Developing a PmSLP3-based vaccine formulation that provides robust long-lasting protection against hemorrhagic septicemia-causing serogroup B and E strains of Pasteurella multocida in cattle.

Background: Pasteurella multocida is a bacterial pathogen that causes a variety of infections across diverse animal species, with one of the most devastating associated diseases being hemorrhagic septicemia. Outbreaks of hemorrhagic septicemia in cattle and buffaloes are marked by rapid progression and high mortality. These infections have particularly harmful socio-economic impacts on small holder farmers in Africa and Asia who are heavily reliant on a small number of animals kept as a means of subsistence for milk and draft power purposes. A novel vaccine target, PmSLP-3, has been identified on the surface of hemorrhagic septicemia-associated strains of P. multocida and was previously shown to elicit robust protection in cattle against lethal challenge with a serogroup B strain. Methods: Here, we further investigate the protective efficacy of this surface lipoprotein, including evaluating the immunogenicity and protection upon formulation with a variety of adjuvants in both mice and cattle. Results: PmSLP-3 formulated with Montanide ISA 61 elicited the highest level of serum and mucosal IgG, elicited long-lasting serum antibodies, and was fully protective against serogroup B challenge. Studies were then performed to identify the minimum number of doses required and the needed protein quantity to maintain protection. Duration studies were performed in cattle, demonstrating sustained serum IgG titres for 3 years after two doses of vaccine and full protection against lethal serogroup B challenge at 7 months after a single vaccine dose. Finally, a serogroup E challenge study was performed, demonstrating that PmSLP-3 vaccine can provide protection against challenge by the two serogroups responsible for hemorrhagic septicemia. Conclusion: Together, these data indicate that PmSLP-3 formulated with Montanide ISA 61 is an immunogenic and protective vaccine against hemorrhagic septicemia-causing P. multocida strains in cattle.

Method for quantifying the Pasteurella multocida antigen adsorbed on aluminum hydroxide adjuvant in swine atrophic rhinitis vaccine.

Swine atrophic rhinitis is a disease caused by Pasteurella multocida and Bordetella bronchiseptica that affects pigs. Inactivated vaccines containing the toxins produced by Pasteurella multocida and Bordetella bronchiseptica have been widely used for the prevention of swine atrophic rhinitis. The efficacy of a vaccine is correlated with the amount of antigen present; however, the protective toxin of P. multocida bound to aluminum hydroxide, which is used as an adjuvant, can hinder the monitoring of the antigen concentration in the vaccine. This study assessed the applicability of a dot immunoassay as an antigen quantification method using monoclonal antibodies. This quantification method was able to detect the antigen with high specificity and sensitivity even when the antigen was bound to the adjuvant, and its application to vaccine products revealed a correlation between the amount of antigen present in the vaccine and the neutralizing antibody titers induced in pigs. The antigen quantification method presented in this study is a simple and sensitive assay capable of quantifying the amount of antigen present in a vaccine that can be used as an alternative quality control measure.

Dendritic cell targeting peptide plus Salmonella FliCd flagellin fused outer membrane protein H (OmpH) demonstrated increased efficacy against infections caused by different Pasteurella multocida serogroups in mouse models.

As the major outer membrane protein (OMP) presents in the Pasteurella multocida envelope, OmpH was frequently expressed for laboratory assessments of its immunogenicity against P. multocida infections, but the results are not good. In this study, we modified OmpH with dendritic cell targeting peptide (Depeps) and/or Salmonella FliCd flagellin, and expressed three types of recombinant proteins with the MBP tag (rDepeps-FliC-OmpH-MBP, rDepeps-OmpH-MBP, rFliC-OmpH-MBP). Assessments in mouse models revealed that vaccination with rDepeps-FliC-OmpH-MBP, rDepeps-OmpH-MBP, or rFliC-OmpH-MBP induced significant higher level of antibodies as well as IFN-γ and IL-4 in murine sera than vaccination with rOmpH-MBP (P < 0.5). Vaccination with the three modified proteins also provided increased protection (rDepeps-FliC-OmpH-MBP, 70 %; rDepeps-OmpH-MBP, 50 %; rFliC-OmpH-MBP, 60 %) against P. multocida serotype D compared to vaccination with rOmpH-MBP (30 %). In mice vaccinated with different types of modified OmpHs, a significantly decreased bacterial strains were recovered from bloods, lungs, and spleens compared to rOmpH-MBP-vaccinated mice (P < 0.5). Notably, our assessments also demonstrated that vaccination with rDepeps-FliC-OmpH-MBP provided good protection against infections caused by a heterogeneous group of P. multocida serotypes (A, B, D). Our above findings indicate that modification with DCpep and Salmonella flagellin could be used as a promising strategy to improve vaccine effectiveness.

Attenuated vaccine PmCQ2Δ4555-4580 effectively protects mice against Pasteurella multocida infection.

Pasteurella multocida type A (PmA) mainly causes respiratory diseases such as pneumonia in bovines, leading to great economic losses to the breeding industry. At present, there is still no effective commercial vaccine against PmA infection. In this study, a mutant strain (PmCQ2Δ4555-4580) with brand-new phenotypes was obtained after serially passaging at 42 °C. Whole genome resequencing and PCR analysis showed that PmCQ2Δ4555-4580 missed six genes, including PmCQ2_004555, PmCQ2_004560, PmCQ2_004565, PmCQ2_004570, PmCQ2_004575, and PmCQ2_004580. Importantly, the virulence of PmCQ2Δ4555-4580 was reduced by approximately 2.8 × 109 times in mice. Notably, live PmCQ2Δ4555-4580 could provide 100%, 100% and 40% protection against PmA, PmB and PmF, respectively; and inactivated PmCQ2Δ4555-4580 could provide 100% and 87.5% protection against PmA and PmB. Interestingly, immune protection-related proteins were significantly upregulated in PmCQ2Δ4555-4580 based on RNA-seq and bioinformatics analysis. Meaningfully, by in vitro expression, purification and in vivo immunization, 12 proteins had different degrees of immune protective effects. Among them, PmCQ2_008205, PmCQ2_010435, PmCQ2_008190, and PmCQ2_004170 had the best protective effect, the protection rates against PmA were 50%, 40%, 30%, and 30%, respectively, and the protective rates against PmB were 62.5%, 42.9%, 37.5%, and 28.6%, respectively. Collectively, PmCQ2Δ4555-4580 is a potential vaccine candidate for the prevention of Pasteurellosis involving in high expression of immune protective related proteins.

Preparation of Escherichia coli ghost of anchoring bovine Pasteurella multocida OmpH and its immunoprotective effect.

Pasteurella multocida is a pathogen that can infect humans and animals. A ghost is an empty bacterial body devoid of cytoplasm and nucleic acids that can be efficiently presented by antigen-presenting cells. To study a novel ghost vector vaccine with cross-immune protection, we used bacteriophage PhiX174 RF1 and Pasteurella multocida standard strain CVCC393 as templates to amplify the split genes E and OmpH to construct a bidirectional expression vector E'-OmpH-pET28a-ci857-E. This is proposed to prepare a ghost Escherichia coli (engineered bacteria) capable of attaching and producing Pasteurella multocida OmpH on the inner membrane of Escherichia coli (BL21). The aim is to assess the antibody levels and the effectiveness of immune protection by conducting a mouse immunoprotective test. The bidirectional expression vector E'-OmpH-pET28a-ci857-E was successfully constructed. After induction by IPTG, identification by SDS-PAGE, western blot, ghost culture and transmission electron microscope detection, it was proven that the Escherichia coli ghost anchored to Pasteurella multocida OmpH was successfully prepared. The immunoprotective test in mice showed that the antibody levels of Pasteurella multocida inactivated vaccine, OmpH, ghost (aluminum glue adjuvant) and ghost (Freund's adjuvant) on day 9 after immunization were significantly different from those of the PBS control group (P < 0.01). The immune protection rates were 100%, 80%, 75%, and 65%, respectively, and the PBS negative control was 0%, which proved that they all had specific immune protection effects. Therefore, this study lays the foundation for the further study of ghosts as carriers of novel vaccine-presenting proteins.

Conformational comparisons of Pasteurella multocida types B and E and structurally related capsular polysaccharides.

Pasteurella multocida, an encapsulated gram-negative bacterium, is a significant veterinary pathogen. The P. multocida is classified into 5 serogroups (A, B, D, E, and F) based on the bacterial capsular polysaccharide (CPS), which is important for virulence. Serogroups B and E are the primary causative agents of bovine hemorrhagic septicemia that is associated with significant yearly losses of livestock worldwide, primarily in low- and middle-income countries. The P. multocida disease is currently managed by whole-cell vaccination, albeit with limited efficacy. CPS is an attractive antigen target for an improved vaccine: CPS-based vaccines have proven highly effective against human bacterial diseases and could provide longer-term protection against P. multocida. The recently elucidated CPS repeat units of serogroups B and E both comprise a N-acetyl-ß-D-mannosaminuronic acid/N-acetyl-ß-D-glucosamine disaccharide backbone with ß-D-fructofuranose (Fruf) side chain, but differ in their glycosidic linkages, and a glycine (Gly) side chain in serogroup B. Interestingly, the Haemophilus influenzae types e and d CPS have the same backbone residues. Here, comparative modeling of P. multocida serogroups B and E and H. influenzae types e and d CPS identifies a significant impact of small structural differences on both the chain conformation and the exposed potential antibody-binding epitopes (Ep). Further, Fruf and/or Gly side chains shield the immunogenic amino-sugar CPS backbone-a possible common strategy for immune evasion in both P. multocida and H. influenzae. As the lack of common epitopes suggests limited potential for cross-reactivity, a bivalent CPS-based vaccine may be necessary to provide adequate protection against P. multocida types B and E.

Immunogenicity and protective efficacy of a multi-epitope recombinant toxin antigen of Pasteurella multocida against virulent challenge in mice.

Pasteurella multocida (P. multocida) infection frequently results in porcine atrophic rhinitis and swine plague, leading to large economic losses for the swine industry worldwide. P. multocida toxin (PMT, 146 kDa) is a highly virulent key virulence factor that plays a vital role in causing lung and turbinate lesions. This study developed a multi-epitope recombinant antigen of PMT (rPMT) that showed excellent immunogenicity and protection in a mouse model. Using bioinformatics to analyse the dominant epitopes of PMT, we constructed and synthesized rPMT containing 10 B-cell epitopes, 8 peptides with multiple B-cell epitopes and 13 T-cell epitopes of PMT and a rpmt gene (1,974 bp) with multiple epitopes. The rPMT protein (97 kDa) was soluble and contained a GST tag protein. Immunization of mice with rPMT stimulated significantly elevated serum IgG titres and splenocyte proliferation, and serum IFN-γ and IL-12 were upregulated by 5-fold and 1.6-fold, respectively, but IL-4 was not. Furthermore, the rPMT immunization group exhibited alleviated lung tissue lesions and a significantly decreased degree of neutrophil infiltration compared with the control groups post-challenge. In the rPMT vaccination group, 57.1% (8/14) of the mice survived the challenge, similar to the bacterin HN06 group, while all the mice in the control groups succumbed to the challenge. Thus, rPMT could be a suitable candidate antigen for developing a subunit vaccine against toxigenic P. multocida infection.

Reverse vaccinology-based identification of a novel surface lipoprotein that is an effective vaccine antigen against bovine infections caused by Pasteurella multocida.

Pasteurella multocida can infect a multitude of wild and domesticated animals, with infections in cattle resulting in hemorrhagic septicemia (HS) or contributing to bovine respiratory disease (BRD) complex. Current cattle vaccines against P. multocida consist of inactivated bacteria, which only offer limited and serogroup specific protection. Here, we describe a newly identified surface lipoprotein, PmSLP, that is present in nearly all annotated P. multocida strains isolated from cattle. Bovine associated variants span three of the four identified phylogenetic clusters, with PmSLP-1 and PmSLP-2 being restricted to BRD associated isolates and PmSLP-3 being restricted to isolates associated with HS. Recombinantly expressed, soluble PmSLP-1 (BRD-PmSLP) and PmSLP-3 (HS-PmSLP) vaccines were both able to provide full protection in a mouse sepsis model against the matched P. multocida strain, however no cross-protection and minimal serum IgG cross-reactivity was identified. Full protection against both challenge strains was achieved with a bivalent vaccine containing both BRD-PmSLP and HS-PmSLP, with serum IgG from immunized mice being highly reactive to both variants. Year-long stability studies with lyophilized antigen stored under various temperatures show no appreciable difference in biophysical properties or loss of efficacy in the mouse challenge model. PmSLP-1 and PmSLP-3 vaccines were each evaluated for immunogenicity in two independent cattle trials involving animals of different age ranges and breeds. In all four trials, vaccination with PmSLP resulted in an increase in antigen specific serum IgG over baseline. In a blinded cattle challenge study with a recently isolated HS strain, the matched HS-PmSLP vaccine showed strong efficacy (75-87.5% survival compared to 0% in the control group). Together, these data suggest that cattle vaccines composed of PmSLP antigens can be a practical and effective solution for preventing HS and BRD related P. multocida infections.

Combinations but Not a Single PlpE Epitope Induces Host Protective Immunity against Pasteurella multocida.

Pasteurella multocida is the causative agent of a wide range of diseases (pasteurellosis) and a zoonotic pathogen in humans. Recombinant subunit vaccines are hot spots in recent pasteurellosis vaccine development. A chimeric vaccine is also constructed for rabbit hemorrhagic disease virus (RHDV) protective antigen VP60 chimeric with fragments of Pasteurella multocida protective antigen PlpE. The protective efficacy of the chimeric vaccine against P. multocida is not as high as that of PlpE, and the reason is not well known. In this study, we analyzed the linear B-cell epitopes of PlpE and then assessed the protective efficacy of these epitopes and their combinations. It was found that the immunodominant region of PlpE was mainly located in the region between the 21st to the 185th amino acids from the N terminus. Overlapping peptide scanning results demonstrated that this region contained six nonoverlapping epitopes, and epitope E was the predominant epitope. Chimeric protein antigens were constructed of single nonoverlapping PlpE epitopes or their combinations chimeric with the RHDV VP60 P domain. Immunization with recombinant antigen chimeric with a single PlpE epitope exhibited poor immunoprotection, whereas immunization with recombinant antigen chimeric with PlpE epitope combinations (epitopes A and E; epitopes C and E; epitopes A, C, and E; and epitopes B, D, and F) exhibited significant immunoprotection. In a word, P. multocida protective antigen PlpE contained six nonoverlapping linear B-cell epitopes, and combinations but not a single epitope induced host protective immunity. Our work will give help for future chimeric vaccine design.

Signal sequence contributes to the immunogenicity of Pasteurella multocida lipoprotein E.

Recombinant Pasterurella multocida lipoprotein E (PlpE) has been shown to protect against fowl cholera. This study aimed to determine if the signal sequence may contribute to the antigenicity and protective efficacy of recombinant PlpE. A small antigenic domain of PlpE (termed truncated PlpE, tPlpE) was constructed with (SP-tPlpE) or without (tPlpE) the signal sequence and evaluated in vitro and in vivo. In vitro, the HEK-Bule hTLR2 Cells were used to evaluate the activation of NF-kB in the test associated with the stimulation of the SP-tPlpE and tPlpE proteins. When chickens were immunized, compared to the tPlpE vaccine group, the SP-tPlpE group showed higher antibody levels and enhanced CD4+ T cell response. In a challenge test, the SP-tPlpE group showed a survival rate of 87.5% (n = 8), compared to 25% for the tPlpE group. It is confirmed that the inclusion of the native signal sequence enhanced protective efficacy against fowl cholera and may act as a vaccine adjuvant. The short SP-tPlpE construct is amenable to further vaccine engineering and has potential to be developed as a fowl cholera vaccine.

Immunogenicity and protective efficacy of the recombinant Pasteurella multocida lipoproteins VacJ and PlpE, and outer membrane protein H from P. multocida A:1 in ducks.

Duck cholera (duck hemorrhagic septicemia) is a highly contagious disease caused by Pasteurella multocida, and is one of the major bacterial diseases currently affecting the duck industry. Type A is the predominant pathogenic serotype. In this study, the genes encoding the lipoproteins VacJ, PlpE, and the outer membrane protein OmpH of P. multocida strain PMWSG-4 were cloned and expressed as proteins in E. coli. The recombinant VacJ (84.4 kDa), PlpE (94.8 kDa), and OmpH (96.7 kDa) proteins were purified, and subunit vaccines were formulated with a single water-in-oil adjuvant, while killed vaccines were prepared using a single oil-coated adjuvant. Antibody responses in ducks vaccinated with recombinant VacJ, PlpE, and OmpH proteins formulated with adjuvants were significantly antigenic (p<0.005). Protectivity of the vaccines was evaluated via the intraperitoneal challenge of ducks with 20 LD50 doses of P. multocida A: 1. The vaccine formulation consisting of rVacJ, rPlpE, rOmpH, and adjuvant provided 33.3%, 83.33%, and 83.33% protection, respectively, the vaccine formulation consisting of three recombinant proteins, rVacJ, rPlpE, rOmpH and adjuvant, was 100% protective, and the killed vaccine was 50% protective. In addition, it was shown through histopathological examination and tissue bacterial load detection that all vaccines could reduce tissue damage and bacterial colonization to varying (p<0.001). These findings indicated that recombinant PlpE or OmpH fusion proteins formulated with oil adjuvants have the potential to be used as vaccine candidates against duck cholera subunits.

Two Bordetella bronchiseptica attenuated vaccine candidates confer protection against lethal challenge with B. Bronchiseptica and Pasteurella multocida toxin in mouse models.

Dermonecrotic toxin (DNT) is an important bacterial virulence factor produced by the zoonotic pathogens Bordetella bronchiseptica and Pasteurella multocida. This study aims to explore the possibility of expressing different fragments of P. multocida toxin (PMT) in the chromosome of attenuated B. bronchiseptica to generate single-component mucosal vaccine candidates. To achieve this, a 954-bp fragment (basepairs 301 âˆ¼ 1254) of the B. bronchiseptica aroA gene was replaced with an N-terminal, 930-bp fragment (basepairs 1-930; PMTN) or a C-terminal, 900-bp fragment (base pairs 2959 âˆ¼ 3858; PMTC) of the PMT encoding gene toxA. The resulting strains, denoted as Bb-PMTN or Bb-PMTC, expressed PMTN and PMTC, as evidenced by ELISA using polyclonal against full-length of PMT. Phenotypical analyses revealed that Bb-PMTN and Bb-PMTC grew much slower than wild type strains in tryptic soy broth. These strains also displayed significantly decreased 161-fold-virulence compared to the wildtype strains in mouse models. Intranasal immunization of Bb-PMTN and Bb-PMTC in mice induced high levels of antibodies against B. bronchiseptica and PMT, as well as IFN-γ and IL-10 in mouse sera, and most importantly, high titers of sIgA in mouse lungs. Vaccination with these two engineering strains provided 100% protection of mice against lethal challenge with B. bronchiseptica and 80%∼100% protection against lethal challenge with PMT, with Bb-PMTN exhibiting 1.25-fold greater immunogenic efficacy over Bb-PMTC. This study highlights the use of B. bronchiseptica attenuated strains as live mucosal vectors to deliver heterologous antigens.

Immunogenicity and protection of a Pasteurella multocida strain with a truncated lipopolysaccharide outer core in ducks.

Pasteurella multocida infection frequently causes fowl cholera outbreaks, leading to huge economic losses to the poultry industry worldwide. This study developed a novel live attenuated P. multocida vaccine strain for ducks named PMZ2 with deletion of the gatA gene and first four bases of the hptE gene, both of which are required for the synthesis of the lipopolysaccharide (LPS) outer core. PMZ2 produced a truncated LPS phenotype and was highly attenuated in ducks with a > 105-fold higher LD50 than the wild-type strain. PMZ2 colonized the blood and organs, including the spleen, liver and lung, at remarkably reduced levels, and its high dose of oral infection did not cause adverse effects on body temperatures and body weights in ducks. To evaluate the vaccine efficacy of the mutant, ducklings were inoculated orally or intranasally with PMZ2 or PBS twice and subsequently subjected to a lethal challenge. Compared with the PBS control, PMZ2 immunization stimulated significantly elevated serum IgG, bile IgA and tracheal IgA responses, especially after the boost immunization in both the oral and intranasal groups, and the induced serum had significant bactericidal effects against the wild-type strain. Furthermore, the two PMZ2 immunization groups exhibited alleviated tissue lesions and significantly decreased bacterial loads in the blood and organs compared with the PBS group post-challenge. All the ducks in the PMZ2 oral and intranasal groups survived the challenge, while 70% of ducks in the PBS group succumbed to the challenge. Thus, the P. multocida mutant with mutation of the gatA gene and part of the hptE gene proved to be an effective live attenuated vaccine candidate for prevention of fowl cholera in ducks.

Ovine Pasteurellosis Vaccine: Assessment of the Protective Antibody Titer and Recognition of the Prevailing Serotypes.

Sheep husbandry is considered one of the most important activities in the socio-economic development in the Middle East region, especially in Iraq and Islamic Republic of Iran (IRI). Therefore this study was designed to evaluate the level of ovine pasteurellosis vaccine protective antibody titer and identification of the prevailing serotypes in Iraq (Basrah, Baghdad, Tikrit, Mosul, Erbil). The vaccine was made from pasteurella multosida Bio-type A and the serotypes of Mannheimia haemolytica. This investigation was performed from September 2021 to January 2022, in Iraq. Sheep blood sera samples were obtained from control unvaccinated and vaccinated sheep after 14, 21 and 28 days post vaccination. The results showed that out of 319 sheep blood sera samples which were evaluated using indirect Haemagglutination (IHA) test to detect Mannheimia haemolytica serotypes, the high prevalence (100 %) of M. haemolytica A2 was found in all the five study regions area, while 96.5 % was M. haemolytica A7 and 88.1 % was M. haemolytica A1. The level of antibody titer was measured by specific serum antibody titer of pasteurella multosida Bio-type A. The results revealed that out of 268 vaccinated blood sera samples the overall antibody titer were 12 (3.8 %), 16 (5%) and 17 (5.3 %) for protective antibody titer of 1:160, 1:80 and 1:40 respectively and for antibody titer of 1:20 were 15 (4.7%) and for antibody titer of 1:10 were 17 (5.3 %), whereas the antibody titer in the control group was 4 (7.8 %). The result of this study indicated that the vaccine administered has limited protective power against pasteurella multocida Bio-type A which lead to researchers for further study on identification of specific strain of pasteurella multosida and development of multivalent vaccine including the most prevalent pasteurella serotypes.

Gamma-Irradiated Fowl Cholera Mucosal Vaccine: Potential Vaccine Candidate for Safe and Effective Immunization of Chicken Against Fowl Cholera.

Fowl cholera (FC) caused by Pasteurella multocida is among the serious infectious diseases of poultry. Currently, formalin inactivated FC (FI-FC) vaccine is widely used in Ethiopia. However, reports of the disease complaint remain higher despite the use of the vaccine. The aim of this study was to develop and evaluate gamma-irradiated mucosal FC vaccines that can be used nationally. In a vaccination-challenge experiment, the performance of gamma-irradiated P. multocida (at 1 kGy) formulated with Montanide gel/01 PR adjuvant was evaluated at different dose rates (0.5 and 0.3 ml) and routes (intranasal, intraocular, and oral), in comparison with FI-FC vaccine in chicken. Chickens received three doses of the candidate vaccine at 3-week intervals. Sera, and trachea and crop lavage were collected to assess the antibody levels using indirect and sandwich ELISAs, respectively. Challenge exposure was conducted by inoculation at 3.5×109 CFU/ml of P. multocida biotype A intranasally 2 weeks after the last immunization. Repeated measures ANOVA test and Kaplan Meier curve analysis were used to examine for statistical significance of antibody titers and survival analysis, respectively. Sera IgG and secretory IgA titers were significantly raised after second immunization (p=0.0001). Chicken survival analysis showed that intranasal and intraocular administration of the candidate vaccine at the dose of 0.3 ml resulted in 100% protection as compared to intramuscular injection of FI-FC vaccine, which conferred 85% protection (p=0.002). In conclusion, the results of this study showed that gamma-irradiated FC mucosal vaccine is safe and protective, indicating its potential use for immunization of chicken against FC.

Effect of methanolic extract of Citrus limetta peel on cellular and humoral immune response in mice.

Citrus limetta is well known for its anti-inflammatory, antimicrobial, antifungal, antidiabetic and antioxidant properties. Methanolic extract of Citrus limetta (MECL) was used to assess cellular and humoral immune responses in mice by carrying out cyclophosphamide-induced neutropenia, delayed-type hypersensitivity (DTH), carbon clearance assay, haemagglutination assay (HA) and mice lethality assay. Methanolic extract of Citrus limetta peel was administered orally to mice in two doses 200mg/kg and 400mg/kg.The extract treated groups showed improvement in neutropenia induced by cyclophosphamide and improvement in the WBC profile. Skin thickness was significantly (P<0.05) higher in 200mg/kg and 400mg/kg groups in comparison to control in DTH. The phagocytic index was significantly (P<0.05) more in 400mg/kg group in carbon clearance assay. Mice were vaccinated with hemorrhagic septicemia vaccine before challenge with Pasteurella multocida for mice lethality test. Percentage mortality was decreased in 400mg/kg treated group in comparison to negative control Antibody titre response to sheep red blood cells was significantly (P<0.05) higher with dose 400mg/kg in HA. Results suggested the effectiveness of the methanolic extract of Citrus limetta as an immunostimulating agent.

Deleting qseC downregulates virulence and promotes cross-protection in Pasteurella multocida.

QseC, a histidine sensor kinase of the QseBC two-component system, acts as a global regulator of bacterial stress resistance, biofilm formation, and virulence. The function of QseC in some bacteria is well understood, but not in Pasteurella multocida. We found that deleting qseC in P. multocida serotype A:L3 significantly down-regulated bacterial virulence. The mutant had significantly reduced capsule production but increased resistance to oxidative stress and osmotic pressure. Deleting qseC led to a significant increase in qseB expression. Transcriptome sequencing analysis showed that 1245 genes were regulated by qseC, primarily those genes involved in capsule and LPS biosynthesis and export, biofilm formation, and iron uptake/utilization, as well as several immuno-protection related genes including ompA, ptfA, plpB, vacJ, and sodA. In addition to presenting strong immune protection against P. multocida serotypes A:L1 and A:L3 infection, live ΔqseC also exhibited protection against P. multocida serotype B:L2 and serotype F:L3 infection in a mouse model. The results indicate that QseC regulates capsular production and virulence in P. multocida. Furthermore, the qseC mutant can be used as an attenuated vaccine against P. multocida strains of multiple serotypes.

The effect of carbohydrates on the adherence of Pasteurella multocida to the nasal respiratory epithelium.

Pasteurella multocida subsp. multocida is responsible for different diseases that generate great economic losses in farm animal. The effectiveness of immunization against those bacteria are variable and the use of antibiotics is questioned; for that reason, we investigated the potential inhibitory effect of different carbohydrates on the adherence in vivo of P. multocida to the rabbit respiratory epithelium as an alternative for the prevention of respiratory infections. Rabbits were intranasally and intratracheally inoculated with a solution containing 200 µl of 1x107 CFU of P. multocida that was previously mixed with 250 µg /200 µl of N-acetylglucosamine, alphamethylglucoside, alphamethylmannoside, N-acetylgalactosamine or sialic acid. The animals that received N-acetylglucosamine, alphamethylglucoside or alphamethylmannoside individually or a mixture of these three carbohydrates plus the bacterium, showed a significant decrease (P <0.05) of the clinical symptoms, microscopic and macroscopic lesions in the nasal septa and in the lungs; also, the number of adhered bacteria to the nasal epithelium were also significantly reduced. This research demonstrates for the first time that such an approach could convert into a method for prevention of P. multocida infection in rabbits that is ecologically and economically safe and effective.

Cross-protection of recombinant Pasteurella multocida toxin proteins against atrophic rhinitis in mice.

Pasteurella multocida (P. multocida) infects the swine respiratory tract and mainly causes atrophic rhinitis (AR). Recently, many commercially inactivated and subunit vaccines have been used as preventive strategies. However, the best antigenic protein portion has not been selected, and the aluminum gel was used as the adjuvant, which may not induce full protection. P. multocida toxin (PMT) is the major virulence factor responsible for AR. PMT is a monomeric 146 kDa protein (approximately 1285 amino acids) encoded by the tox A gene. In this study, we expressed different fragments of recombinant PMT proteins, combined them with a water-in-oil-in-water adjuvant, and evaluated mice's immune response. The results indicated that the rPMT-C-immunized group showed significantly higher levels (p < 0.05) of IgG, IgG2a antibody and interferon-γ, IL-12 cytokine expression than other groups. Furthermore, vaccination with rPMT-C recombinant protein can provide homologous and heterologous protection against P. multocida challenge. In conclusion, our approach may be feasible for developing an effective subunit vaccine against atrophic rhinitis with a cost-down simple ingredient.

In silico Analysis of Pasteurella multocida PlpE Protein Epitopes As Novel Subunit Vaccine Candidates.

Background: Pasteurella multocida is a Gram-negative, non-motile, non-spore forming, and aerobic/anaerobic cocobacillus known as the causative agent of human and animal diseases. Humans can often be affected by cat scratch or bite, which may lead to soft tissue infections and in rare cases to bacteremia and septicemia. Commercial vaccines against this agent include inactivated, live attenuated, and non-pathogenic bacteria. Current vaccines have certain disadvantages such as reactogenicity or reversion to virulence. Therefore, the aim of this study was to reach a multi-epitope vaccine candidate that could be serotype independent and covers most incident serotypes of P. multocida. Methods: In this study, reverse vaccinology strategy was used to identify potentially immunogenic and protective epitopes. First, multiple alignments of different sequences of Pasteurella lipoprotein E (PlpE) from various serotypes of P. multocida were analyzed to identify the conserved regions. Bioinformatics tools were then applied to predict and select epitopes for further studies. Results: Three different conserved immunogenic regions were selected according to the selected criteria, and their various sequential orders were evaluated structurally by in silico tools to find the best order. Conclusion: In searching the epitopes of PlpE to design a new vaccine candidate against pasteurellosis, we found the region 1 + region 2 + region 3 (without any linker between regions) of epitope, including the regions of PlpE protein of P. multocida, as the appropriate serotype independent vaccine candidate against pasteurellosis.