Abstract:
The present invention declaims the use of  Pasteurella  lipoprotein E (PlpE) as a subunit vaccine and the use of vaccines containing PlpE to protect animals from diseases caused by  P. multocida . The results of vaccination and challenge experiments showed that mice and chickens immunized with PlpE were completely protected animals from challenge infection with 10 1 -10 3  LD 50  of  P. multocida  and no adverse effect was observed.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention relates to the use of  Pasteurella  lipoprotein E (PlpE) in a subunit vaccine, and the use of vaccines containing PlpE to protect animals from diseases caused by  P. multocida.    
       BACKGROUND OF THE INVENTION 
       [0002]      Pasterurella multocida  is classified into family Pasteurellaceae, genus  Pasteurella , and  multocida  means “causing many kinds of animal disease” in Latin. In the past, the taxonomic situation of Pasteurellaceae is ambiguous and arguable. However, according to the sequence analysis of 16s-RNA, Pasteurellaceae is classified into a gamma subgroup of purple bacteria, which is Gram-negative facultative anaerobe, and homologous to Enterobacteriaaceae.  Pasteurella multocida  is an important pathogen of domestic animals and an opportunistic pathogen of humans. Human infections with  P. multocida  largely arise from the bite of an infected carnivore, but other types of infections are occasionally reported (Hubbert W T, et al., Am J Public Health 1970; 60:1109-17).  P. multocida  has wide host range, and is the causative agent of fowl cholera in domestic birds, haemorrhagic septicaemia in cattle or sheep, and atrophic rhinitis in pigs (Hunt M L, et al., Vet Microbiol 2000, 72:3-25). Most  P. multocida  strains are highly pathogenic to murine and rabbits, which can result in acute septic symptoms at an infection dose of 1-10 CFU. 
         [0003]    In domestic birds,  P. multocida  causes acute septic disorders in turkey, chicken, duck, and goose, which may lead to a great loss in economy. Of the five capsular serotypes (A, B, D, E and F) and 16 LPS serotypes, fowl cholera is mainly caused by serotypes A:1, A:3 and A:4 (Glisson J R. In: Saif Y M, editor. Diseases of poultry. Iowa State University Press, Ames, Iowa, 2003:657-90). Although both live-attenuated vaccines and bacterins are available, outbreaks of fowl cholera continue to occur. Live-attenuated vaccines have the disadvantage of reversion to virulence, while bacterins do not protect hosts against heterologous challenge (Bierer B W, Derieux W T, Poult Sci 1972; 51:408-16; and Rebers P A, Heddleston K L, Avian Dis 1977; 21:50-56). These disadvantages call for the development of a new type of vaccine for  P. multocida.    
         [0004]    In a previous report, a lipoprotein, designated  Pasteurella  lipoprotein E (PlpE), from  Mannheimia haemolytica  (formerly known as  Pasteurella haemolytica ), was found to be highly immunogenic in cattle (Confer A W, et al., Vaccine 2003; 21:2821-9). PlpE is a lipid-modified, surface-exposed outer membrane protein that is important in complement-mediated killing of  M. haemolytica  (Pandher K, et al., Infect Immun 1998; 66:5613-9). Addition of recombinant PlpE to the commercial  M. haemolytica  vaccine markedly enhanced the vaccine-induced resistance against experimental challenge with serotypes 1 and 6. A bioinformatics-based sequence search showed that a gene annotated PlpE is present in the published genome sequence of  P. multocida  strain pm-70 (serotype A:3) (May B J, et al., Proc Natl Acad Sci USA 2001; 98:3460-5). This gene has the potential to encode a lipoprotein of 335 amino acids that has 24.3% sequence identity with PlpE of  M. haemolytica  and 19.1% identity with OmlA of  A. pleuropneumoniae . It has not yet been determined whether the PlpE of  P. multocida  could serve as a vaccine antigen by using mice and/or chicken as animal models. 
         [0005]    Therefore, the present invention first finds out a new use of  Pasteurella  lipoprotein E (PlpE) in controlling infective diseases caused by  P. multocida , or related disorders thereof, and further develops a subunit vaccine for protecting animal from diseases caused by  P. multocida . The subunit vaccine of present invention is characterized by using recombinant PlpE protein as active antigen, which has no adverse side effect of forming fibrosarcoma. Additionally, the present subunit vaccine of  P. multocida  can provide a predominant protective effect against the challenge of homologous and/or heterologous serotypes in immunized animals over traditional inactivated or live-attenuated vaccines. 
       SUMMARY OF THE INVENTION 
       [0006]    In one aspect, the present invention provides a subunit vaccine for protecting animal from diseases caused by  P. multocida , which is characterized by comprising  Pasteurella  lipoprotein E (PlpE) as antigen, and a veterinary acceptable adjuvant. In one embodiment, the  Pasteurella  lipoprotein E is a protein having the amino acid sequence as listed in SEQ ID EF219452-EF219457 (SEQ ID No. 1 to 6), or an amino acid sequence with similarity of more than 90% to the amino acid sequence as listed in SEQ ID EF219452-EF219457 (SEQ ID No. 1 to 6). In another embodiment, the disease caused by  P. multocida  is fowl cholera in domestic birds, haemorrhagic septicaemia in cattle, or atrophic rhinitis in pigs. 
         [0007]    In another aspect, the present invention provides a use of  Pasteurella  lipoprotein E in controlling infective diseases caused by  P. multocida , or related disorders thereof. The disease caused by  P. multocida  infection may be fowl cholera in domestic birds, haemorrhagic septicaemia in cattle, or atrophic rhinitis in pigs. In other animals, such as mice, rabbits, cats, or dogs,  P. multocida  infection may cause haemorrhagic septicaemia. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0008]      FIG. 1 . Expression and purification of recombinant PlpE (r-PlpE) and recombinant PlpB (r-PlpB) in  E. coli . Note that r-PlpB was used as the control antigen in this report. (A) Coomassie blue-stained SDS-PAGE of recombinant proteins from crude extract or purified samples. Lane M represents the molecular mass markers. The lanes marked control contain crude extract of  E. coli  that harbored no recombinant plasmid. (B) Immunoblot of duplicated gel probed with mouse anti-hexa-histidine monoclonal antibody. The bands corresponding to r-PlpB, r-PlpE, and their processed products are indicated by arrows. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0009]    The present invention will be further defined by reference to the following examples, which are set forth to assist in understanding the invention and should not be construed as specifically limiting the invention. Therefore, any modification or derivative made without departing from the spirit of this invention will be considered to fall within the scope of the invention. 
       Example 
     Example 1 
     Preparation of Recombinant Lipoprotein E (r-PlpE) of  P. multocida  in  E. coli    
       [0010]    A. Bacterial Strains and Genomic DNA Extraction of  P. multocida    
         [0011]      P. multocida  standard strains X-73 (A:1), P-1059 (ATCC 15742) (A:3), and P-1662 (A:4) were grown at 37° C. in brain-heart infusion (BHI) broth (Difco Laboratories, MI, USA) for 18-24 hours. Bacterial genomic DNA was isolated using the DNeasy tissue kit (Qiagen, Hilden, Germany). 
         [0000]    B. Genetic Cloning and Expression Vector Construction for Recombinant Lipoprotein E (r-PlpE) 
         [0012]    One set of primers, P1/P2, was used to amplify the plpE gene from  P. multocida  strain X-73. The amplified genes were then used for expressing recombinant PlpE (r-PlpE) in  E. coli . The sequences of primers P1/P2 were as follows. P1: 5′-CCA TGG GCA TGA AAT TAA CAA AAC TTT T-3′, and P2: 5′ AAG CTT CCA ACC TTT AAC TAC ACC ACC-3′. These primers contained restriction enzyme (NcoI or XhoI) cutting sites at their 5′-ends (underlined sequences), followed by sequences specific to plpE. The PCR product was cloned into the expression vector pET28a according to the manufacturer&#39;s instructions (Novagen, Inc. Madison, Wis.) to obtained a plasmid designated as pX73-PlpE. The identity of the insert in pET28a was verified by DNA sequence analysis. The amino sequence of r-PlpE protein is described as in SEQ ID 1. 
         [0000]    C. Expression and Purification of Recombinant Lipoprotein E (r-PlpE) 
         [0013]    Recombinant plasmid pX73-PlpE obtained in section B was transformed into  E. coli  strain BL21 (DE3) and recombinant protein was purified by nickel chromatography as previously described (Chang P C, et al., 2002, Avian Dis 46:570-80). In brief,  E. coli  strain BL21 (DE3) harboring the recombinant plasmid was cultured in LB medium at 37° C. until absorbance at 600 nm reached 0.6. Isopropylthio-β-D-thiogalactose (IPTG) was added to a final concentration of 0.4 mM, and the culture was grown for another 3 hrs. Cells were pelleted by centrifugation at 3000×g for 20 min, and resuspended in 2 ml of binding buffer (20 mM pH 7.9 Tris, 5 mM imidazole, 500 mM NaCl). The suspension was sonicated and centrifuged at 12,000×g for 40 min. The supernatant was collected and loaded into a column containing 2.5 ml of “His-bind” resin (Novagen). The column was washed with 25 ml of binding buffer and 15 ml of washing buffer (20 mM pH 7.9 Tris, 50 mM imidazole, 500 mM NaCl) to remove the unbound proteins. The bound protein was eluted with 15 ml of eluting buffer (20 mM pH 7.9 Tris, 250 mM imidazole, 500 mM NaCl), only the first 3 ml of the elute was collected. Protein concentration was determined using a “Protein Assay” kit (BIO-RAD, Hercules, Calif., USA). 
         [0014]    The expression product and purity of the recombinant protein was observed by SDS-PAGE and Western blotting analysis, respectively. The results were showed in  FIG. 1 . The r-PlpB in  FIG. 1  was a noneffective subunit vaccine used as a control in the experiments. The plpB and plpE genes were cloned from  P. multocida  strain X-73 (serotype A:1) and then expressed in  E. coli  as recombinant proteins. The recombinant PlpB (r-PlpB) and PlpE (r-PlpE) contained a hexa-histidine-tag attached at their carboxyl termini. 
         [0015]    As showed in  FIG. 1 , the calculated molecular masses of r-PlpB and r-PlpE were 31.5 and 38.7 kDa, respectively. Both r-PlpB and r-PlpE contained a signal peptide of 20 amino acid residues at their amino termini, and after cleavage of the signal peptide, the matured r-PlpB and r-PlpE had molecular masses of 29.3 and 36.3 kDa, respectively. As shown in  FIG. 1A , r-PlpB and r-PlpE, with the expected molecular masses, were highly expressed in  E. coli  and were purified using nickel chromatography ( FIG. 1A ). 
         [0016]    Western blot analyses using anti-hexa-histidine monoclonal antibody showed that this monoclonal antibody reacted with r-PlpB and r-PlpE ( FIG. 1B ); moreover, both r-PlpB and r-PlpE produced two bands on the blot, the major band having the molecular mass corresponding to the full-length r-PlpB or PlpE (31.5 or 38.7 kDa), whereas the minor band had the molecular mass of the mature form (29.3 or 36.3 kDa) ( FIG. 11B ). This result suggests that some processing of r-PlpB and r-PlpE occurred in  E. coli . The bands corresponding to r-PlpB, r-PlpE and their processed products are indicated by arrows. 
       Example 2 
     Evaluation of Protective Effects of r-PlpE Subunit Vaccine in BALB/c Mice Model 
       [0017]    Three experiments were conducted in BALB/c mice. In experiments 1 and 2, groups of 6-week-old mice were immunized subcutaneously with 10 microgram of purified r-PlpB or r-PlpE in aluminum hydroxide adjuvant (Sigma-Aldrich Co., MO, USA), either alone or together with a bacterin composed of 1.25×10 7  or 2.5×10 7  CFU of formalin-inactivated  P. multocida  X-73 (A:1). Two weeks after immunization, mice were challenged with subcutaneous injection of 10-20 LD 50  of strain X-73. In experiment 3, mice were immunized as described for experiments 1 and 2. Two weeks after immunization, mice were challenged with subcutaneous injection of 10 LD 50  of strains P-1059 (A:3) or P-1662 (A:4), or strain T2A5 (which is a designated challenge strain used in drug inspection in Taiwan). All mice challenged were observed for 10 days and their survival rates were recorded. The results are summarized in Table 1. For statistical analysis, the survival rates were compared by Chi-squared tests using SAS software (SAS Institute Inc., Cary, N.C., USA). The mean times to death were compared using the GLM procedure in the same software. Differences were considered significant when p&lt;0.05. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Results of immunization and challenge tests in BALB/c mice. 
               
             
          
           
               
                   
                 Challenge strain 
                   
               
               
                 Immunized with 1   
                 and dose 2   
                 % survival 3   
               
               
                   
               
             
          
           
               
                 Exp. 1 
                 X-73 (A:1) 
                   
                   
               
               
                 Control 
                 30 CFU 
                 0 
                 (0/6) a   
               
               
                 r-PlpB (10 microgram) 
                 30 CFU 
                 0 
                 (0/10) a   
               
               
                 r-PlpE (10 microgram) 
                 30 CFU 
                 100 
                 (10/10) b   
               
               
                 Inactivated X-73 (2.0 × 10 8  CFU) 
                 30 CFU 
                 100 
                 (6/6) b   
               
               
                 Inactivated X-73 (1.25 × 10 7  CFU) 
                 30 CFU 
                 17 
                 (1/6) a   
               
               
                 Inactivated X-73 (1.25 × 10 7  CFU) + 
                 30 CFU 
                 30 
                 (3/10) a   
               
               
                 r-PlpB (10 microgram) 
               
               
                 Inactivated X-73 (1.25 × 10 7  CFU) + 
                 30 CFU 
                 10 
                 (10/10) b   
               
               
                 r-PlpE (10 microgram) 
               
               
                 Exp. 2 
                 X-73 (A:1) 
               
               
                 Control 
                 60 CFU 
                 0 
                 (0/6) a   
               
               
                 r-PlpB (10 microgram) 
                 60 CFU 
                 10 
                 (1/10) a   
               
               
                 r-PlpE (10 microgram) 
                 60 CFU 
                 80 
                 (8/10) bc   
               
               
                 Inactivated X-73 (2.5 × 10 7  CFU) 
                 60 CFU 
                 50 
                 (3/6) ab   
               
               
                 Inactivated X-73 (2.5 × 10 7  CFU) + 
                 60 CFU 
                 40 
                 (4/10) ab   
               
               
                 r-PlpB (10 microgram) 
               
               
                 Inactivated X-73 (2.5 × 10 7  CFU) + 
                 60 CFU 
                 90 
                 (9/10) c   
               
               
                 r-PlpE (10 microgram) 
               
               
                 Exp. 3 
                 P-1059 (A:3) 
               
               
                 Control 
                 35 CFU 
                 0 
                 (0/5) a   
               
               
                 r-PlpE (10 microgram) 
                 35 CFU 
                 100 
                 (10/10) b   
               
               
                   
                 P-1662 (A:4) 
               
               
                 Control 
                 30 CFU 
                 0 
                 (0/5) a   
               
               
                 r-PlpE (10 microgram) 
                 30 CFU 
                 100 
                 (10/10) b   
               
               
                   
                 T2A5 (A:1) 
               
               
                 Control 
                 3 CFU 
                 0 
                 (0/5) a   
               
               
                 r-PlpE (10 microgram) 
                 3 CFU 
                 80 
                 (8/10) b   
               
               
                   
               
               
                   1 Mice in the control group were not immunized. 
               
               
                   2 The LD 50  of strains X-73 and P1662 in mice was &lt;3 CFU, and that of P-1059 was 3.5 CFU. 
               
               
                   3 Different alphabetical characters indicate significant difference (p &lt; 0.05) between groups. 
               
             
          
         
       
     
         [0018]    In experiment 1, mice immunized with 10 microgram of purified r-PlpE were completely protected (100% survival) (Table 1, experiment 1). In contrast, mice immunized with 10 microgram of purified r-PlpB were not protected (0% survival) against challenge infection with 30 CFU (&gt;10 LD 50 ) of X-73 (serotype A:1). Mice immunized with a bacterin composed of 2×10 8  CFU of formalin-inactivated X-73 were completely protected (100% survival), whereas those immunized with a bacterin composed of a lower dose (1.25×10 7  CFU) of X-73 were not protected (17% survival) (Table 1, experiment 1). To investigate whether r-PlpB or r-PlpE could enhance the protective efficacy of the bacterin, mice were immunized with a bacterin composed of 1.25×10 7  CFU of X-73 supplemented with 10 microgram r-PlpB or r-PlpE. The results showed that r-PlpB did not significantly enhance the protective efficacy of the bacterin (30% survival, p&gt;0.05) whereas r-PlpE did (100% survival, p&lt;0.05) (Table 1, experiment 1). 
         [0019]    In experiment 2, the challenge dose of X-73 was increased to 60 CFU (&gt;20 LD 50 ) and a bacterin composed of 2.5×10 7  CFU of X-73 was used. The results showed that mice immunized with 10 microgram of r-PlpB were not protected (10% survival) whereas those with 10 microgram of r-PlpE were significantly protected (80% survival, p&lt;0.05) (Table 1, experiment 2). Mice immunized with a bacterin composed of 2.5×10 7  CFU of X-73 were moderately protected (50% survival). Mice immunized with the same bacterin supplemented with r-PlpB showed a survival rate of 40%, which was similar to that with the bacterin alone. In contrast, mice immunized with the bacterin supplemented with r-PlpE showed a survival rate of 90%, which was significantly higher than that with the bacterin alone (p&lt;0.05) (Table 1, experiment 2). 
         [0020]    In experiment 3, strains P-1059 (serotype A:3) and P-1662 (serotype A:4) were used as the challenge strains. The results showed that mice immunized with 10 microgram of r-PlpE were completely protected against challenge infection with 10 LD 50  of P-1059 or &gt;10 LD 50  of P-1662 (Table 1, experiment 3). This result showed that r-PlpE, which was derived from X-73 (serotype A:1), conferred cross protection on mice against challenge with strains of serotypes A:3 and A:4. Additionally, mice immunized with 10 microgram of r-PlpE showed a survival rate of 90% when challenge with strain T2A5 (A:1), which was up to the proof inspection standard (survival rate of 60%) (p&lt;0.05) (Table 1, experiment 3). 
       Example 3 
     Evaluation of Protective Effects of r-PlpE Subunit Vaccine in SPF Chicken Model 
       [0021]    Three experiments in SPF chickens were conducted. In experiment 1, groups of 3-week-old SPF chickens were immunized subcutaneously with 100 microgram of purified r-PlpB or r-PlpE in complete Freund&#39;s adjuvant (Sigma-Aldrich). Three weeks after the primary immunization, a booster immunization was conducted, and three weeks after booster immunization, chickens were challenged with intramuscular injection of 3.6×10 3  CFU of strain X-73 or 5.5×10 8  CFU of strain P-1662. In experiments 2 and 3, chickens were immunized subcutaneously twice with 125 microgram of a crude extract of r-PlpE in a double emulsion adjuvant with a 3-week interval between immunizations. The crude extract was prepared by sonicating the pellet of  E. coli  that expressed r-PlpE. The double emulsion adjuvant contained Marcol 52 oil (63%), Arlacel A (7%), and Tween 80 (1.5%). Three weeks after booster immunization, chickens were challenged by intramuscular injection of 3.6×10 3 −3.6×10 6  CFU of strain X-73 or 5.5×10 7 −5.5×10 9  CFU of strain P-1662. All chickens challenged were monitored for 10 days and the survival rates were recorded. The results are summarized in Table 2. For statistical analysis, the survival rates were compared by Chi-squared tests using SAS software (SAS Institute Inc., Cary, N.C., USA). The mean times to death were compared using the GLM procedure in the same software. Differences were considered significant when p&lt;0.05. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Results of immunization and challenge tests in SPF chickens 
               
             
          
           
               
                   
                 Challenge strain 
                   
                 Mean time to 
               
               
                 Immunized with  1   
                 and dose  2   
                 % survival  3   
                 death (days)  3   
               
               
                   
               
             
          
           
               
                 Exp. 1 
                 X-73 (A:1) 
                   
                   
                   
               
               
                 Control 
                 3.6 × 10 3  CFU 
                 30 
                 (3/10) a   
                 3.3 a   
               
               
                 Purified r-PlpB (100 microgram) 
                 3.6 × 10 3  CFU 
                 50 
                 (5/10) a   
                 4.2 a   
               
               
                 Purified r-PlpE (100 microgram) 
                 3.6 × 10 3  CFU 
                 100 
                 (10/10) b   
                 NA 
               
               
                   
                 P-1662 (A:4) 
               
               
                 Control 
                 5.5 × 10 8  CFU 
                 13 
                 (1/8) a   
                 4.1 a   
               
               
                 Purified r-PlpB (100 microgram) 
                 5.5 × 10 8  CFU 
                 13 
                 (1/8) a   
                 5.4 a   
               
               
                 Purified r-PlpE (100 microgram) 
                 5.5 × 10 8  CFU 
                 63 
                 (5/8) b   
                 5.7 a   
               
               
                 Exp. 2 
                 X-73 (A:1) 
               
               
                 Control 
                 3.6 × 10 3  CFU 
                 25 
                 (2/8) a   
                 2.7 a   
               
               
                   
                 3.6 × 10 4  CFU 
                 0 
                 (0/8) a   
                 1.9 a   
               
               
                   
                 3.6 × 10 5  CFU 
                 0 
                 (0/8) a   
                 2.0 a   
               
               
                 Crude extract r-PlpE 
                 3.6 × 10 3  CFU 
                 100 
                 (8/8) b   
                 NA 
               
               
                 (125 microgram) 
                 3.6 × 10 4  CFU 
                 75 
                 (6/8) b   
                 7.0 b   
               
               
                   
                 3.6 × 10 5  CFU 
                 75 
                 (6/8) b   
                 5.0 b   
               
               
                   
                 3.6 × 10 6  CFU 
                 88 
                 (7/8) b   
                 4.0 b   
               
               
                 Exp. 3 
                 P-1662 (A:4) 
               
               
                 Control 
                 5.5 × 10 7  CFU 
                 25 
                 (2/8) a   
                 5.7 a   
               
               
                   
                 5.5 × 10 8  CFU 
                 13 
                 (1/8) a   
                 4.1 a   
               
               
                   
                 5.5 × 10 9  CFU 
                 0 
                 (0/8) b   
                 2.9 a   
               
               
                 Crude extract r-PlpE 
                 5.5 × 10 7  CFU 
                 50 
                 (4/8) a   
                 4.8 a   
               
               
                 (125 microgram) 
                 5.5 × 10 8  CFU 
                 50 
                 (4/8) a   
                 4.8 a   
               
               
                   
                 5.5 × 10 9  CFU 
                 50 
                 (4/8) a   
                 5.3 a   
               
               
                   
               
               
                   1  Immunization was conducted twice with a 3-week interval. Chickens in the control group were not immunized. 
               
               
                   2, 3  Different alphabetical characters indicate significant difference (p &lt; 0.05) between immunization and control groups challenged with the same dose of X-73 or P-1662. 
               
             
          
         
       
     
         [0022]    In experiment 1, chickens immunized twice with 100 microgram of purified r-PlpB showed a survival rate of 50% against challenge with X-73, but this survival rate was not significantly higher than that of the control group (30% survival, p&gt;0.05). In contrast, chickens immunized twice with 100 microgram of purified r-PlpE showed a survival rate of 100%, which was significantly higher than that of the control group (p&lt;0.05) (Table 2. experiment 1). This result suggests that r-PlpE but not r-PlpB conferred protection on chickens. A similar conclusion was reached when strain P-1662 was used as the challenge strain (Table 2, experiment 1). 
         [0023]    In experiments 2 and 3, a crude extract of r-PlpB and r-PlpE ( FIG. 1A ), instead of the purified one, was used as the antigen. Moreover, a double emulsion adjuvant, instead of Freund&#39;s complete adjuvant, was used as the emulsifying agent. These modifications were carried out to reduce the cost and labor required for preparation and administration of the antigen. The results showed that chickens immunized twice with 125 microgram of crude extract of r-PlpE had a survival rate of 75-100% against challenge with 3.6×10 3 −3.6×10 6  CFU of strain X-73. These rates were significantly higher than those of the control group (p&lt;0.05) (Table 2, experiment 2). Moreover, the mean time to death of chickens immunized with r-PlpE was significantly longer than that of the control group (p&lt;0.05) (Table 2, experiment 2). 
         [0024]    In experiment 3, P-1662 was used as the challenge strain. The results showed that chickens immunized with 125 microgram of crude extract of r-PlpE had a survival rate of 50% against challenge with 5.5×10 7 −5.5×10 9  CFU of strain P-1662. These rates were not significantly higher than those of the control groups (p&gt;0.05), except when the challenge dose of P-1662 was 5.5×10 9  (p&lt;0.05) (Table 2, experiment 3). The mean times to death of immunized chickens were not significantly longer than those of the control groups (p&gt;0.05) (Table 2, experiment 3). 
       Example 4 
     Nucleotide Sequences of plpE from Reference Strains of  P. multocida    
       [0025]    Two primers, P3 and P4, were used to amplify the plpE genes from different reference strains of  P. multocida , X-73 (A:1), pm-70 (A:3), P-470 (A:3), P-61 (D:3), P-1059 (A:3), P-1662 (A:4), and ATCC 12948 (D:11). The two primers were designed on the basis of the published genome sequence of  P. multocida  strain pm-70. P3 and P4 amplified the 1.0 kb DNA fragment containing the plpE gene. The sequences of primers P3 and P4 were as follows. P3: 5′-ATG AAA CAA ATC GTT TTA AA-3′, and P4: 5′-TTA TTG TGC TTG GTG ACT TT-3′. The PCR products were purified with a QIAquick gel extraction kit (Qiagen) and sequenced from both directions using a Big Dye Terminator cycle sequencing kit (Applied Biosystems, Foster City, Calif.) in an automatic sequencer (ABI-3730XL DNA Analizer, Applied Biosystems). Sequences were compiled using the Seqman program in the LASERGENE package (DNASTAR Inc. Madison, Wis., USA). Open reading frames prediction and antigenic index assay were performed using the GeneQuest and Protean programs from the same package. Nucleotide and protein sequences were searched for homology in GenBank using the BLAST program provided by NCBI, USA. 
         [0026]    The nucleotide sequences of the plpE gene determined in this study are available in GenBank under the accession numbers EF219452-EF219457 (corresponding to the SEQ ID Nos. 1 to 6 in the appending sequence listing). All these plpE genes were found to contain an open reading frame of 1008-1019 nt, encoding a PlpE protein of 37.4-37.7 kDa. Pair-wise sequence comparison showed that these PlpE proteins had 90.8-100% sequence identity with each other, suggesting that PlpE might serve as a cross-protective antigen. This is the first report of a recombinant  P. multocida  antigen that confers cross protection on animals. Therefore, a protein having the amino acid sequence as listed in SEQ ID No. 1 to 6, or an amino acid sequence with similarity of more than 90% to the amino acid sequence as listed in SEQ ID No. 1 to 6, is considered to exhibit highly similar protective effects, and contemplates to be included in the subunit vaccine of present invention. 
         [0027]    The above examples are given by way of illustration only, and should not be construed as specifically limiting the scope of present invention. Any variation of the invention described and claimed herein, including the substitution of all equivalents, which would be within the purview of those skilled in the art, is to be considered to fall within the scope of the invention incorporated herein. 
         [0028]    The strain  E. coli  BL21 (DE3) containing the recombinant vector X73-plpE of the invention was deposited with the Agricultural Research Service Culture Collection (NARRL), on Feb. 29, 2008, as Deposit No. NARRL B-50117.