Reverse vaccinology-based identification of a novel surface lipoprotein that is an effective vaccine antigen against bovine infections caused by Pasteurella multocida
Epshita A. IslamJamie E. FeganTakele AbaynehDavid M. CurranRegula WaeckerlinDixon NgSang Kyun AhnChun Heng Royce LaiQuynh Huong NguyenMegha ShahLiyuwork TesfawKassaye AdamuWubet W. MedhinAbinet LegesseGetaw DeresseBelayneh GetachewNeil RawlykBrock EvansAndrew PotterAnthony B. SchryversScott D. Gray‐OwenTrevor F. Moraes
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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.Keywords:
Bovine Respiratory Disease
Bovine respiratory disease is the leading cause of loss to the North American cattle industry. The disease is characterized by a severe fibrinous pneumonia or a suppurative bronchitis and bronchopneumonia. The bacteria most commonly associated with these cases of pneumonia and deaths are Pasteurella haemolytica (PH) and Pasteurella multocida (PM). Attempts to immunize calves with killed bacterins or extracts of these bacteria have been ineffective. Recently several investigators have used live cultures of PH and PM to immunize calves against respiratory disease. The purpose of these investigations was to develop vaccines that effectively immunize calves against pneumonic pasteurellosis. Two vaccines have been developed that contain viable Pasteurella haemolytica (PRECON-PH) or Pasteurella multocida (BOVICON-PM) (A.H. Robins Corporation, Richmond, Va.).
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A total of one hundred and seventy one indigenous birds from smallholder farms and those traded in market centers in Nairobi were examined for the presence of Pasteurella multocida . Of these, 135 were farmed and 36 were market birds. They comprised of 117 indigenous chickens and 54 ducks. Three hundred and forty two oropharyngeal and cloacal swabs were collected from them and cultured onto blood agar and other media. The recovered isolates were characterized using colonial morphology, biochemical and other tests. Twenty three P. multocida isolates were recovered: 11/135 (8%) from farm and 12/36 (33%) from the market birds. Majority of the P. multocida isolates were Pasteurella multocida gallicida 11/23 (48%), followed by Pasteurella multocida multocida 7/23 (30%) and Pasteurella multocida septica 5/23 (22%). Pasteurella multocida gallicida isolates were encountered more in the market birds, while Pasteurella multocida multocida isolates were more in farm birds. Ducks had more isolates than chickens. The concentration of the birds at market areas appeared to favor the maintenance of P. multocida in the cages, crates and pens. Market birds may, therefore, play a major role in the spreading of P. multocida. The Kenya Veterinarian Vol. 29 2005: pp. 45-47
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Pasteurella multocida infections. II. Pasteurella multocida infection in man unrelated to animal bite. W T Hubbert, and M N RosenCopyRight https://doi.org/10.2105/AJPH.60.6.1109 Published Online: August 29, 2011
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The objective of this study was to develop a sensitive and specific PCR method for the detection of Pasteurella multocida from porcine.A pair of primers were designed according to the plpE genes of Pasteurella multocida published in GenBank.Then the specific PCR method on the basis of the plpE gene was developed and optimized for rapid detection of Pasteurella multocida.The sensitivity of the method were evaluated with the constructed recombinant plasmid of Pasteurella multocida and other strains.Finally the PCR method established was employed on the detection of 64 suspected samples collected from Sichuan province.Results showed the method based on the plpE gene can amplify the genome of Pasteurella multocida only,not for others and the detection limitation reached 5×101 copies.Thirty-six samples were infected with Pasteurella multocida and the positive rate reached 56.2%.These results indicate that the PCR method on the basis of the plpE gene is specific,repeatable and sensitive and can be used for the detection of Pasteurella multocida.
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Pasteurella multocida is an animal-associated Gram-negative member of the Pasteurellaceae family. It is an opportunistic pathogen and is one of the principal bacterial species contributing to bovine respiratory disease complex (BRDC) in feedlot cattle.
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Abstract Srivastava, S.K. 1998. Imrnunogenicity of Pasteu rella multocida grown in iron restricted medium. J. Appl. Anim. Res., 13: 137–144. Pasteurella multocida semtype B:2 was gmwn under imn-restriction condition to enhance the growth of outer membrane protein (OMP) and tested for immunogenicity. Sodium dodecyl sulphate polyacrylamide gel electmphresis revealed enhanced production of an 84 KDa protein but decrease in pmduction of a pmtein with a molecular weight of 16 KDa. No difference in the quantity of OMP produced by the cells grown under iron- restriction and iron-sufficient conditions was observed as deduced by Congo red binding assay. Vaccines prepared fmm P. multocida cells gmwn in these 2 conditions did not differ from each other in conferring protection in mice and mbbits. However, the antibody titres detected in rabbits were significantly higher with the vaccine consisting of P. multocida cells gmwn under restriction than those grown under iron-sufficient conditions. Similar results were also observed in cattle suggesting an improved immunogenicity of the former. It was concluded that a vawine consisting of P. multocida cells grown under imn-restriction could be more effective in animals than the vaccine prepared from cells gmwn conventionally.
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This work develops a loop-mediated isothermal amplification (LAMP) assay that detects the presence of bacterial pathogens (Pasteurella multocida, Mannheimia haemolytica, and Histophilus somni) for bovine respiratory disease complex (BRD) in crude nasal samples. Diagnosing BRD involves a physical examination of cattle expressing physical symptoms linked to the disease. Unfortunately, these symptoms are not unique to BRD alone and do not identify specific pathogens to treat. Nucleic acid-based diagnostics, like polymerase chain reaction (PCR), identify BRD pathogens by amplifying species-specific genes present in samples. However, PCR-based approaches require (i) expensive equipment for successful operation and (ii) DNA extractions to remove PCR inhibitors in samples. LAMP offers an accurate, inhibitor-resistant approach to detecting BRD pathogens in a point-of-care format. This developed LAMP assay is 97% accurate in pure DNA samples, 99% sensitive, and 89% specific in DNA-spiked bovine nasal samples (with 104 DNA copies/reaction).
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