Pathogenesis and Virulence of Pasteurella Haemolytica in Cattle: An Analysis of Current Knowledge and Future Approaches

Confer, Anthony W.; Clinkenbeard, Kenneth D.; Murphy, George L.

doi:10.1007/978-1-4899-0978-7_5

Cited by 19 publications

(16 citation statements)

References 42 publications

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“…PCR primers were synthesized at the Laboratory Services Division. A typical 50-l reaction mixture contained 10 to 100 ng of template, 50 to 100 pmol of each primer, a 0.2 mM concentration of each deoxynucleoside triphosphate, and 2 to 4 mM MgSO 4 in the PCR buffer supplied by the manufacturer. The reaction included a hot start of 2 to 5 min at 95°C, followed by the addition of 1 U of Taq polymerase (Roche Diagnostics, Laval, Quebec, Canada) or Pwo polymerase (Roche Diagnostics) and then 30 cycles of 95°C for 1 min, 45 to 65°C for 1 min, and 72°C for 2 min.…”

Section: Methodsmentioning

confidence: 99%

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Towards Development of an Edible Vaccine against Bovine Pneumonic Pasteurellosis Using Transgenic White Clover Expressing aMannheimia haemolyticaA1 Leukotoxin 50 Fusion Protein

Lee¹,

Strommer

Hodgins

et al. 2001

Infect Immun

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Development of vaccines against bovine pneumonia pasteurellosis, or shipping fever, has focused mainly on Mannheimia haemolytica A1 leukotoxin (Lkt). In this study, the feasibility of expressing Lkt in a forage plant for use as an edible vaccine was investigated. Derivatives of the M. haemolytica Lkt in which the hydrophobic transmembrane domains were removed were made. Lkt66 retained its immunogenicity and was capable of eliciting an antibody response in rabbits that recognized and neutralized authentic Lkt. Genes encoding a shorter Lkt derivative, Lkt50, fused to a modified green fluorescent protein (mGFP5), were constructed for plant transformation. Constructs were screened by Western immunoblot analysis for their ability to express the fusion protein after agroinfiltration in tobacco. The fusion construct pBlkt50-mgfp5, which employs the cauliflower mosaic virus 35S promoter for transcription, was selected and introduced into white clover by Agrobacterium tumefaciens-mediated transformation. Transgenic lines of white clover were recovered, and expression of Lkt50-GFP was monitored and confirmed by laser confocal microscopy and Western immunoblot analysis. Lkt50-GFP was found to be stable in clover tissue after drying of the plant material at room temperature for 4 days. An extract containing Lkt50-GFP from white clover was able to induce an immune response in rabbits (via injection), and rabbit antisera recognized and neutralized authentic Lkt. This is the first demonstration of the expression of an M. haemolytica antigen in plants and paves the way for the development of transgenic plants expressing M. haemolytica antigens as an edible vaccine against bovine pneumonic pasteurellosis.Mannheimia haemolytica A1 is the principal microorganism responsible for bovine pneumonia pasteurellosis, or shipping fever, a major cause of sickness, death, and economic loss in the feedlot cattle industry (12, 33). Traditional immunization approaches using needle injection of various vaccine preparations have provided some degree of protection. However, needle injection requires the herding and restraint of the animals, inducing additional stress as well as incurring a substantial labor cost. As an alternative, we propose to develop a noninvasive means of delivery of the vaccine via the oral route by using transgenic plants expressing recombinant immunogens. Recent advances in the understanding of transgene expression and recombinant protein accumulation, stability, and processing in plants have allowed the development of novel strategies such as using edible plants for delivery of antigens for active immunization (for reviews, see references 24, 28, and 30).The leukotoxin (Lkt) of M. haemolytica A1 is one of its major virulence factors (26). Lkt is secreted by M. haemolytica A1 and acts as a pore-forming cytolysin that inserts into the membrane of target cells (3), resulting in osmotic imbalance and cell lysis. This initiates a cascading effect that leads to tissue damage, pneumonia, and death of the animals (1, 4). Lkt is a...

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Section: Methodsmentioning

confidence: 99%

“…Lkt is secreted by M. haemolytica A1 and acts as a pore-forming cytolysin that inserts into the membrane of target cells (3), resulting in osmotic imbalance and cell lysis. This initiates a cascading effect that leads to tissue damage, pneumonia, and death of the animals (1,4). Lkt is a member of the RTX family of cytolysins (31,32).…”

mentioning

confidence: 99%

Towards Development of an Edible Vaccine against Bovine Pneumonic Pasteurellosis Using Transgenic White Clover Expressing aMannheimia haemolyticaA1 Leukotoxin 50 Fusion Protein

Lee¹,

Strommer

Hodgins

et al. 2001

Infect Immun

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“…We used a biologically inactive mutant leukotoxin ( Bovine pneumonic pasteurellosis (BPP) caused by Mannheimia (Pasteurella) haemolytica serotype 1 remains a major economic problem for the beef and dairy cattle industries in North America and Western Europe (2, 10, 14, 47). The leukotoxin (LktA) produced by this bacterium is the primary virulence factor that contributes to the pathogenesis of the fibrinonecrotizing pleuropneumonia and death characteristic of this disease (7,8,43,44). A large body of evidence indicates that much of the lung injury in this disease is caused by inflammatory mediators released from alveolar leukocytes by LktA-induced activation and cytolysis (11,34,41,48).…”

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confidence: 99%

Mannheimia haemolyticaLeukotoxin Activates a Nonreceptor Tyrosine Kinase Signaling Cascade in Bovine Leukocytes, Which Induces Biological Effects

et al. 2001

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The leukotoxin (LktA) produced by Mannheimia haemolytica binds to bovine lymphocyte function-associated antigen 1 (LFA-1) and induces biological effects in bovine leukocytes in a cellular and species-specific fashion. We have previously shown that LktA also binds to porcine LFA-1 without eliciting any effects. These findings suggest that the specificity of LktA effects must entail both binding to LFA-1 and activation of signaling pathways which are present in bovine leukocytes. However, the signaling pathways leading to biological effects upon LktA binding to LFA-1 have not been characterized. In this context, several reports have indicated that ligand binding to LFA-1 results in activation of a nonreceptor tyrosine kinase (NRTK) signaling cascade. We designed experiments with the following objectives: (i) to determine whether LktA binding to LFA-1 leads to activation of NRTKs, (ii) to examine whether LktA-induced NRTK activation is target cell specific, and (iii) to determine whether LktA-induced NRTK activation is required for biological effects. We used a biologically inactive mutant leukotoxin ( Bovine pneumonic pasteurellosis (BPP) caused by Mannheimia (Pasteurella) haemolytica serotype 1 remains a major economic problem for the beef and dairy cattle industries in North America and Western Europe (2, 10, 14, 47). The leukotoxin (LktA) produced by this bacterium is the primary virulence factor that contributes to the pathogenesis of the fibrinonecrotizing pleuropneumonia and death characteristic of this disease (7,8,43,44). A large body of evidence indicates that much of the lung injury in this disease is caused by inflammatory mediators released from alveolar leukocytes by LktA-induced activation and cytolysis (11,34,41,48).LktA is a member of a family of gram-negative RTX (repeats in toxin) cytolysins (12,20). Unlike most other RTX cytolysins, leukotoxins produced by Actinobacillus actinomycetemcomitans (LtxA) and M. haemolytica (LktA) demonstrate cell-type-specific and species-specific biological effects. The LtxA of A. actinomycetemcomitans, which causes dental caries in humans, interacts only with cells of the lymphocytic and monomyelocytic lineages of humans and some nonhuman primates and provokes biological effects (29); the LktA of M. haemolytica interacts only with ruminant leukocytes and platelets and induces biological effects (6,25,37). A study by Lally et al. (30) reported that human lymphocyte function-associated antigen 1 (LFA-1), a member of the ␤2 integrins, is a target cell receptor for LtxA of A. actinomycetemcomitans. Three studies have identified bovine CD18, the ␤ subunit of all three bovine ␤2 integrins (LFA-1, Mac-1, and p150,95), as a receptor for M. haemolytica LktA (3,33,42). However, in these studies no specific member of the ␤2 integrin family was identified as an LktA receptor. We have extended these observations and have shown that bovine LFA-1, but not other members of the ␤2 integrin family, is a receptor for M. haemolytica LktA (23).LFA-1 is a heterodimeric glycoprotein co...

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“…Mannheimia haemolytica A1 is the principal microorganism associated with shipping fever, a major cause of sickness and death and significant economic loss in feedlots of North America. Leukotoxin (Lkt), a virulence factor, has been identified as an important protective antigen (Confer et al, 1995). We have demonstrated that an Lkt fragment (Lkt50) synthesized as a fusion protein with green fluorescent protein (GFP) in transgenic clover can induce an immune response in rabbits by eliciting antibodies that neutralize authentic Lkt (Lee et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

Transformation of alfalfa with a bacterial fusion gene, Mannheimia haemolytica A1 leukotoxin50-gfp: Response with Agrobacterium tumefaciens strains LBA4404 and C58

Ziauddin

Lee

et al. 2004

Plant Cell Tiss Organ Cult

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Alfalfa transformed with a portion of the leukotoxin gene from Mannheimia haemolytica was produced to test the feasibility of developing an edible vaccine capable of protecting cattle from pneumonic pasteurellosis. Leukotoxin (Lkt), has been identified as an important protective antigen of M. haemolytica, and a fragment, Lkt50, was shown to produce toxin-neutralizing antibodies in rabbits. The construct chosen for introduction into alfalfa carried lkt50 fused to a green fluorescent protein reporter gene, mgfp5-ER. The fusion gene was driven by either the cauliflower mosaic virus 35S promoter (35S) or the promoter from a rubisco small subunit (rbcS-3A) gene of pea. The constructs were introduced into alfalfa RSY27 germplasm using two Agrobacterium tumefaciens strains, LBA4404 and C58, producing a number of transformed lines with both A. strains. Although strain C58 had a slower initial response and produced less callus than strain LBA4404, it resulted in higher numbers of transformed embryos and plants. In total, 30 alfalfa lines (91% of those analyzed), each derived from a separate transformation event, produced detectable levels of Lkt50-GFP. Western analysis with anti-Lkt+66 antiserum revealed the presence of both full-length and truncated polypeptides in plants kept in magenta boxes, while plants transferred to the greenhouse produced only the full-length product. Immunoblotting with anti-GFP antiserum provided evidence that part of the GFP moiety was lost in the truncated protein. Southern blot analysis indicated a low number of insertion sites per event.

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Pathogenesis and Virulence of Pasteurella Haemolytica in Cattle: An Analysis of Current Knowledge and Future Approaches

Cited by 19 publications

References 42 publications

Towards Development of an Edible Vaccine against Bovine Pneumonic Pasteurellosis Using Transgenic White Clover Expressing aMannheimia haemolyticaA1 Leukotoxin 50 Fusion Protein

Towards Development of an Edible Vaccine against Bovine Pneumonic Pasteurellosis Using Transgenic White Clover Expressing aMannheimia haemolyticaA1 Leukotoxin 50 Fusion Protein

Mannheimia haemolyticaLeukotoxin Activates a Nonreceptor Tyrosine Kinase Signaling Cascade in Bovine Leukocytes, Which Induces Biological Effects

Transformation of alfalfa with a bacterial fusion gene, Mannheimia haemolytica A1 leukotoxin50-gfp: Response with Agrobacterium tumefaciens strains LBA4404 and C58

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