Immunogenicity of iron-regulated outer membrane proteins of Pasteurella multocida B:2 in mice model

Transcription

1 Indian Journal of Experimental Biology Vol. 48, December 2010, pp Immunogenicity of iron-regulated outer membrane proteins of Pasteurella multocida B:2 in mice model Subhash Kharb & Shiv Charan* Department of Veterinary Microbiology, CCS Haryana Agricultural University, Hisar , India Received 12 April 2010; revised 28 June 2010 The present study was conducted to investigate the role of iron-regulated outer membrane proteins (IROMP) of Pasteurella multocida B:2 in mice as potential immunogens. Outer membrane proteins extracted from P. multocida B:2 grown under normal (OMP) and iron-deficient (IROMP) conditions were subjected to discontinuous SDS-PAGE. Nine polypeptides of MW ranging from 85.1 to 16.7 kda from OMP preparations and two additional polypeptides of MW 95.4 and 89.1 kda from IROMP preparations were observed with bands of MW 37.2 and 34.7 kda as major proteins. Mice were immunized twice with OMP, IROMP-enriched fractions and whole cell lysate (WCL) via subcutaneous route at day 0 and 21. Antibody titers were determined from sera collected at weekly interval and protection was studied against challenge using 10 2 cfu of P. multocida two weeks after secondary immunization via intranasal and subcutaneous routes. IROMP and OMP immunized mice provoked significant antibody responses and IROMP induced higher antibody responses. IROMP and OMP immunized mice showed protection (100%) upon intranasal challenge and a protection (84%) following subcutaneous challenge as compared to high mortality (84%) in control mice. These results indicate that OMP enriched with IROMP fractions can be superior means of immunization. Keywords: Iron-regulated proteins, Immunogenicity, Outer membrane proteins, Pasteurella multocida B:2, Mice Pasteurella multocida is a Gram-negative organism, which has been classified into five serogroups on the basis of capsular antigens 1-3 and 1-16 serotypes on the basis of somatic antigens 4. P. multocida serotypes B and E cause haemorrhagic septicaemia (HS) in cattle and buffaloes in south-east Asia and Africa, respectively which is a disease of major economic importance. It is a septicaemic form of pasteurellosis with short incubation period. Killed vaccines are generally used for the control of HS in endemic areas. These vaccines have certain limitations viz. alum precipitated vaccines induce immunity for shorter period (4 to 6 months), whereas oil adjuvanted vaccines being too viscous are difficult to inject in animals particularly during herd vaccination 5,6. Significant outbreaks are still occurring in endemic areas after vaccination. Live vaccines have also been used in some areas but outbreaks attributed to vaccine strains 7 and sometimes reversal to virulent form limit the use of these vaccines 8,9. Live vaccine using B:3,4 strain has been reported to be unsafe for primary vaccination in young calves 10. *Correspondent author Telephone: ; Fax: shivcharan55@yahoo.com Various workers aimed at developing more effective vaccines by identifying potential protective antigen(s) have shown that capsule is a poor immunogen in animals 11,12. However, capsular polysaccharide induced higher level of protection in rabbits when conjugated with bovine serum albumin 13. LPS has been found to be partially immunogenic in mammals, but major immunogen in birds. Mice, cattle and rabbits have not been readily protected against P. multocida infection following immunization with LPS 14,15. Various studies on outer membrane proteins (OMP) have shown that OMP are major immunogens against homologous challenge in mammals 15,16. It has been observed that OMP that are expressed in vivo under iron-deficient conditions known as ironregulated outer membrane proteins (IROMP) are involved in protective immunity and these IROMP have been found to be more immunogenic than OMP. IROMPs of P. multocida type:a have been found to be immunogenic not only against homologous challenge but also against heterologous challenge in mice, birds, rabbits and calves 15, Since HS outbreaks are alarming it as major devastating disease in Asian countries, the present study has been

2 1182 INDIAN J EXP BIOL, DECEMBER 2010 undertaken in a mouse model to exploit the benefits of the IROMPs as immunogens in field animals eventually. Materials and Methods Bacterial strain and growth conditions P. multocida B:2 (P 52 - vaccine strain) maintained in the department was grown on brain heart infusion (BHI) agar enriched with 7% (v/v) sheep blood by incubating at 37ºC for 18 h. Stock culture was purified and passaged in mice by inoculating the cultured BHI broth via subcutaneous route as per the standard protocol 20. Purity of the culture was confirmed by Gram staining and biochemical tests. For preparation of IROMP, bacteria were grown in BHI broth containing 2,2'-bipyridyl, an iron chelator (Fluka Chemicals Co., USA) at a concentration of mm 19. Whole cell lysates (WCL) were prepared by centrifugation at 3,000 g for 30 min at 4 0 C and sonication of the suspended bacterial pellet (4x, 30 sec, 4ºC). Extraction of OMP and IROMP-enriched fractions OMP and IROMP-enriched fractions were extracted from bacterial culture 21. Briefly, cells were harvested by centrifuging at 3,000 g for 30 min at 4ºC, washed thrice and re-suspended in 10 mm, HEPES buffer (ph 7.4). The suspended cells were sonicated 4x, 30 sec at 4ºC. Intact cells and debris were removed by centrifugation at 1,700 g for 20 min. The supernatant was collected and ultracentrifuged at 100,000 g for 60 min at 4ºC. The pellets, which contained the total membranes, obtained after removing supernatant were treated with 1% sodium N-laurylsarcosine (Sarkosyl; Sigma Chemicals Co., USA) in 10 mm, HEPES buffer for 1 h at 37ºC. The detergent insoluble membranes were harvested by ultracentrifugation at 100,000 g for 60 min at 4ºC. The pellets were resuspended in PBS (ph 7.4), detergent insoluble membranes were aliquoted and stored at -20ºC. The protein concentrations of OMP, IROMP-enriched fractions and WCL were quantitated 22 using GeNei TM Protein Estimation Kit (Bangalore Genei Limited, India). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis The proteins were characterized by SDS-PAGE employing 12.5% separation gel 23. A concentration of 20 µg/well from each preparation was used for characterization of the protein profiles. The MW of the polypeptide bands was interpolated from the graph drawn between relative fronts (R f values) vs. log MW of markers run in parallel with proteins. Animals and immunogens Swiss-Webster female mice of 6-8 week age were used in experiments after obtaining permission from Institutional Animal Ethical Committee (IAEC). The immunogens (IROMP-enriched fractions, OMP and WCL) were prepared as water in oil emulsion using Freund s Complete Adjuvant (FCA; Bangalore Genei Limited, India) for primary immunization and Freund s Incomplete Adjuvant (FIA; Bangalore Genei Limited, India) for secondary immunization. Immunization schedule To determine the antibody response in mice to IROMP, OMP and WCL, forty eight Swiss-Webster mice were divided into four groups as IROMP group, OMP group, WCL group and a control group (12 mice/group). Mice were immunized at day 0 with 100 µg of protein preparations in a volume of 200 µl per mouse and at day 21 with 50 µg of protein preparations via subcutaneous route. Sera were collected at weekly interval from tail vein and stored at -20ºC for further analysis. Challenge studies To study whether IROMPenriched fractions and OMP protect the animals from challenge, mice were challenged with 10 2 cfu of live P. multocida by further dividing each group into two sub-groups (6 mice/sub-group). After 2 weeks of secondary immunization (day 35), one sub-group was challenged via intranasal route with 10 2 cfu of live P. multocida employing 20 µl/mouse. Second sub-group of mice was challenged via subcutaneous route with 10 2 cfu of live P. multocida by injecting 200 µl/mice. The mortality pattern in all the four groups of mice was recorded following challenge. Sera were collected at day 5 and 10 post-challenge. Indirect-enzyme linked immunosorbent assay (ELISA) ELISA was performed to determine antibody response in mice immunized with IROMPenriched fractions, OMP and WCL 24. Briefly, OMP (OMP-ELISA) and WCL (WCL-ELISA) were coated on the microtitre plate wells (Maxisorb 96-well flat bottom ELISA plate, Nunc, Denmark) at a concentration of 1 µg/well. Test sera were diluted 1:100 in PBST and bound antibody was then detected using 1:2500 dilution of affinity purified goat antimouse IgG-horse radish peroxidase (HRPO) conjugate (Sigma Chemicals Co., USA) in PBST. Enzymatic reaction was assayed using orthophenylene diamine. Concentrations of coating

3 KHARB & CHARAN: IMMUNOGENICITY OF IRON-REGULATED MEMBRANE PROTEINS 1183 antigen, test sera and IgG-HRPO were determined by checkerboard titration. Antibody responses were expressed as ELISA Units (EUs). One EU is considered as highest dilution of hyper-immune serum giving an absorbance value equal to mean of negative sera with addition of double the standard deviation (Mean ± 2SD). A standard curve was generated by plotting O.D. values of hyper-immune sera (two-fold serial diluted) vs. EUs (on log 2 basis) and a straight line was drawn from the curve. EUs of test sera were calculated using regression equation 25. EUs were converted from log 2 basis to normal values using Microsoft Office Excel Worksheet. Determination of bacterial load in organs To measure the bacterial load in different organs of immunized and control mice after challenge, mice from each group were euthanized at day 5 and 10 post-challenge. Spleen, liver and lung were collected individually and ten-fold dilutions were cultured on 1.5% BHI agar. The titers of bacteria were expressed as cfu per gram of tissue. Statistical analysis Mean antibody responses to IROMP-enriched fractions, OMP and WCL were compared using one way analysis of variance (ANOVA) between the groups and antibody responses at various intervals within the group were compared using Student s t test 26. Chi-square test was used for comparing protection in groups after challenge 16. Results Characterization of OMP and IROMP Protein profiles of OMP and IROMP were compared employing 12.5% SDS-PAGE. The Coomassie brilliant blue (CBB-250) stained gel revealed nine OMP bands of MW 85.1, 61.7, 57.5, 46.2, 40.7, 37.2, 34.7, 28.2, and 16.7 kda. On the basis of band thickness and stain intensity, bands of MW 37.2 and 34.7 kda appeared to be major proteins of OMP and IROMP preparations. Two extra bands of MW 95.4 and 89.1 kda approx. were also seen in IROMPenriched fractions along with nine OMP (Fig. 1). Antibody response studies To determine the immunogenic potential of IROMP, OMP, and WCL in mice, antibody responses against OMP and WCL of P. multocida were monitored employing preoptimized indirect ELISA. In OMPELISA, the antibody responses were increased significantly at day 7 (P < 0.05, Student s t test) in IROMP group with mean EU 5.6 and at day 14 in OMP and WCL groups (P < 0.05) with mean EU 5.7 and 7.1 as compared to control group with mean EU 1.9. Antibody titers were further increased significantly on 21 days postimmunization (DPI) in all immunized groups (P<0.01). Anamenestic antibody response was observed 7 days post-secondary immunization (28 DPI) in all immunized groups of mice whereas control group of mice did not show any antibody response. Antibody responses continued to increase on 35 DPI with mean EU 170, 96, and 62.6 in IROMP, OMP and WCL groups respectively. Following intranasal and subcutaneous challenge employing 10 2 cfu of P. multocida, antibody titers decreased on 40 DPI in IROMP and OMP immunized mice, however titers were not lower than peaks of antibody responses after primary immunization (Fig. 2). In WCL-ELISA, statistically significant rise in antibody titers in all immunized groups was observed at day 14 with mean EU 6.3, 4.5, and 6.8 respectively Fig. 1 Coomassie Briliant Blue stained SDS-PAGE (12.5%) profile of outer membrane proteins of P. multocida, grown under normal and iron-deficient conditions along with standard MW markers. (Lane A-molecular markers; Lane B-polypeptide profile of OMP and Lane C-polypeptide profile of IROMP)

4 1184 INDIAN J EXP BIOL, DECEMBER 2010 (P < 0.05) when compared with control (EU 2.2). The antibody response significantly increased on 21 DPI. A significantly enhanced antibody response was observed in all immunized groups following secondary immunization with mean EU 34.4, 19.6, and 42.6 in IROMP, OMP, and WCL immunized mice respectively. After intranasal and subcutaneous challenge with live bacteria employing 10 2 cfu, significantly enhanced antibody response was observed in WCL immunized mice as compared to IROMP and OMP immunized mice (Fig. 2). However, among the mice immunized with IROMP and OMP, a significantly higher antibody response was detected in the mice immunized with IROMP. Challenge studies Protective ability of various preparations of P. multocida viz. IROMP, OMP and WCL was studied and mortality patterns in different immunized groups of mice and control mice were observed following challenge with 10 2 cfu of P. multocida. Following intranasal challenge, IROMP and OMP immunized groups showed a protection (100%), but WCL group showed a protection of 84% as compared to high mortality (84%) in control mice. Statistical analysis employing Chi-square test showed that immunized mice were protected significantly as compared to control mice (IROMP and OMP groups having P < 0.01 and WCL group having P < 0.05). However, differences in protection between the immunized groups were not significant. Following subcutaneous challenge, immunized mice showed protection (84%). Chi-square analysis of results showed significant protection (P<0.05) when compared with control group (Table 1). Bacterial titration in different organs To study the efficacy of different preparations of P. multocida in inhibiting the multiplication of bacteria, bacterial titration in lung, liver and spleen of mice from all groups was performed. Following intranasal challenge, no mortality was observed in IROMP and OMP immunized groups of mice, however, in WCL immunized group of mice, one out of six mice died. High titers of bacteria were found in lung, liver and spleen of this mouse as well as control mice which died after challenge, while all other mice immunized with IROMP, OMP and WCL, which survived challenge, no organism was detected in their organs when titrated on day 5 and day 10 post-challenge. Similarly, mice unable to survive following subcutaneous challenge from the immunized groups, showed high titers of bacteria in lung, liver and spleen as in the control group of mice (Table 2). In all the immunized mice, which survived of challenge, no Fig. 2 Antibody responses against OMP and WCL of P. multocida in groups of mice immunized with IROMP, OMP and WCL followed by intranasal or subcutaneous challenge. (A)-Antibody responses against OMP following intranasal challenge; (B)-Antibody responses against OMP following subcutaneous challenge; (C)-Antibody responses against WCL following intranasal challenge; and (D)- Antibody responses against WCL following subcutaneous challenge

5 KHARB & CHARAN: IMMUNOGENICITY OF IRON-REGULATED MEMBRANE PROTEINS 1185 Table 1 Protection in mice immunized with IROMP-enriched fractions, OMP and WCL of P. multocida Groups Immunization schedule Challenge dose Post-challenge survival Primary a Secondary b (cfu) Intranasal challenge Subcutaneous challenge (µg) (µg) (%) (%) IROMP (6/6) d 84 (5/6) d OMP (6/6) 84 (5/6) WCL (5/6) 84 (5/6) Control c NSS NSS (1/6) 16 (1/6) a Primary immunization was carried out on day 0 with 100 µg/mouse in FCA via subcutaneous route. b Secondary immunization was carried out on day 21 with 50 µg/mouse in FIA via subcutaneous route. c NSS was injected in control mice with FCA and FIA at day 0 and 21, respectively. d Number of mice survived out of total mice in each group. Table 2 Bacterial titration in immunized mice after intranasal and subcutaneous challenge with 10 2 cfu of P. multocida Groups Titers on death Titers on day 5 post-challenge Titers on day 10 post-challenge Lung Liver Spleen Lung Liver Spleen Lung Liver Spleen Upon intranasal challenge with 10 2 cfu of P. multocida Group A (IROMP) No mortality occurred Nil Nil Nil Nil Nil Nil Group B (OMP) No mortality occurred Nil Nil Nil Nil Nil Nil Group C (WCL) Nil Nil Nil Nil Nil Nil Group D (Control) Mouse# Mice died within 72 h of challenge Mice died within 72 h of challenge Mouse# Mouse# Upon subcutaneous challenge with 10 2 cfu of P. multocida Group A (IROMP) Nil Nil Nil Nil Nil Nil Group B (OMP) Nil Nil Nil Nil Nil Nil Group C (WCL) Nil Nil Nil Nil Nil Nil Group D (Control) Mouse# Mice died within 72 h of challenge Mice died within 72 h of challenge Mouse# Mouse# bacteria demonstrated in their organs on day 5 and 10 post-challenge. Discussion While investigating the role of LPS, capsular polysaccharides, OMP and various extracellular enzymes, OMP have been shown to play a predominant role in protective immunity 15,16,27. Iron is a key nutrient for microorganisms as it acts as cofactor or prosthetic group in several enzyme systems. Under iron-deficient conditions, P. multocida modifies its gene expression profile and genes encoding proteins involved in iron transport get up-regulated leading to expression of high MW proteins (IROMP) on the surface of bacteria 28,29. Role of IROMP in improving the immunogenicity has been suggested to be mediated through involvement of antibodies by blocking the receptors associated with iron uptake leading to the starvation of bacteria for iron and rendering them non-viable 30. IROMP have

6 1186 INDIAN J EXP BIOL, DECEMBER 2010 been demonstrated to be immunogens of significant interest with respect to protection against fowl cholera caused by P. multocida serotype A in poultry. These IROMP have been reported to work even against the heterologous challenge 15,17,30. In order to improve the efficacy of currently used killed vaccines (Alum or Oil adjuvanted vaccines), the potentials of IROMP have been investigated in mice. When P. multocida B:2 was grown under irondeficient conditions to express IROMP, two extra polypeptide bands of MW 95.4 and 89.1 kda were expressed in addition to nine polypeptide bands of OMP on SDS-PAGE. Various workers have reported OMPs bands with some variation in number and MW, which may be due to difference in serotypes, strains and laboratory conditions. Two extra proteins of high MW 104 and 101 kda were reported in IROMPenriched fractions of P. multocida B:2 31. In poultry, OMPs extracted from the bacteria grown under ironrestricted medium of P. multocida type A revealed additional protein of 97.8 kda 17, while in P. multocida employing a bovine isolate of serogroup A:3 (Pm232), five IROMPs including one 96 kda immunodominant protein have been reported 19. Overall these studies suggest that OMP and IROMP expressed under different conditions have some variations and there is increased expression of some higher MW proteins on surface of bacteria. These extra polypeptides expressed in response to environmental stress are likely to have immunogenic potential, however, their clear-cut role in enhancing the immunity against HS is not well established. In the present study, it has been observed that the mice immunized with IROMP-enriched fractions provoked higher antibody responses vis-á-vis OMP immunized mice. The elicited responses in IROMP immunized mice were further enhanced after secondary immunization. The enhanced response in IROMP immunized mice may be because of additional high MW bands present in IROMPenriched fractions as compared to OMP. Similarly, in rabbits vaccinated intranasally with IROMP- and OMP-enriched fractions of P. multocida type A:3 found that IROMP and OMP stimulated enhanced serum and nasal antibody responses in rabbits. However, intranasal bacterial count was reduced significantly in IROMP immunized animals as compared to OMP, which suggested that incorporation of IROMPs was useful in improving the efficacy of conventional vaccines 18. Immunization studies on cattle with OMP and IROMP-enriched fractions of P. multocida A:1 reported significant induction of antibody responses in both groups with no significant correlation between lung lesion scores and antibody response. However, a significant correlation between lung lesion scores and antibody responses to 96 kda IROMP band was observed suggesting the role of antibodies against IROMPs in decreasing the severity of disease 19. In the present study, mice immunized with IROMP, OMP and WCL were challenged with 10 2 cfu of P. multocida via intranasal and subcutaneous routes, it was observed that the group of mice challenged with intranasal route was fully protective (100%). However, following subcutaneous challenge, 84% protection was observed in mice suggesting protective role of these immunogens in natural infection (Table 1). Although in terms of per cent protection, IROMP was found as good as OMP, however, IROMP induced higher antibody responses than OMP at most of the time points (Fig. 2). Expressions of additional proteins under irondeficient conditions have also been reported in various other bacteria. Under iron-deficient conditions, Yersinia species expressed various ironregulated proteins. Among them, two high MW IROMP are synthesized only in highly virulent phenotypes suggesting that these IROMP play a significant role in various microbial mechanisms 32. IROMP of Salmonella typhi has been found to protect animals against challenge in conjugation with Vi polysaccharide as least colonization of bacteria in spleen, liver, and peyer s patches was observed after immunization with IROMP and Vi antigen 33. In conclusion IROMP-enriched fractions induced enhanced antibody responses in mice. As antibodies mediate the protective immunity in HS, the vaccines incorporating IROMP are likely to provide more effective protection. Although the exact mechanism of IROMP antibodies is not well known, it is reasonable to presume that antibodies against IROMP(s) molecules will provide additional handling sites on bacteria for clearing the organism more efficiently by the host immune system. Acknowledgement Authors thank Dr S K Batra in the department and Dr B R Gulati of the National Research Centre for Equines, Hisar for critically reading the manuscript and providing ultracentrifuge facility respectively.

7 KHARB & CHARAN: IMMUNOGENICITY OF IRON-REGULATED MEMBRANE PROTEINS 1187 References 1 Carter G R, Studies on P. multocida: A haemagglutination test for identification of serological types, A J Vet Res, 16 (1955) Carter G R, A new serological type of Pasteurella multocida from Central Africa, Vet Rec, 73 (1961) Rimler R B & Rhoades K R, Serogroup-F, a new capsule serogroup of Pasteurella multocida, J Clin Microbiol, 25 (1987) Heddleston K L, Gallagher J E & Rebers P A, Fowl Cholera: Gel diffusion precipitin test for serotyping Pasteurella multocida from avian species, Avian Dis, 16 (1972) De Alwis M C L, Haemorrhagic Septicaemia A general review, Br Vet J, 148 (1992) OIE, Haemorrhagic Septicaemia. Manual of diagnostic tests and vaccines for terrestrial animals (Cambridge University Press), OIE, Manual of Standards for Diagnostic tests and Vaccines Verma R & Jaiswal T N, Haemorrhagic septicemia vaccines, Vaccine, 16 (1998) Boyce J D & Adler B, Acapsular Pasteurella multocida B:2 can stimulate protective immunity against Pasteurellosis, Infect Immun, 69 (2001) Myint A & Carter G R, Field use of live haemorrhagic septicemia vaccine, Vet Rec, 126 (1990) Dhanda M R, Immunization of cattle against haemorrhagic septicaemia with purified capsular antigens, Indian Vet J, 36 (1959) Confer A W, Immunogens of Pasteurella, Vet Microbiol, 37 (1993) Somarajan S R, Chaturvedi V K, Kumar S, Kumar B, Nair B C, Gupta P K & Pandey K D, Immunogenicity of capsular polysaccharide of Pasteurella multocida B:2 conjugated to protein carrier in rabbits, Indian J Comp Micobiol Immunol Infect Dis, 27 (2007) Rimler R B & Rhoades K R, Pasteurella multocida in Pasteurella and Pasteurellosis edited by C Adlam & J M Rutters (Academy Press Limited, London) 1989, Adler B, Chanellor R, Homchampa P, Hunt M, Ruffolo C G, Strugnell R & Wapling D, Immunity and vaccine development in Pasteurella multocida infections, J Biotechnol, 44 (1996) Pati U S, Srivastava S K, Roy S C & More T, Immunogenicity of outer membrane protein of Pasteurella multocida in buffalo calves, Vet Microbiol, 52 (1996) Chawak M M, Verma K C & Kataria J M, Characterization and immunizing properties of outer membrane proteins of Pasteurella multocida grown in iron-sufficient and ironrestricted media, Indian J Comp Microbiol Immunol Infect Dis, 22 (2001) Confer A W, Suckow M A, Montelongo M, Dabo S M, Miloscio L J, Gillespie A J & Meredita G L, Intranasal vaccination of rabbits with Pasteurella multocida A:3 outer membranes that express iron-regulated proteins, Am J Vet Res, 62 (2001) Prado M E, Dabo S M & Confer A W, Immunogenicity of IROMP of Pasteurella multocida A:3 in cattle: Molecular characterization of the immunodominant heme acquisition system receptor (HasR) proteins, Vet Microbiol, 105 (2005) Bain R V S, Studies on haemorrhagic septicaemia of cattle. IV. A preliminary examination of antigens of Pasteurella multocida type I, Br Vet J, 111 (1955) Choi-Kim K, Maheswaran S K, Felice L J & Mlolitor T W, Relationship between the iron regulated outer membrane proteins and the outer membrane proteins of in vivo grown Pasteurella multocida, Vet Microbiol, 28 (1991) Lowry O H, Rosebrough N J, Farr A L & Randall R J, Protein estimation with the folin-phenol reagent, J Biol Chem, 193 (1951) Laemmli U K, Cleavage of structural proteins during the assembly of the head of bacteriophage T 4, Nature, 227 (1970) Law B, Malone M D & Biddlecombe R A, Enzyme linked immunosorbent assay: Development and optimization. in Immunoassay: A practical guide, edited by B Law (Tayler & Francis) 1996, Carpenter A B, Enzyme-Linked Immunoassays, in Mannual of clinical immunology, edited by N R Rose, E C de Macario, J D Folds, H C Lane & R M Nakamura (ASM, Washington DC) 1997, Confer A W, Nutt S H, Dabo S M & Paniera R J, Antibody responses of cattle to outer membrane proteins of Pasteurella multocida A:3, Am J Vet Res, 57 (1996) Lu Y S, Lai W C, Pakes, S P & Stephanu C, The outer membrane of Pasteurella multocida 3:A protects rabbits against homologous challenge, Infect Immun, 59 (1991) Paustian M L, May B J & Kapur V, Pasteurella multocida gene expression in response to iron limitation, Infect Immun, 69 (2001) Boyce J D & Adler B, How does Pasteurella multocida respond to the host environment? Curr Opin Microbiol, 9 (2006) Ruffolo C G, Helen Jost B & Adler B, Iron-regulated outer membrane proteins of Pasteurella multocida and their role in immunity, Vet Microbiol, 59 (1998) Filia G, Jand S K, Arora A K & Oberoi M S, Comparative analysis of outer membrane protein profile of Pasteurella multocida serotype B:2 grown under iron regulated conditions, Indian J Anim Sci, 76 (2006) Carniel E, Mazigh D & Mollaret H H, Expression of ironregulated proteins in Yersinia species and their relation to virulence, Infect Immun, 55 (1987) Chibber S & Bhardwaj S B, Protection in a mouse peritonitis model mediated by iron-regulated outer-membrane protein of Salmonella typhi coupled to its Vi antigen, J Med Microbiol, 53 (2004) 705.