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http://repositorio.inger.gob.mx/jspui/handle/20.500.12100/17105| Título : | Two outer membrane lipoproteins from histophilus somni are Immunogenic in rabbits and sheep and induce protection against bacterial challenge in mice |
| Autor: | CAROLINA GUZMAN BRAMBILA ARGELIA ESPERANZA ROJAS MAYORQUIN BEATRIZ FLORES SAMANIEGO DANIEL ORTUÑO SAHAGUN |
| Palabras clave : | BIOLOGÍA Y QUÍMICA;Ciencias de la vida;Biología animal (zoología);Patología animal;Enfermedad respiratoria bovina;Biological science disciplines;Zoology;Pasteurellaceae;Histophilus somni;Bovine respiratory disease |
| Fecha de publicación: | 12-sep-2012 |
| Editorial : | American Society for Microbiology |
| Descripción : | Abstract: Histophilus somni is an economically important pathogen of cattle and other ruminants and is considered one of the key components of the bovine respiratory disease (BRD) complex, the leading cause of economic loss in the livestock industry. BRD is a multifactorial syndrome, in which a triad of agents, including bacteria, viruses, and predisposing factors or “stressors,” combines to induce disease. Although vaccines against H. somni have been used for many decades, traditional bacterins have failed to demonstrate effective protection in vaccinated animals. Hence, the BRD complex continues to produce strong adverse effects on the health and well-being of stock and feeder cattle. The generation of recombinant proteins may facilitate the development of more effective vaccines against H. somni, which could confer better protection against BRD. In the present study, primers were designed to amplify, clone, express, and purify two recombinant lipoproteins from H. somni, p31 (Plp4) and p40 (LppB), which are structural proteins of the outer bacterial membrane. The results presented here demonstrate, to our knowledge for the first time, that when formulated, an experimental vaccine enriched with these two recombinant lipoproteins generates high antibody titers in rabbits and sheep and exerts a protective effect in mice against septicemia induced by H. somni bacterial challenge. Discussion: BRD is generally detected in cattle raised on farms with poor or nonexistent cattle health management plans (20), but many feedlots, calf raising facilities, and other facilities with excellent management also still have a considerable BRD problem (14, 15, 20, 33, 47). Therefore, the use of vaccines as a preventative measure is important to prevent the spread of this disease (40). Although several commercial vaccines were developed prior to the 1990s (reviewed in reference 41), most caused secondary adverse reactions (12), emphasizing the need to develop recombinant vaccines in which the presence of more than one antigen may increase efficacy. The results presented here demonstrate that a recombinant experimental vaccine containing protein fragments of two OMP H. somni antigens, p31 (Plp4) and p40 (LppB), induced high antibody titers in mice and sheep. Furthermore, this vaccine formulation protected mice from septicemia after bacterial challenge. In addition, the use of Al(OH)3 as an adjuvant potentiated the immune response, thereby maximizing the potency and efficacy of the antigens, which are generated in limited amounts (13), and enhancing the immune response (46). Protective effects of antibodies against the H. somni OMPs p31 (Plp4) and p40 (LppB).The immunogenic potential of some H. somni proteins has been previously assessed, demonstrating that they can produce partial protection against bacterial infection. Among antigens most probably involved in stimulating host defense as well as immunopathology, OMPs are relevant as virulence factors (6, 9). Proteins on the cell surface undoubtedly play an important role in H. somni virulence and host immunity (reviewed in reference 44). Because Gram-negative bacteria exhibit a high degree of genomic variability in some of its proteins, conserved OMPs became relevant immunogenic targets that are able to elicit cellular mechanisms of host defense involving the antigen-induced release of cytokines from lymphocytes and the resulting activation of macrophages with the ability to kill the pathogen. For example, a 40-kDa OMP from H. somni was proposed as a candidate protective protein against pneumonia in calves following active immunization (19). However, according to a recent analysis of genes and gene products putatively involved in H. somni strain 2336 virulence, this protein corresponds to a different OMP than the p40 (LppB) used here (see Table 2 in reference 44). A 78-kDa OMP antigen that was also shown to be consistently and intensely immunoreactive in Western blots of H. somnus WC reacted with convalescent-phase serum from cattle with experimental H. somnus pneumonia (26). However, this antigen failed to elicit protective effects (19). An immunoglobulin binding protein A (IbpA) containing a Fic motif involved in the virulence of several pathogens (42, 53) was recently described (30, 55) as a viable vaccine candidate in the bovine host. Immunization with the IbpA DR2 subunit from H. somni was demonstrated to partially protect against bacterial infection in a natural host (16, 30). However, further studies will be necessary to determine the immunogenic properties of the different virulence factors involved in H. somni infection, since the evidence for protection against pneumonia by current vaccines remains controversial (29, 48). Although further immunogenic studies in beef cattle are required, the results presented here demonstrate for the first time that the antibody response in rabbits and in sheep against two H. somni OMPs, p31 (Plp4) and p40 (LppB), is relevant. In addition, when combined with a commercial vaccine for other bacterial diseases (Biovac 7 vías), recombinant fragments of p31 (Plp4) and p40 (LppB), which appear to be conserved structural proteins of the outer bacterial membrane, exert a protective effect against H. somni bacterial challenge in mice. p31 and p40 structure and possible functional implications.The electrophoretic mobility observed for the p40 fusion protein corresponded to that originally reported for this protein (8, 50), although it was slightly higher than theoretically expected. This protein is rich in proline (8%), asparagine (8.0%), and isoleucine (9.7%) compared to the average amino acid composition of vertebrate proteins (54), a profile that may influence its electrophoretic mobility. The fusion protein also contains some putative peptidoglycan binding sites (32), which may modify its relative molecular mobility. In addition, the increased electrophoretic mobility described here may be partially due to the addition of a histidine tag and the factor Xa recognition site. Nonetheless, when the clones were sequenced they precisely matched the reported sequence, and thus the induced fusion protein corresponds to p40. It is of interest to determine whether the organization of the distinct domains in these two proteins are implicated in the effects of H. somni on endothelial cells and in the aggregation of platelets to form thrombi in blood vessels (19). These phenomena induce endothelial cell proinflammatory responses and platelet internalization (27), and they trigger cytoskeletal alterations that increase the permeability of the endothelium (4), resulting in the redistribution of PECAM 1 on the surfaces of bronchial endothelial cells (51). Further studies are thus required with antibodies that identify p31 (Plp4) and p40 (LppB), such as those described here, that can be used to study their effects at the cellular level (e.g., by analyzing the antibody neutralization of these two proteins in vitro). Finally, the basis of viral/bacterial synergism and the manner in which cattle respond to the virulence strategies of bacterial pathogens remain poorly understood (10). The ability of H. somni to resist leukocytes while creating a proinflammatory and procoagulation environment at the endothelial cell surface and the ability of M. haemolytica to circumvent leukocyte antibacterial activity via its leukotoxin LKTA probably contribute to the intense inflammation that characterizes BRD (10). As such, it seems feasible to propose that a vaccine that combines antigenic surface proteins from M. haemolytica (e.g., different fragments of LktA and Plp) (7, 22) and H. somni (e.g., p31 and p40, as demonstrated here) may be useful in preventing infection and reducing the incidence of BRD. Further experiments would be needed to test this hypothesis. |
| URI : | http://repositorio.inger.gob.mx/jspui/handle/20.500.12100/17105 |
| Aparece en las colecciones: | 1. Artículos |
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