells. The serum samples of all animals in the trial were also diluted in 1:50. Antibodies against IRPs were also tested by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting according to the method described by Donachie and Gilmour (1988).
3.1. IHA test results
3.2. ELISA test results The mean OD readings of the positive control for the OMPs and 35 kDa protein are shown in Tables 1 and 2. The mean OD readings of the negative control for the OMPs was 0.185 and for the 35 kDa protein was 0.021. Reading double that of the negative control was considered as positive (Voller et al., 1979).
Table 3 Number of sera collected 2 weeks after vaccination tested with IHA showing sero-conversion against P. haemolytica serotypes and the mean titre Serotype tested
A1a A2 T3 A6 A7 A9
Serum dilutions (no. of sera reacting) 0
1/4
1/8
5 3 7 3 1 3
3 4 3 7 5 3
2 3 0 0 4 4
Percentage of positive sera tested
Mean titre and S.E.
20 30 0 0 40 40
2.8 4 1.2 2.88 5.2 4.4
± ± ± ± ± ±
1.04 1.03 0.61 0.366 0.85 1.107
Positive control was rabbit anti-sera which gave a titre (>1:256) for each serotype tested; positive titre considered from (1:8); S.E., standard error. a Ten serum samples were tested.
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Table 4 Number of sera collected 4 weeks after first vaccination tested with IHA showing sero-conversion against P. haemolytica serotypes and the mean titre Serotype tested
Serum dilution (no. of sera reacting)
A1a A2 T3 A6 A7 A9
0
1/4
1/8
1/16
0 0 5 0 0 0
4 5 3 6 3 4
4 3 2 4 7 4
2 2
2
Percentage of positive sera tested
Mean titre and S.E.
60 50 20 40 70 60
8 7.6 2.8 5.6 6.8 8
± ± ± ± ± ±
1.46 1.51 1.04 0.65 0.611 1.46
Positive control was rabbit anti-sera which gave a titre (>1:256) for each serotype tested; positive titre considered from (1:8); SE, standard error. a Ten serum samples were tested.
In order to interpret the immunological response of each vaccinated lamb, the OD reading at day 0 of the pre-vaccination sera was subtracted from other OD readings of sera collected at 2, 4 and 8 weeks of the IRP vaccine and control groups. The OD reading of the control group showed no change between pre-vaccination and the 8 weeks readings. The subtracted OD readings were compared with the positive and negative control. The strength of the immunological response was interpreted by comparing it to the mean of OD reading of the positive control at different dilutions (Tables 1 and 2). In the vaccinated group, the immunological response against the outer membrane IRP was interpreted as follows: 2 weeks after the first vaccination, two lambs showed an intermediate response, three
showed a weak positive, three showed an unclear reaction and two lambs showed negative. After 4 weeks from the first vaccination one lamb exhibited an intermediate response, seven were weakly positive and two were unclear. Four weeks following revaccination, six lambs showed weak positive and four intermediate immunological response (Table 6). In the vaccinated group, the immunological response against the 35 kDa OMP was as follows: Two weeks after the first vaccination, eight lambs showed unclear results and two were negative. After 4 weeks, five lambs showed weak positive results. But after 4 weeks following the booster dose, all lambs showed immunological responses to the 35 kDa protein (Table 7).
Table 5 Number of sera collected 8 weeks after first vaccination tested with IHA showing sero-conversion against P. haemolytica serotypes and the mean titre Serotype tested
A1a A2 T3 A6 A7 A9
Serum dilutions (no. of sera reacting) 0
1/4
1/8
1/16
0 0 0 0 0 0
2 4 3 2 2 1
3 3 2 5 4 4
5 3 4 2 2 2
1/32
1 1 2 3
Percentage of positive sera tested
Mean titre and S.E.
80 60 70 80 80 60
11.2 08.8 10 11.2 13.6 8
± ± ± ± ± ±
1.66 1.66 2.9 2.65 3.33 1.46
Positive control was rabbit anti-sera which gave a titre (>1:256) for each serotype tested; positive titre were considered from (1:8); S.E., standard error. a Ten serum samples were tested.
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Table 6 The subtracted optical density (OD) readings of sera from 10 lambs (IRP vaccine group) tested by ELISA against P. haemolytica serotype A2 outer membrane iron regulating proteins at different times after vaccination Tag no.
OD/day 0
OD/2 weeks
Intera
OD/4 weeks
Inter
OD/8 weeks
Inter
405 407 408 417 419 428 431 437 439 447
0 0 0 0 0 0 0 0 0 0
0.598 0.439 0.327 0.191 0.304 0.296 1.058 0.194 0.880 0.782
+ + ± − ± ± ++ − ++ +
0.607 0.453 0.414 0.351 0.433 0.391 0.958 0.277 0.813 0.727
+ + + ± + + ++ ± + +
0.878 0.469 0.493 0.468 0.525 0.473 1.357 0.399 1.132 1.134
++ + + + + + ++ + ++ ++
a
Inter, interpretation of OD reading according to the scheme (Table 1).
Table 7 The subtracted optical density (OD) readings of sera from 10 lambs (IRP vaccine group) tested by ELISA against the 35 kDa protein at different time after vaccination Tag no.
OD/day 0
OD/2 weeks
Intera
OD/4 weeks
Inter
OD/8 weeks
Inter
405 407 408 417 419 428 431 437 439 447
0 0 0 0 0 0 0 0 0 0
0.075 0.041 0.042 0.073 0.079 0.055 0.176 0.053 0.026 0.072
± ± ± ± ± ± + ± − ±
0.124 0.034 0.071 0.067 0.100 0.128 0.113 0.177 0.043 0.072
+ − ± ± + + + + ± ±
0.251 0.292 0.224 0.160 0.441 0.308 0.240 0.548 0.177 0.373
+ + + + ++ ++ + +++ + ++
a
Inter, interpretation of OD readings according to the scheme (Table 2).
3.3. SDS-PAGE immunoblotting
4. Discussion
The immunoblotting profile of the iron regulating OMP of P. haemolytica A2 reacted with sera from vaccinated lambs showed bands of major OMPs which were distinct from those sera of the control group or sera from the test group collected pre-vaccination, at regions 30, 42 and 55 kDa. Additional bands at 70 and 100 kDa were more prominent especially after 4 weeks following the booster in the vaccinated sera, but either absent or weakly expressed in the unvaccinated or pre-vaccination sera. No protein bands were detected in the SPF lamb from the control group sera.
Potentially, the vaccine tested may be useful because many of the serotypes of P. haemolytica present in the vaccine are known to cause pneumonic pasteurellosis in sheep in Jordan (Al-Tarazi, 1995). P. haemolytica is a commensal organism of the nasopharynx of ruminant animals (Carter et al., 1995). This raises the possibility of the presence of antibodies against these organisms in conventional animals used in vaccine trials that may complicate the interpretation of the results. To overcome this problem the use of SPF lambs is desirable in vaccine trials and in challenge experiments. Other complexities in conducting
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challenge experiments are the need for a high challenge dose of P. haemolytica with aerosol exposure for not <15 min within a chamber and pre-infection by other biological pre-disposing factors such as viruses (i.e. parainfluenza 3), mycoplasmas or chlamydiae. Pre-disposing environmental factors such as stress due to transportation or cold may also be needed (Jones et al., 1989). Due to the lack of facilities in Jordan to conduct a vaccine trial which involved experimental challenge with P. haemolytica, the current investigation of sero-conversion against some virulence factors of P. haemolytica, i.e. soluble surface antigens (the capsule) and iron regulating OMPs was carried out. Studies of the specificity of immunity to P. haemolytica in mice have shown that soluble surface capsular antigens play the major role in determining the specificity of the immune response (Biberstein and Thompson, 1965). In the current study, after the first vaccination a negative or very low titre was detected by IHA test. This may indicate that one inoculation is not enough to induce a high antibody titre. The development of a higher titre with mean positive values after the booster dose against all serotypes tested indicates that the vaccine induced sero-conversion against the capsular antigens of P. haemolytica. These results are in agreement with those of Jones et al. (1989) where a low antibody titre was detected by IHA test in six lambs, following three injections 28 days apart with a vaccine containing heat killed and SSE of P. haemolytica A2. Similarly, Purdy and Foster (1991) reported that 32 days after intra-bronchial inoculation of 10 goats with 106 CFU/ml P. haemolytica A1, a low IHA antibody titres was detected and also that inoculated animals were protected from challenge with the homologous serotype. However, the low titer detected in this study may be attributed to an age factor. Donachie (1994) reported that a vaccine prepared from the capsule and OMPs from serotype A2 inoculated subcutaneously into ewes stimulated high titres of anti-capsular antibodies while serum collected from similarly vaccinated 12-week-old lambs did not show high anti-capsular antibodies. Another interpretation for the low IHA titre detected could be due to the lack of sensitivity of the test, as Burrells et al. (1979) showed that the ELISA test was 160 times more sensitive than the IHA test in measuring antibody titre against P. haemolytica A1 in sera of vaccinated SPF lambs.
The results of the ELISA showed that the same sera collected 2 and 4 weeks after the first vaccination gave equivocal titers against the outer membrane IRPs-A2 and 35 kDa protein. Four weeks after the booster dose all lambs showed a positive titre against the outer membrane IRP-A2 and 35 kDa protein. The results of the ELISA are in agreement with the results of the SDS immunoblot shown in this study, where the outer membrane IRPs of 70 and 100 kDa molecular weight were seen after 2 and 4 weeks of the first vaccination and more clearly after the booster dose. In addition, clear bands of the major OMPs of 30 and 42 kDa were detected and also a band of 55 kDa. The IRPs had been considered as virulence factors of P. haemolytica A2 (Donachie and Gilmour, 1988) and the presence of antibodies to IRPs significantly enhanced protection against challenge with P. haemolytica A2 in the SPF lambs which indicates that these IRPs are immunogenic factors (Gilmour et al., 1991). In addition, the OMPs have been found to induce synthesis of protective serum antibodies to P. haemolytica serotypes A2, A7 and A9 (Mosier et al., 1989 and Sabri et al., 2000). Therefore, in the present study, the development of antibody titers against OMPs and IRPs in lambs of the vaccinated group could be considered as a useful indication for the efficacy of the vaccine. The current study describes the detection of antibodies against two IRPs of 70 and 100 kDa and against the major OMPs of 30 and 42 kDa. In addition, a band was detected at 55 kDa. These findings are in agreement with those of Squire et al. (1984) and Donachie and Gilmour (1988) who described immunoblots of OMPs of P. haemolytica A2 recovered directly from the pleural fluid of an infected sheep. This was reacted with pooled sera taken from lambs which had recovered from P. haemolytica infection and expressed protein bands of the same molecular weight as those protein bands detected in this study. Also, the finding of the 30 kDa protein are in agreement with those of Craven et al. (1991) who showed that antibodies detectable by Western immunoblot developed in cattle against the 30 kDa OMP when they were vaccinated with live P. haemolytica A1. In addition, Craven et al. (1991) found that the high antibody response to the 30 kDa protein significantly correlated with resistance to challenge by the homologous serotype. The authors concluded that the 30 kDa
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protein may be important in inducing immunity to P. haemolytica. Failure to detect the 35 kDa protein in the immunoblotting of sera from vaccinated lambs against A2-OMP was due to the need for the whole cell antigen to be used in the immunoblot, as the 35 kDa protein is mainly located in the periplasmic space (Lainson et al., 1991). Nevertheless, serum obtained from one vaccinated lamb 4 weeks after the booster dose showed a clear band at the 35 kDa and the presence of antibodies against the 35 kDa protein in sera of vaccinated lambs was confirmed in the ELISA results of this study. The 35 kDa protein has been found to be expressed in natural infections and is immunogenic in sheep and mice (Lainson et al., 1991). Use of the IRPs-A2 outer membrane in ELISA and in the SDS immunoblot to detect antibodies against IRPs in all A serotypes present in the tested vaccine is appropriate, because the major OMPs profile of all A serotypes are similar (Donachie et al., 1984). The IRPs profile to T serotypes is different and is expressed at 37 and 78 kDa. However, T serotypes of P. haemolytica are of secondary importance in causing pneumonic pasteurellosis in sheep and goats (Murry et al., 1992). This study showed that the vaccine induced sero-conversion against surface soluble capsular antigens, major OMPs and IRPs of P. haemolytica in the vaccinated lambs. Further investigations are needed to evaluate the occurrence of pneumonic pasteurellosis in vaccinated and unvaccinated groups of animals by recording the clinical and lung lesions score (Donachie et al., 1986; Jones et al., 1989) after post-mortem examination. However, this approach would only be meaningful if pneumonic pasteurellosis can be confirmed in the flock by microbiological and pathological methods.
5. Conclusion This investigation showed that the tested vaccine induced sero-conversion against surface soluble antigen (the capsule), major OMPs and IRPs of P. haemolytica in vaccinated lambs. Also it showed that one injection of the vaccine is not enough to induce a high antibody titre. A repeat dose 1 month after the first vaccination appears to be needed to develop higher titre against the capsule, OMPs and IRPs.
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References Al-Tarazi, Y.H.M., 1995. Some Aspects of Pneumonia in Sheep and Goats in Jordan with Special Reference to Pneumonic Pasteurellosis. Ph.D. Thesis, Royal Veterinary College, University of London, pp. 121–167. Biberstein, E.L., Thompson, D.A., 1965. Type specificity of immunity to Pasteurella haemolytica infection in mice. J. Comp. Pathol. 75, 331–337. Burrells, C., Wells, D.W., Dawson, A. Mcl., 1979. The quantitative estimation of antibody to Pasteurella haemolytica in sheep sera using a microenzyme linked immunosorbent (ELISA). Vet. Microbiol. 3, 291–301. Carter, G.R., Chengappa, M.M., Roberts, A.W., William Claus, G., Rikihisa, Y., 1995. In: Pasteurella and Francisella, Essential of Veterinary Microbiology, 5th Edition. Williams and Wilkins, Baltimore, USA, pp. 171–179. Craven, R.C., Confer, A.W., Centry, M.J., 1991. Cloning and expression of a 30 kDa surface antigen of Pasteurella haemolytica. Vet. Microbiol. 27, 63–78. Donachie, W., 1994. In: Proceedings of the International Conference on Abstract Book and Programme: Vaccine Development Against Pasteurella haemolytica Infections in Sheep, Haemophilus, Actinobacillus and Pasteurella (H.A.P.), Edinburgh, 31 July–4 August 1994, pp. 121–167. Donachie, W., Gilmour, N.J.L., 1988. Sheep antibody response to cell wall antigens expressed in vivo by P. haemolytica serotype A2. FEMS Microbiol. Lett. 56, 271–276. Donachie, W., Jones, G.E., 1982. The ELISA, Enzyme Linked Immunosorbent Assay in Veterinary Research and Diagnosis. In: Wardley, R.C., Crowther, J.R. (Eds.), The Hague: Martinus Nijhoff, pp. 102. Donachie, W., Fraser, J., Quirie, M., Gilmour, N.J.L., 1984. Studies on strains of Pasteurella haemolytica not typable by the indirect haemagglutination test. Res. Vet. Sci. 37, 188–193. Donachie, W., Burrells, C., Sutherland, A.D., Gilmour, J.S., Gilmour, N.J.L., 1986. Immunity of specific pathogen-free lambs to challenge with an aerosol of Pasteurella haemolytica biotype A serotype. Part 2. Pulmonary antibody and cell responses to primary and secondary infections. Vet. Immunol. Pathol. 11, 265–279. Fraser, J., Donachie, W., Quirie, M., Gilmour, N.J.L., 1983. Rapid indirect haemagglutination test for serotyping Pasteurella haemolytica. J. Clin. Microbiol. 18, 206–207. Gilmour, N.J.L., Martin, W.B., Sharp, J.M., Thompson, D.A., Wells, P.W., Donachie, W., 1983. Experimental immunisation of lambs against pneumonic pasteurellosis. Res. Vet. Sci. 35, 80–86. Gilmour, N.J.L., Donachie, W., Sutherland, A.D., Gilmour, J.S., Jones, G.E., Quirie, M., 1991. Vaccine containing iron-regulated proteins of P. haemolytica A2 enhances protection against experimental pasteurellosis in lambs. Vaccine 9, 137–140. Jones, G.E., Donachie, W., Sutherland, A.D., Knox, Gilmour, 1989. Protection of lambs against experimental pneumonic pasteurellosis by transfer of immune serum. Vet. Microbiol. 20, 59–71.
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Lainson, F.A., Harkins, D.C., Wilson, C.F., Sutherland, A.D., Murray, J.E., Donachie, W., Baird, G.D., 1991. Identification and localisation of an iron regulated 35 kDa protein of P. haemolytica serotype A2. J. Gen. Microbiol. 137, 219–226. Mosier, D.A., Simon, K.R., Confer, A.W., Panciera, R.J., Clinkenbeard, 1989. Serum IgG and IgM antibody response in cattle to antigens of Pasteurella haemolytica. Vet. Immunol. Immunopathol. 22, 53–65. Murry, J.E., Davies, R.C., Lainson, F.A., Wilson, C.F., Donachie, W., 1992. Antigenic analysis of iron-regulated proteins in Pasteurella haemolytica A and T biotypes by immunoblotting reveals biotype-specific epitopes. J. Gen. Microbiol. 138, 283– 288. Purdy, C.W., Foster, G.S., 1991. Comparison of four immune variables and pulmonary lesions of goats with intrapulmonary
exposure and subsequent intrathoracic challenge exposure with Pasteurella haemolytica. Am. J. Vet. Res. 52, 1214– 1220. Sabri, M.Y., Zamri-Saad, M., Mutalib, A.R., Israf, D.A., Muniandy, N., 2000. Efficacy of an outer membrane protein of pasteurella haemolytica A2, A7 or A9-enriched vaccine against intratracheal challenge exposure in sheep. Vet. Microbiol. 73 (1), 13–23. Squire, P.G., Smiley, D.W., Crosskell, R.B., 1984. Identification and extraction of Pasteurella haemolytica membrane proteins. Infect. Immun. 45, 667–673. Voller, A., Bidwell, D.E., Bartlett, A., 1979. In: The Enzyme Linked Immunosorbent Assay (ELISA): Practical Aspects, Sponsored and Available From: Dynatech Europe, Borough House. Rue du Pre, Guernsey, GB, pp. 30–33.
The efficacy of an experimental Pasteurella hemolytica vaccine as measured by sero-conversion in Awassi lambs in Jordan Y.H.M. Al-Tarazi∗ Department of Veterinary Basic Sciences, Faculty of Veterinary Medicine, Jordan University of Science and Technology, P.O. Box 3030, Irbid 22110, Jordan
Abstract An inactivated iron regulating protein (IRP) vaccine containing nine Pasteurella haemolytica serotypes was used. Two groups of Awassi lambs were used during the experiment. One group of 10 lambs was vaccinated and the second unvaccinated group of 10 lambs considered as negative control. Blood samples were collected on day 0, 2 and 4 weeks (post-vaccination) and 4 weeks after a revaccination. Antibody titers against capsular antigen of P. haemolytica serotypes A1, A2, A6, A7 and T3 was evaluated for both groups of lambs by indirect haemagglutination test (IHT). Antibody titers against P. haemolytica A2 outer membrane IRPs and the 35 kDa proteins was evaluated by enzyme linked immunosorbent assay (ELISA) and by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Results showed that the vaccine used induced sero-conversion against the capsular antigen, major outer membrane proteins (OMPs) and IRPs of P. haemolytica in the vaccinated lambs. Results also showed that one inoculation of the vaccine was insufficient to induce high antibody titers. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Pasteurella haemolytica; Vaccine; Lambs; ELISA; Jordan
1. Introduction Vaccines against pasteurellosis in sheep have been available for many years, but their efficacy has been equivocal (Donachie, 1994). Vaccine research on pneumonic pasteurellosis in sheep has primarily focused on Pasteurella haemolytica serotype A2 (Donachie et al., 1986). In the early 1980s, vaccine research showed that specific pathogen free (SPF) lambs could be protected against pneumonic pasteurellosis caused by serotypes A1, A6 and A9 using a vaccine containing a sodium salicylate extract (SSE) of these serotypes (Gilmour et al., 1983). It showed a high degree of protection which was ∗ Tel.: +962-2-7201000x22009; fax: +962-2-709-5123. E-mail address: [email protected] (Y.H.M. Al-Tarazi).
serotype-specific and antibodies were raised against bacterial cell surface antigens such as outer membrane proteins (OMPs), lipopolysaccharide and the capsule (Donachie et al., 1984). A similar vaccine comprising serotype A2 did not show protection exceeding 50% (Gilmour et al., 1983). Research towards an effective P. haemolytica A2 vaccine, arose from studies using SPF lambs which were experimentally infected by aerosol with the A2 serotype. These lambs recovered following antibiotic treatment and were subsequently immune to challenge with the Parainfleunza Type 3 (PI3) virus and P. haemolytica serotype A2 (Donachie et al., 1986). The sera of these lambs were found to contain antibodies against different virulence factors of P. haemolytica, including antibodies against the leukotoxin, the capsule and the lipopolysaccharide.
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Other research work has been carried on the iron regulating protein (IRP) antigens, which are present in the outer membrane of P. haemolytica A2 recovered from the pleural fluid of SPF lambs experimentally infected with A2. They can also be detected in P. haemolytica strains grown on iron depleted media. They are absent in the OMPs of P. haemolytica grown in nutrient broth (Donachie and Gilmour, 1988). Vaccines prepared from IRPs and SSE of P. haemolytica significantly enhance protection against A2 challenge, confirming its role in inducing immunity (Gilmour et al., 1991). In the current investigation, a newly developed vaccine available from a commercial company was used because anecdotal evidence suggested that pneumonic pasteurellosis in sheep and goats in Jordan was not prevented by available vaccines.
2. Materials and methods 2.1. Iron regulating protein vaccine This was an experimental, IRP vaccine, containing killed P. haemolytica of serotypes A1, A2, T3, T4, A6, A7, A9, T10 and T15. The viable counts for each serotype used in the vaccine was 5 × 108 CFU/ml. They had been grown in iron deprived media. The vaccine contained aluminium hydroxide as an adjuvant. 2.2. Animals Two groups of Awassi lambs were used, one group of 10 animals were vaccinated with the experimental vaccine and 10 unvaccinated animals served as a control group. The 3-month-old lambs were obtained from ewes which had not been vaccinated against P. haemolytica and were chosen at random. The animals were kept under a closed rearing system in separate pens at the Tafila fattening station in Jordan. 2.3. Vaccination schedules
2.4. Blood sampling This was done on day 0, 2 and 4 weeks before second vaccination and 4 weeks after the booster from all lambs included in the study. 2.5. Antibody measurements The antibody titers were measured for P. haemolytica serotypes A1, A2, T3, A6, A7 and A9 for both groups of lambs by the indirect haemagglutination test (IHT), which was performed in microtiter plates by a method modified by Fraser et al. (1983). The antibody titers against P. haemolytica serotype A1 outer membrane IRPs and the 35 kDa protein were measured by an enzyme linked immunosorbent assay (ELISA) as described by Donachie and Jones (1982). The microtiter plates were coated with 100 mg per well outer membrane IRPs and with 100 mg per well 35 kDa protein. The positive control used was anti-P. haemolytica serotype A1 anti-serum from a convalescent sheep. The positive control sera were used at dilutions of 1:10 to 1:50,000 each dilution in two wells. The optical density (OD) was read at 492 nm. The mean of the OD of two readings for each dilution was calculated and allocated “∗” or “+” rating. The scheme started from the highest reading which was designated Table 1 The optical density (OD) of the positive control P. haemolytica anti-A1 sheep serum tested against outer membrane iron regulating proteins by ELISA Serum dilutions
Mean of OD (from duplicate readings)
Interpretation
1:10 1:50 1:100 1:500 1:1000 1:5000 1:10000 1:50000
Over 2.303 2.022 1.083 0.704 0.270 0.178 0.117
∗a ++++b +++c ++d +e ±f Negative Negative
a
Two injections of 2 ml each of the vaccine were given subcutaneously, on day 0 and 4 weeks later to one group, while the other group remained unvaccinated as controls.
Highly positive. Very strong. c Positive. d Intermediate. e Weak positive. f Unclear. b
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Table 2 The optical density (OD) readings of the positive control P. haemolytica anti-A1 sheep serum tested against 35 kDa protein by ELISA
3. Results
Serum dilution
Mean of OD (from duplicate readings)
Interpretation
1:10 1:50 1:100 1:500 1:1000 1:5000 1:10000 1:50000
Over 2.235 1.674 0.669 0.412 0.209 0.077 0.017
∗a +++++b ++++c +++d ++e +f ±g Negative
No antibody titers were detected in the sera of the IRP vaccine group on day 0 nor in the sera from day 0 and 2, 4 and 8 weeks of the unvaccinated group for all P. haemolytica serotypes tested. In the vaccinated group, a titre 1:8 was detected against serotypes A1, A2, A7 and A9 2 weeks post-vaccination (Table 3). Other serum samples either did not show sero-conversion or gave a value of 1:4. The arithmetic mean for the antibody titre for all sera tested against different serotypes was <8. Four weeks after vaccination more animals showed sero-conversion for all serotypes tested, with higher means than those found after 2 weeks (Table 4). Eight weeks after the first vaccination and 4 weeks following re-vaccination, 80% of the sera tested for serotypes A1, A6, A7, and 70, 60 and 60% for serotypes T3, A2 and A9 showed a titres <1:32, respectively. The mean of the antibody titre for all serotypes tested ranged from 8.8 to 13.6 (Table 5).
a
Highly positive. Very strong positive. c Very strong. d Positive. e Intermediate. f Weak positive. g Unclear. b
as ∗ (highly positive) through +++++ (very strong positive) to negative as illustrated in Tables 1 and 2. The negative control was sera from a SPF lamb derived from a non-vaccinated ewe and used at dilutions of 1:50 in eight wells. The serum samples of all animals in the trial were also diluted in 1:50. Antibodies against IRPs were also tested by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting according to the method described by Donachie and Gilmour (1988).
3.1. IHA test results
3.2. ELISA test results The mean OD readings of the positive control for the OMPs and 35 kDa protein are shown in Tables 1 and 2. The mean OD readings of the negative control for the OMPs was 0.185 and for the 35 kDa protein was 0.021. Reading double that of the negative control was considered as positive (Voller et al., 1979).
Table 3 Number of sera collected 2 weeks after vaccination tested with IHA showing sero-conversion against P. haemolytica serotypes and the mean titre Serotype tested
A1a A2 T3 A6 A7 A9
Serum dilutions (no. of sera reacting) 0
1/4
1/8
5 3 7 3 1 3
3 4 3 7 5 3
2 3 0 0 4 4
Percentage of positive sera tested
Mean titre and S.E.
20 30 0 0 40 40
2.8 4 1.2 2.88 5.2 4.4
± ± ± ± ± ±
1.04 1.03 0.61 0.366 0.85 1.107
Positive control was rabbit anti-sera which gave a titre (>1:256) for each serotype tested; positive titre considered from (1:8); S.E., standard error. a Ten serum samples were tested.
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Table 4 Number of sera collected 4 weeks after first vaccination tested with IHA showing sero-conversion against P. haemolytica serotypes and the mean titre Serotype tested
Serum dilution (no. of sera reacting)
A1a A2 T3 A6 A7 A9
0
1/4
1/8
1/16
0 0 5 0 0 0
4 5 3 6 3 4
4 3 2 4 7 4
2 2
2
Percentage of positive sera tested
Mean titre and S.E.
60 50 20 40 70 60
8 7.6 2.8 5.6 6.8 8
± ± ± ± ± ±
1.46 1.51 1.04 0.65 0.611 1.46
Positive control was rabbit anti-sera which gave a titre (>1:256) for each serotype tested; positive titre considered from (1:8); SE, standard error. a Ten serum samples were tested.
In order to interpret the immunological response of each vaccinated lamb, the OD reading at day 0 of the pre-vaccination sera was subtracted from other OD readings of sera collected at 2, 4 and 8 weeks of the IRP vaccine and control groups. The OD reading of the control group showed no change between pre-vaccination and the 8 weeks readings. The subtracted OD readings were compared with the positive and negative control. The strength of the immunological response was interpreted by comparing it to the mean of OD reading of the positive control at different dilutions (Tables 1 and 2). In the vaccinated group, the immunological response against the outer membrane IRP was interpreted as follows: 2 weeks after the first vaccination, two lambs showed an intermediate response, three
showed a weak positive, three showed an unclear reaction and two lambs showed negative. After 4 weeks from the first vaccination one lamb exhibited an intermediate response, seven were weakly positive and two were unclear. Four weeks following revaccination, six lambs showed weak positive and four intermediate immunological response (Table 6). In the vaccinated group, the immunological response against the 35 kDa OMP was as follows: Two weeks after the first vaccination, eight lambs showed unclear results and two were negative. After 4 weeks, five lambs showed weak positive results. But after 4 weeks following the booster dose, all lambs showed immunological responses to the 35 kDa protein (Table 7).
Table 5 Number of sera collected 8 weeks after first vaccination tested with IHA showing sero-conversion against P. haemolytica serotypes and the mean titre Serotype tested
A1a A2 T3 A6 A7 A9
Serum dilutions (no. of sera reacting) 0
1/4
1/8
1/16
0 0 0 0 0 0
2 4 3 2 2 1
3 3 2 5 4 4
5 3 4 2 2 2
1/32
1 1 2 3
Percentage of positive sera tested
Mean titre and S.E.
80 60 70 80 80 60
11.2 08.8 10 11.2 13.6 8
± ± ± ± ± ±
1.66 1.66 2.9 2.65 3.33 1.46
Positive control was rabbit anti-sera which gave a titre (>1:256) for each serotype tested; positive titre were considered from (1:8); S.E., standard error. a Ten serum samples were tested.
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Table 6 The subtracted optical density (OD) readings of sera from 10 lambs (IRP vaccine group) tested by ELISA against P. haemolytica serotype A2 outer membrane iron regulating proteins at different times after vaccination Tag no.
OD/day 0
OD/2 weeks
Intera
OD/4 weeks
Inter
OD/8 weeks
Inter
405 407 408 417 419 428 431 437 439 447
0 0 0 0 0 0 0 0 0 0
0.598 0.439 0.327 0.191 0.304 0.296 1.058 0.194 0.880 0.782
+ + ± − ± ± ++ − ++ +
0.607 0.453 0.414 0.351 0.433 0.391 0.958 0.277 0.813 0.727
+ + + ± + + ++ ± + +
0.878 0.469 0.493 0.468 0.525 0.473 1.357 0.399 1.132 1.134
++ + + + + + ++ + ++ ++
a
Inter, interpretation of OD reading according to the scheme (Table 1).
Table 7 The subtracted optical density (OD) readings of sera from 10 lambs (IRP vaccine group) tested by ELISA against the 35 kDa protein at different time after vaccination Tag no.
OD/day 0
OD/2 weeks
Intera
OD/4 weeks
Inter
OD/8 weeks
Inter
405 407 408 417 419 428 431 437 439 447
0 0 0 0 0 0 0 0 0 0
0.075 0.041 0.042 0.073 0.079 0.055 0.176 0.053 0.026 0.072
± ± ± ± ± ± + ± − ±
0.124 0.034 0.071 0.067 0.100 0.128 0.113 0.177 0.043 0.072
+ − ± ± + + + + ± ±
0.251 0.292 0.224 0.160 0.441 0.308 0.240 0.548 0.177 0.373
+ + + + ++ ++ + +++ + ++
a
Inter, interpretation of OD readings according to the scheme (Table 2).
3.3. SDS-PAGE immunoblotting
4. Discussion
The immunoblotting profile of the iron regulating OMP of P. haemolytica A2 reacted with sera from vaccinated lambs showed bands of major OMPs which were distinct from those sera of the control group or sera from the test group collected pre-vaccination, at regions 30, 42 and 55 kDa. Additional bands at 70 and 100 kDa were more prominent especially after 4 weeks following the booster in the vaccinated sera, but either absent or weakly expressed in the unvaccinated or pre-vaccination sera. No protein bands were detected in the SPF lamb from the control group sera.
Potentially, the vaccine tested may be useful because many of the serotypes of P. haemolytica present in the vaccine are known to cause pneumonic pasteurellosis in sheep in Jordan (Al-Tarazi, 1995). P. haemolytica is a commensal organism of the nasopharynx of ruminant animals (Carter et al., 1995). This raises the possibility of the presence of antibodies against these organisms in conventional animals used in vaccine trials that may complicate the interpretation of the results. To overcome this problem the use of SPF lambs is desirable in vaccine trials and in challenge experiments. Other complexities in conducting
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challenge experiments are the need for a high challenge dose of P. haemolytica with aerosol exposure for not <15 min within a chamber and pre-infection by other biological pre-disposing factors such as viruses (i.e. parainfluenza 3), mycoplasmas or chlamydiae. Pre-disposing environmental factors such as stress due to transportation or cold may also be needed (Jones et al., 1989). Due to the lack of facilities in Jordan to conduct a vaccine trial which involved experimental challenge with P. haemolytica, the current investigation of sero-conversion against some virulence factors of P. haemolytica, i.e. soluble surface antigens (the capsule) and iron regulating OMPs was carried out. Studies of the specificity of immunity to P. haemolytica in mice have shown that soluble surface capsular antigens play the major role in determining the specificity of the immune response (Biberstein and Thompson, 1965). In the current study, after the first vaccination a negative or very low titre was detected by IHA test. This may indicate that one inoculation is not enough to induce a high antibody titre. The development of a higher titre with mean positive values after the booster dose against all serotypes tested indicates that the vaccine induced sero-conversion against the capsular antigens of P. haemolytica. These results are in agreement with those of Jones et al. (1989) where a low antibody titre was detected by IHA test in six lambs, following three injections 28 days apart with a vaccine containing heat killed and SSE of P. haemolytica A2. Similarly, Purdy and Foster (1991) reported that 32 days after intra-bronchial inoculation of 10 goats with 106 CFU/ml P. haemolytica A1, a low IHA antibody titres was detected and also that inoculated animals were protected from challenge with the homologous serotype. However, the low titer detected in this study may be attributed to an age factor. Donachie (1994) reported that a vaccine prepared from the capsule and OMPs from serotype A2 inoculated subcutaneously into ewes stimulated high titres of anti-capsular antibodies while serum collected from similarly vaccinated 12-week-old lambs did not show high anti-capsular antibodies. Another interpretation for the low IHA titre detected could be due to the lack of sensitivity of the test, as Burrells et al. (1979) showed that the ELISA test was 160 times more sensitive than the IHA test in measuring antibody titre against P. haemolytica A1 in sera of vaccinated SPF lambs.
The results of the ELISA showed that the same sera collected 2 and 4 weeks after the first vaccination gave equivocal titers against the outer membrane IRPs-A2 and 35 kDa protein. Four weeks after the booster dose all lambs showed a positive titre against the outer membrane IRP-A2 and 35 kDa protein. The results of the ELISA are in agreement with the results of the SDS immunoblot shown in this study, where the outer membrane IRPs of 70 and 100 kDa molecular weight were seen after 2 and 4 weeks of the first vaccination and more clearly after the booster dose. In addition, clear bands of the major OMPs of 30 and 42 kDa were detected and also a band of 55 kDa. The IRPs had been considered as virulence factors of P. haemolytica A2 (Donachie and Gilmour, 1988) and the presence of antibodies to IRPs significantly enhanced protection against challenge with P. haemolytica A2 in the SPF lambs which indicates that these IRPs are immunogenic factors (Gilmour et al., 1991). In addition, the OMPs have been found to induce synthesis of protective serum antibodies to P. haemolytica serotypes A2, A7 and A9 (Mosier et al., 1989 and Sabri et al., 2000). Therefore, in the present study, the development of antibody titers against OMPs and IRPs in lambs of the vaccinated group could be considered as a useful indication for the efficacy of the vaccine. The current study describes the detection of antibodies against two IRPs of 70 and 100 kDa and against the major OMPs of 30 and 42 kDa. In addition, a band was detected at 55 kDa. These findings are in agreement with those of Squire et al. (1984) and Donachie and Gilmour (1988) who described immunoblots of OMPs of P. haemolytica A2 recovered directly from the pleural fluid of an infected sheep. This was reacted with pooled sera taken from lambs which had recovered from P. haemolytica infection and expressed protein bands of the same molecular weight as those protein bands detected in this study. Also, the finding of the 30 kDa protein are in agreement with those of Craven et al. (1991) who showed that antibodies detectable by Western immunoblot developed in cattle against the 30 kDa OMP when they were vaccinated with live P. haemolytica A1. In addition, Craven et al. (1991) found that the high antibody response to the 30 kDa protein significantly correlated with resistance to challenge by the homologous serotype. The authors concluded that the 30 kDa
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protein may be important in inducing immunity to P. haemolytica. Failure to detect the 35 kDa protein in the immunoblotting of sera from vaccinated lambs against A2-OMP was due to the need for the whole cell antigen to be used in the immunoblot, as the 35 kDa protein is mainly located in the periplasmic space (Lainson et al., 1991). Nevertheless, serum obtained from one vaccinated lamb 4 weeks after the booster dose showed a clear band at the 35 kDa and the presence of antibodies against the 35 kDa protein in sera of vaccinated lambs was confirmed in the ELISA results of this study. The 35 kDa protein has been found to be expressed in natural infections and is immunogenic in sheep and mice (Lainson et al., 1991). Use of the IRPs-A2 outer membrane in ELISA and in the SDS immunoblot to detect antibodies against IRPs in all A serotypes present in the tested vaccine is appropriate, because the major OMPs profile of all A serotypes are similar (Donachie et al., 1984). The IRPs profile to T serotypes is different and is expressed at 37 and 78 kDa. However, T serotypes of P. haemolytica are of secondary importance in causing pneumonic pasteurellosis in sheep and goats (Murry et al., 1992). This study showed that the vaccine induced sero-conversion against surface soluble capsular antigens, major OMPs and IRPs of P. haemolytica in the vaccinated lambs. Further investigations are needed to evaluate the occurrence of pneumonic pasteurellosis in vaccinated and unvaccinated groups of animals by recording the clinical and lung lesions score (Donachie et al., 1986; Jones et al., 1989) after post-mortem examination. However, this approach would only be meaningful if pneumonic pasteurellosis can be confirmed in the flock by microbiological and pathological methods.
5. Conclusion This investigation showed that the tested vaccine induced sero-conversion against surface soluble antigen (the capsule), major OMPs and IRPs of P. haemolytica in vaccinated lambs. Also it showed that one injection of the vaccine is not enough to induce a high antibody titre. A repeat dose 1 month after the first vaccination appears to be needed to develop higher titre against the capsule, OMPs and IRPs.
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