Protective immunity conferred by recombinant Pasteurella multocida lipoprotein E (PlpE)

Vaccine 25 (2007) 4140–4148

Protective immunity conferred by recombinant Pasteurella multocida lipoprotein E (PlpE) Jin-Ru Wu a , Jui-Hung Shien b , Happy K. Shieh b , Chih-Feng Chen c , Poa-Chun Chang a,∗ a

Graduate Institute of Veterinary Microbiology, National Chung Hsing University, Taichung 402, Taiwan b Department of Veterinary Medicine, National Chung Hsing University, Taichung 402, Taiwan c Department of Animal Science, National Chung Hsing University, Taichung 402, Taiwan Received 1 February 2007; received in revised form 5 March 2007; accepted 5 March 2007 Available online 20 March 2007

Abstract The genes encoding Pasteurella multocida lipoprotein E (PlpE) and lipoprotein B (PlpB) were cloned from P. multocida strain X-73 (serotype A:1) and expressed in Escherichia coli. The protective immunity conferred by recombinant PlpE (r-PlpE) and PlpB (r-PlpB) on mice and chickens was evaluated. The results showed that mice immunized with 10 ␮g of purified r-PlpE were protected (80–100% survival rate) against challenge infection with 10 or 20 LD50 of P. multocida strains X-73 (serotype A:1), P-1059 (serotype A:3) and P-1662 (serotype A:4). In contrast, mice immunized with r-PlpB were not protected. Chickens immunized with 100 ␮g of purified r-PlpE were protected (63–100% survival rate) against lethal challenge infection with strains X-73 and P-1662, whereas those immunized with r-PlpB were not. Sequence analyses showed that PlpE from different strains of P. multocida exhibited 90.8–100% sequence identity to 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. © 2007 Elsevier Ltd. All rights reserved. Keywords: Lipoprotein E; PlpE; Lipoprotein B; PlpB; Pasteurella multocida

1. Introduction Pasteurella multocida is an important pathogen of domestic animals and an opportunistic pathogen of humans. It is the causative agent of fowl cholera in domestic birds, haemorrhagic septicaemia in cattle, and atrophic rhinitis in pigs [1]. Human infections with P. multocida largely arise from the bite of an infected carnivore, but other types of infections are occasionally reported [2,3]. Of the five capsular serotypes (A, B, D, E and F) and 16 LP serotypes, fowl cholera is mainly caused by serotypes A:1, A:3 and A:4 [4]. Although both liveattenuated vaccines and bacterins are available, outbreaks of fowl cholera continue to occur. Live-attenuated vaccines have the disadvantage of reversion to virulence, while bacterins ∗

Corresponding author. Tel.: +886 4 2286 0196; fax: +886 4 2285 1741. E-mail address: [email protected] (P.-C. Chang).

0264-410X/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2007.03.005

do not protect hosts against heterologous challenge [5–9]. These disadvantages call for the development of a new type of vaccine for P. multocida [7]. Rimler [6,10] has reported that P. multocida from infected turkey tissues expresses a unique immunogen called the cross-protection antigen. The cross-protection antigen induces cross protection in turkeys against challenge infection with strains of heterologous serotypes. Recently, Rimler [11] has purified a protein of approximately 39 kDa that was believed to be one of the cross-protection antigens. In addition to Rimler’s work, Ali et al. [12] have also purified from P. multocida a 39 kDa protein that was believed to be a cross-protective antigen, but it remains unknown whether the 39 kDa proteins purified by Rimler [11] and by Ali et al. [12] are the same protein. The 39 kDa protein prepared by Rimler [11] was identified to be Pasteurella lipoprotein B (PlpB) using a peptide mass fingerprinting assay [13]; however,

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it has not been shown yet whether PlpB confers cross protection. In addition to PlpB, another lipoprotein, designated Pasteurella lipoprotein E (PlpE), from Mannheimia haemolytica (formerly known as Pasteurella haemolytica), was found to be highly immunogenic in cattle [14]. PlpE is a lipidmodified, surface-exposed outer membrane protein that is important in complement-mediated killing of M. haemolytica [15]. Addition of recombinant PlpE to the commercial M. haemolytica vaccine markedly enhanced the vaccine-induced resistance against experimental challenge with serotypes 1 and 6 [14,16,17]. Pandher et al. [15] reported that the PlpE of M. haemolytica has sequence homology (18% identity) with the OmlA of Actinobacillus pleuropneumoniae. OmlA is a lipid-modified outer membrane protein that confers protection against experimental challenge with A. pleuropneumoniae on pigs [18]. Because A. pleuropneumoniae, M. haemolytica and P. multocida are all members of the family Pasteurellaceae, it is intriguing to investigate whether P. multocida contains a protein with sequence homology to PlpE or OmlA, and whether this protein could serve as a protective antigen. 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) [19]. 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. The aims of this study were to express recombinant proteins PlpB and PlpE of P. multocida in Escherichia coli, and to determine whether or not recombinant PlpB and PlpE of P. multocida are antigens that protect mice and chickens against infection with different serotypes of P. multocida.

2. Materials and methods 2.1. Bacterial strains and DNA extraction P. multocida P-1059 (ATCC 15742) was obtained from the Bioresource Collection and Research Center in Hsinchu, Taiwan. Other strains were obtained from Dr. Chin Chen at the National Animal Research Institute in Taipei, Taiwan. Of these strains, X-73 (A:1) was isolated from chickens; P-1059 (A:3), P-1662 (A:4), CU (A:3,4), P-1072 (A:5,16), P-2100 (A:10), P-2237 (A:15) and P-2723 (A:16) were from turkeys; P-470 (A:3), P-61 (D:3), P-903 (A:11), and 12948 (D:11) were from swine; P-1573 (A:12) and P-1591 (A:13) were from humans; M-1404 (B:2) was from bison. The identities of all these strains were confirmed using species-specific and capsule-specific PCR methodology [20,21]. All strains were grown at 37 ◦ C in brain–heart infusion (BHI) broth (Difco Laboratories, MI, USA). Bacterial DNA was isolated using the DNeasy tissue kit (Qiagen, Hilden, Germany).

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2.2. Expression and purification of recombinant PlpB and PlpE Two sets of primers, P1/P2 and P3/P4, were used to amplify the plpB and plpE genes from P. multocida strain X-73. The amplified genes were then used for expressing recombinant PlpB and PlpE in E. coli. The sequences of primers P1/P2 and P3/P4 were as follows. P1: 5 -CCA TGG GCA TGA AAT TAA CAA AAC TTT T-3 , P2: 5 -AAG CTT CCA ACC TTT AAC TAC ACC ACC-3 , P3: 5 -CCA TGG GCA TGA AAC AAA TCG TTT TAA A-3 and P4: 5 -AAG CTT TTG TGC TTG GTG ACT TTT TTC-3 . These primers contained restriction enzyme (NcoI or XhoI) cutting sites at their 5 -ends (underlined sequences), followed by sequences specific to plpB or plpE. The PCR products were cloned into the expression vector pET28a according to the manufacturer’s instructions (Novagen, Inc. Madison, WI). The identity of the insert in pET28a was verified by DNA sequence analysis. Recombinant plasmids were transformed into E. coli strain BL21 (DE3) and recombinant proteins were purified by nickel chromatography as previously described [22]. 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-␤-d-thiogalactose (IPTG) was added to a final concentration of 0.4 mM, and the culture was grown for another 3 h. 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, CA, USA). 2.3. PCR amplification and sequence analysis of the plpE gene Two primers, P5 and P6, were used to amplify the plpE genes from 15 reference strains of P. multocida. The two primers were designed on the basis of the published genome sequence of P. multocida strain pm-70 [19]. P5 and P6 amplified the 1.0 kb DNA fragment containing the plpE gene. The sequences of primers P5 and P6 were as follows. P5: 5 ATG AAA CAA ATC GTT TTA AA-3 , and P6: 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, CA) in an automatic sequencer (ABI-3730XL DNA Analizer,

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Applied Biosystems). Sequences were compiled using the Seqman program in the LASERGENE package (DNASTAR Inc. Madison, WI, 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 [23]. The nucleotide sequences of the plpE gene determined in this study are available in GenBank under the accession numbers EF219452–EF219457. 2.4. Western blot Chicken anti-PlpB and anti-PlpE sera were prepared by subcutaneous immunization of 3-week-old SPF chickens with 100 ␮g of purified r-PlpB or r-PlpE in complete Freund’s adjuvant (Sigma–Aldrich). Three weeks after the primary immunization, a booster immunization was conducted, and 2 weeks after booster immunization, sera were collected. For each type of antigen, sera were collected from three chickens and pooled before Western blot analyses. The antigens were mixed with an equal volume of gel-loading buffer containing 62.5 mM Tris–HCl pH 6.8, 2% SDS, 5% 2mercaptoethanol, 25% glycerol, and 0.01% Bromophenol Blue. The samples were heated at 100 ◦ C for 10 min and then subjected to SDS-PAGE using the “Mini-PROTEIN 3” system (BIO-RAD). The proteins were transferred onto nitrocellulose membrane using the “SEMI-DRY TRANSFER” system (BIO-RAD). The membrane was blocked with 3% skimmed milk and probed with mouse anti-hexa-histidine monoclonal antibodies at 1:2000 dilution (Amersham Biosciences, Piscataway, NJ), or with chicken anti-PlpB at 1:5000 dilution, or with chicken anti-PlpE sera at 1:1000 dilution. For competitive binding assay, 10 ␮l of chicken antiPlpE sera were adsorbed with 10 ␮g of purified r-PlpE or with 40 ␮g of E. coli proteins at 37 ◦ C for 2 h. The E. coli proteins were from supernatants of crude extracts of E. coli that harbored no recombinant plasmid. Immune complexes were detected using alkaline phosphotase labeled antimouse IgG at 1:2000 dilution or anti-chicken IgG at 1:5000 dilution (Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA). 2.5. Vaccination and challenge studies in BALB/c mice and SPF chickens Three experiments were conducted in BALB/c mice. In experiments 1 and 2, groups of 6-week-old mice were immunized subcutaneously with 10 ␮g 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 × 107 or 2.5 × 107 CFU of formalin-inactivated P. multocida X-73 (A:1). Two weeks after immunization, mice were challenged with subcutaneous injection of 10–20 LD50 of strain X-73. In experiment 3, mice were immunized as described for experiments 1 and 2. Two weeks after immu-

nization, mice were challenged with subcutaneous injection of 10 LD50 of strains P-1059 (A:3) or P-1662 (A:4). The LD50 was determined by the method of Reed and Muench [24]. All mice challenged were observed for 10 days and their survival rates were recorded. Three experiments in SPF chickens were conducted. In experiment 1, groups of 3-week-old SPF chickens were immunized subcutaneously with 100 ␮g of purified r-PlpB or r-PlpE in complete Freund’s adjuvant (Sigma–Aldrich). Three weeks after the primary immunization, a booster immunization was conducted, and 3 weeks after booster immunization, chickens were challenged with intramuscular injection of 3.6 × 103 CFU of strain X-73 or 5.5 × 108 CFU of strain P-1662. In experiments 2 and 3, chickens were immunized subcutaneously twice with 125 ␮g 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-PlpB or r-PlpE. The double emulsion adjuvant contained Marcol 52 oil (63%), Arlacel A (7%), and Tween 80 (1.5%) [25]. Three weeks after booster immunization, chickens were challenged by intramuscular injection of 3.6 × 103 –3.6 × 106 CFU of strain X-73 or 5.5 × 107 –5.5 × 109 CFU of strain P-1662. All chickens challenged were monitored for 10 days and the survival rates were recorded. The animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at National Chung Hsing University. The approval numbers were IACUC 95-40 and 95-56. For statistical analysis, the survival rates were compared by Chi-squared tests using SAS software (SAS Institute Inc., Cary, NC, USA). The mean times to death were compared using the GLM procedure in the same software. Differences were considered significant when p < 0.05.

3. Results 3.1.1. Expression and purification of recombinant PlpB and PlpE 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. The calculated molecular masses of rPlpB 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). Western blot analyses using anti-hexa-histidine monoclonal antibody showed that this monoclonal antibody reacted with r-PlpB and rPlpE (Fig. 1B); moreover, both r-PlpB and r-PlpE produced

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Fig. 1. Expression and purification of r-PlpB and r-PlpE in E. coli. (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.

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. 1B). This result suggests that some processing of r-PlpB and r-PlpE occurred in E. coli.

3.2. Immunization and challenge studies in BALB/c mice Three vaccination and challenge experiments in BALB/c mice were conducted and the results are summarized in Table 1. In experiment 1, mice immunized with 10 ␮g of purified r-PlpB were not protected (0% survival) against chal-

Table 1 Results of immunization and challenge tests in BALB/c mice Immunized witha

Challenge strain and doseb

% Survivalc

X-73 (A:1) 30 CFU 30 CFU 30 CFU 30 CFU 30 CFU 30 CFU 30 CFU

0 (0/6) a 0 (0/10) a 100 (10/10) b 100 (6/6) b 17 (1/6) a 30 (3/10) a 10 (10/10) b

X-73 (A:1) 60 CFU 60 CFU 60 CFU 60 CFU 60 CFU 60 CFU

0 (0/6) a 10 (1/10) a 80 (8/10) bc 50 (3/6) ab 40 (4/10) ab 90 (9/10) c

Control r-PlpE (10 ␮g)

P-1059 (A:3) 35 CFU 35 CFU

0 (0/5) a 100 (10/10) b

Control r-PlpE (10 ␮g)

P-1662 (A:4) 30 CFU 30 CFU

0 (0/5) a 100 (10/10) b

Experiment 1 Control r-PlpB (10 ␮g) r-PlpE (10 ␮g) Inactivated X-73 (2.0 × 108 CFU) Inactivated X-73 (1.25 × 107 CFU) Inactivated X-73 (1.25 × 107 CFU) + r-PlpB (10 ␮g) Inactivated X-73 (1.25 × 107 CFU) + r-PlpE (10 ␮g) Experiment 2 Control r-PlpB (10 ␮g) r-PlpE (10 ␮g) Inactivated X-73 (2.5 × 107 CFU) Inactivated X-73 (2.5 × 107 CFU) + r-PlpB (10 ␮g) Inactivated X-73 (2.5 × 107 CFU) + r-PlpE (10 ␮g) Experiment 3

a b c

Mice in the control group were not immunized. The LD50 of strains X-73 and P1662 in mice was <3 CFU, and that of P-1059 was 3.5 CFU. Different alphabetical characters indicate significant difference (p < 0.05) between groups.

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lenge infection with 30 CFU (>10 LD50 ) of X-73 (serotype A:1). In contrast, mice immunized with 10 ␮g of purified r-PlpE were completely protected (100% survival) (Table 1, experiment 1). Mice immunized with a bacterin composed of 2 × 108 CFU of formalin-inactivated X-73 were completely protected (100% survival), whereas those immunized with a bacterin composed of a lower dose (1.25 × 107 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 × 107 CFU of X-73 supplemented with 10 ␮g r-PlpB or r-PlpE. The results showed that r-PlpB did not significantly enhance the protective efficacy of the bacterin (30% survival, p > 0.05) whereas r-PlpE did (100% survival, p < 0.05) (Table 1, experiment 1). In experiment 2, the challenge dose of X-73 was increased to 60 CFU (>20 LD50 ) and a bacterin composed of 2.5 × 107 CFU of X-73 was used. The results showed that mice immunized with 10 ␮g of r-PlpB were not protected (10% survival) whereas those with 10 ␮g of r-PlpE were significantly protected (80% survival, p < 0.05) (Table 1, experiment 2). Mice immunized with a bacterin composed of 2.5 × 107 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 < 0.05) (Table 1, experiment 2). 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 ␮g of r-PlpE were completely protected against challenge infection with 10 LD50 of P-1059 or >10 LD50 of P-1662 (Table 1, experiment 3). This result showed that r-PlpE, which was derived from X73 (serotype A:1), conferred cross protection on mice against challenge with strains of serotypes A:3 and A:4. 3.3. Immunization and challenge studies in SPF chickens Three immunization and challenge experiments in SPF chickens were conducted (Table 2). In experiment 1, chickens immunized twice with 100 ␮g 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 > 0.05). In contrast, chickens immunized twice with 100 ␮g of purified r-PlpE showed a

Table 2 Results of immunization and challenge tests in SPF chickens Immunized witha

Challenge strain and dose

% Survivalb

Mean time to death (days)c

Control Purified r-PlpB (100 ␮g) Purified r-PlpE (100 ␮g)

X-73 (A:1) 3.6 × 103 CFU 3.6 × 103 CFU 3.6 × 103 CFU

30 (3/10) a 50 (5/10) a 100 (10/10) b

3.3 a 4.2 a NA

Control Purified r-PlpB (100 ␮g) Purified r-PlpE (100 ␮g)

P-1662 (A:4) 5.5 × 108 CFU 5.5 × 108 CFU 5.5 × 108 CFU

13 (1/8) a 13 (1/8) a 63 (5/8) b

4.1 a 5.4 a 5.7 a

X-73 (A:1) 3.6 × 103 CFU 3.6 × 104 CFU 3.6 × 105 CFU

25 (2/8) a 0 (0/8) a 0 (0/8) a

2.7 a 1.9 a 2.0 a

Crude extract r-PlpB (125 ␮g)

3.6 × 103 CFU

40 (2/5) a

4.0 a

Crude extract r-PlpE (125 ␮g)

3.6 × 103

CFU 3.6 × 104 CFU 3.6 × 105 CFU 3.6 × 106 CFU

100 (8/8) b 75 (6/8) b 75 (6/8) b 88 (7/8) b

NA 7.0 b 5.0 b 4.0 b

P-1662 (A:4) 5.5 × 107 CFU 5.5 × 108 CFU 5.5 × 109 CFU

25 (2/8) a 13 (1/8) a 0 (0/8) b

5.7 a 4.1 a 2.9 a

5.5 × 107 CFU 5.5 × 108 CFU 5.5 × 109 CFU

50 (4/8) a 50 (4/8) a 50 (4/8) a

4.8 a 4.8 a 5.3 a

Experiment 1

Experiment 2 Control

Experiment 3 Control

Crude extract r-PlpE (125 ␮g)

a

Immunization was conducted twice with a 3-week interval. Chickens in the control group were not immunized. alphabetical characters indicate significant difference (p < 0.05) between immunization and control groups challenged with the same dose of X-73 or P-1662. b ,c Different

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survival rate of 100%, which was significantly higher than that of the control group (p < 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). 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 [25], instead of Freund’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 ␮g of crude extract of r-PlpB showed a survival rate of 40% against challenge with 3.6 × 103 CFU of X-73, but this survival rate was not significantly higher than that of the control group challenged with the same dose of X-73 (25% survival, p > 0.05) (Table 2, experiment

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2). In contrast, chickens immunized twice with 125 ␮g of crude extract of r-PlpE had a survival rate of 75–100% against challenge with 3.6 × 103 –3.6 × 106 CFU of strain X-73. These rates were significantly higher than those of the control group (p < 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 < 0.05) (Table 2, experiment 2). In experiment 3, P-1662 was used as the challenge strain. The results showed that chickens immunized with 125 ␮g of crude extract of r-PlpE had a survival rate of 50% against challenge with 5.5 × 107 –5.5 × 109 CFU of strain P-1662. These rates were not significantly higher than those of the control groups (p > 0.05), except when the challenge dose of P-1662 was 5.5 × 109 (p < 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 > 0.05) (Table 2, experiment 3).

Fig. 2. Western blot analyses demonstrating presence of PlpB and PlpE in type strains of P. multocida. (A) The blot of whole-cell lysates probed with chicken anti-PlpB sera. The name of each strain is shown on the top of each lane and the loading was the same as determined by Coomassie Blue staining. (B) The same as in (A), except that the blot was probed with chicken anti-PlpE sera. (C) The blot of strain X-73 and purified r-PlpE probed with various forms of chicken anti-PlpE sera. Lanes 1 and 2, anti-PlpE serum with no adsorption; lanes 3 and 4, anti-PlpE serum absorbed with purified PlpE; lanes 5 and 6, anti-PlpE serum absorbed with E. coli proteins. The bands corresponding to PlpE or r-PlpE are indicated by arrows. (D) The same as in (C), except that strain P-1662 was used.

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3.4. Distribution of PlpB and PlpE in reference strains of P. multocida The distribution of PlpB and PlpE antigens in 15 reference strains of P. multocida was investigated by Western blot analyses using sera collected from chickens immunized with

r-PlpB or r-PlpE. The results showed that chicken anti-PlpB sera reacted with a 28.3 kDa protein in all strains examined (Fig. 2A). Because the calculated molecular mass of processed PlpB, on the basis of the plpB sequence [19], was also 28.3 kDa, it was concluded that the PlpB antigen was present in all strains examined. Note that the molecular mass

Fig. 3. Amino acid sequence alignment of PlpE from seven type strains of P. multocida and one strain of M. haemolytica [15], and OmlA from one strain of A. pleuropneumoniae [17]. The signal peptide cleavage site is indicated with a vertical arrow. The identical amino acids are boxed and residues conserved in all sequences are indicated by asterisks.

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of PlpB (28.3 kDa) is smaller than that of processed r-PlpB (29.3 kDa); this is because the latter contains a hexa-histidine tag at its carboxyl terminus. The chicken anti-PlpE sera reacted with three protein bands with molecular mass of 63.0, 35.3 and 28.0 kDa in all strains examined (Fig. 2B). Because the calculated molecular mass of processed PlpE, on the basis of the plpE sequence [19], was 35.3 kDa, it is possible that the 35.3 kDa protein is the PlpE protein. To test this hypothesis, we conducted competitive binding assay of Western blot using anti-PlpE sera adsorbed with purified r-PlpE or with E. coli proteins. The result showed the intensity of the 35.3 kDa protein was greatly reduced when the serum was adsorbed with r-PlpE but its intensity remained essentially the same when the serum was adsorbed with E. coli proteins (Fig. 2C and D). It was therefore concluded that the 35.3 kDa protein is PlpE. The same conclusion was achieved when strains of X-73 and P-1662 were used in the assay (Fig. 2C and D). 3.5. Nucleotide sequences of plpE from reference strains of P. multocida PCR using primers annealed to the 5 and 3 ends of the coding region of plpE was able to amplify the expected 1.0 kb PCR product from all 15 reference strains of P. multocida examined, suggesting that plpE was present in all of these strains. The complete nucleotide sequences of plpE from six reference strains of P. multocida were determined (accession numbers EF219452–EF219457). 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. Antigenic index assay showed that these PlpE proteins have putative surface exposure regions randomly distributed throughout the protein; this feature is very similar to those found in PlpE proteins of M. haemolytica and OmlA of A. pleuropneumoniae. However, the hexapeptide repeats found in the PlpE of M. haemolytica [15] and the dipeptide repeats found in OmlA from A. pleuropneumoniae [18] were not present in the PlpE of P. multocida. The alignment of amino acid sequences of PlpE from P. multocida, together with those of PlpE from M. haemolytica and OmlA from A. pleuropneumoniae, are shown in Fig. 3. The alignment showed that all PlpE and OmlA have a putative signal peptide of 19–20 residues, followed by a consensus lipoprotein processing site (LVAC) (Fig. 3) [26]. Moreover, the second residue of the mature PlpE and OmlA is a serine or glycine, which allows the targeting of PlpE and OmlA for the outer membrane [27]. Sequence alignment also revealed that all PlpE and OmlA had a stretch of glycine or serine residues after the signal peptidase cleavage site (Fig. 3). Beyond this, sequences of PlpE from P. multocida were poorly aligned with PlpE from M. haemolytica and

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OmlA of M. haemolytica. Pair-wise sequence comparison showed that PlpE from P. multocida had 23.0–24.3% identity with PlpE of M. haemolytica and 16.8–19.1% identity with OmlA of A. pleuropneumoniae.

4. Discussion Extensive efforts have been made to identify the crossprotective antigens of P. multocida [1,7]. Two laboratories have independently identified a 39 kDa protein, purified either from the capsular extract or from the outer membrane of P. multocida, to be the cross-protective antigen [11,12]. One of the 39 kDa proteins was identified to be PlpB by protein mass analysis [13]. However, it has not yet been demonstrated whether PlpB is the cross-protective antigen. Data presented in this study show that PlpB is unlikely to be the 39 kDa cross-protective antigen. First, r-PlpB failed to confer any protection against challenge infection on mice or chickens. Second, the molecular mass of PlpB, based on Western blot analyses or sequencing of the plpB gene, is only 28 kDa, which differs from that of the 39 kDa protein. It is therefore concluded that PlpB is not the 39 kDa cross-protective antigen. PlpE appears more likely to be the cross-protection antigen. Immunization with a single dose of purified r-PlpE conferred protection on mice against challenge infection with strains X-73 (A:1), P-1059 (A:3) and P-1662 (A:4). To our knowledge, this is the first report of a P. multocida recombinant antigen that provides cross protection in mice. In addition to mice, r-PlpE also conferred good protection against challenge infection with X-73 on chickens, and gave some protection against P-1662. The reduction in resistance of r-PlpE-immunized chickens to P1662 may have resulted from the high challenge dose used (5.5 × 107 –5.5 × 109 CFU). The high dose was chosen because the LD50 of P-1662 in chickens was found be about 107 CFU. However, it is possible that the antibody titers elicited by 100 or 125 ␮g of r-PlpE were not sufficient to give good protection against challenge with this high dose. Experiments are underway to investigate whether a higher antigen dose could provide chickens with better protection against P-1662. The mechanism by which the PlpE of P. multocida confers protection remains unknown. However, it could be inferred from research on the PlpE of M. haemolytica [14–17,28] and the OmlA of A. pleuropneumoniae [18] that the PlpE of P. multocida is a surface-exposed outer membrane protein that elicits protective immunity against P. multocida. Sequence alignment shows that PlpEs and OmlA have one unique feature in common: all of them contain a stretch of serine and glycine residues at their amino termini. Site-directed mutagenesis or nested deletion at the amino termini of these proteins is required to investigate whether these residues are important for the function of PlpE and OmlA. Sequence comparison showed that P. multocida PlpE

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has low amino acid sequence homology with M. haemolytica PlpE; moreover, P. multocida PlpE lacks tandem repeats, which is a characteristic of M. haemolytica PlpE. Therefore, a new name should be proposed for P. multocida PlpE, if future work demonstrates that the two proteins have distinct functions. PCR and Western blot analyses showed that genes and antigens of PlpE are present in all 15 reference strains of P. multocida examined. These strains are known to cause a variety of diseases in different animals, including fowl cholera in birds, haemorrhagic septicaemia in cattle, and atrophic rhinitis in pigs. Data presented in this study show that r-PlpE from strain X-73 confers protection on mice and chickens against challenge with strains that cause fowl cholera. This result might encourage studies on the use of r-PlpE as an antigen to protect animals against P. multocida strains that cause other diseases, such as haemorrhagic septicaemia and atrophic rhinitis.

Acknowledgments This investigation was supported by grants 91-2313-B005-125 and 95-2745-B-005-002 from the National Science Council, Taiwan, Republic of China.

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