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Pfister, Kurt
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Pfister, Kurt
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- PublicationAccès libreAnalysis of "Anaplasma marginale" strains grown "in vitro"(2015)
A. marginale is an obligatory intracellular bacterium that multiplies only within tick cells or ruminants' erythrocytes. Many differences among A. marginale strains have emerged, which were probably driven by continuous exposure to different host immune systems during the transition of bacteria between ticks and vertebrates. The vast majority of studies aiming at elucidating differences between strains were conducted on the genomic level, and little is known about protein expression. Thus, this thesis investigates differences in protein regulation among A. marginale strains.
A. marginale cultivated in vitro are in general an excellent source of organisms for experimentation. Furthermore, culture-derived organisms offer an alternative to the use of experimental animals.
Many studies require intracellular organisms free from host cell debris. Therefore the use of Percoll gradients for the separation of A. marginale was evaluated. Bacteria isolated in this way contained only minimal amounts of IDE8 cell stroma but most importantly they retained their viability. A. marginale purified this way can be used directly for proteomic studies or for vaccination trials.
In this thesis three geographical A. marginale strains grown in vitro have been partially characterized by gene and serological analyses. The differences on the proteomic level have been assessed by the 2D-DIGE technique, indicating that many antigenic membrane proteins are differentially regulated among the strains examined. Some of these proteins are also known to be virulence-associated.
Increasing the number of strains in continuous in vitro cultivation, and improving purification methods for rickettsia, allow researchers to investigate differences in protein expression between A. marginale strains, and therefore identify proteins which could be incorporated into an improved vaccine against anaplasmosis. - PublicationAccès libreThe immune response to live vaccines against bovine anaplasmosis(2015)
L’infection précédente avec UFMG1 n'a pas significativement réduit la sévérité de la maladie causée par la souche inoculée avec le Gonen, alors que l'infection par A. centrale a fournit une protection croisée contre le Gonen. Par rapport à la réaction des anticorps à l’infection UFMG1, la réaction à l'infection par A. centrale avait des niveaux globaux plus élevés d'IgG et a montré une réactivité croisée ultérieure à l'antigène de la souche Gonen. La réaction des anticorps à A. centrale avait également des niveaux plus élevés d'IgG2, et a montré une activité plus opsonophagocytaire. Toutes ces caractéristiques sont en corrélation significative avec la protection contre la maladie lors de l'épreuve. Comprendre comment l'infection par A. centrale stimule cette réponse immunitaire efficace serait une piste de recherche vaccinale prometteuse., Anaplasma marginale causes bovine anaplasmosis, a hemolytic disease which is a serious problem for the cattle industry in tropical and sub-tropical regions worldwide. A. centrale is a less pathogenic species which is closely related to A. marginale and used as a live vaccine against anaplasmosis. Unfortunately the A. centrale vaccine can only be produced from the blood of infected cattle, risking contamination with other pathogens. This project compared the A. centrale live vaccine with A. marginale UFMG1, a low pathogenicity Brazilian strain which has been proposed as a potentially safer live vaccine derived from cell culture. Calves were infected with UFMG1 or A. centrale, and then challenged with the pathogenic Israeli A. marginale Gonen strain.
Previous infection with UFMG1 did not significantly reduce the severity of disease caused by challenge with the Gonen strain, whereas A. centrale infection did provide cross-protection against Gonen. In comparison to the antibody response to UFMG1 infection, the response to A. centrale infection had higher overall levels of IgG and showed higher cross-reactivity to Gonen strain antigen. The antibody response to A. centrale also had higher levels of IgG2, and showed more opsonophagocytic activity. All of these characteristics correlated significantly with protection from disease upon challenge. Understanding how A. centrale infection stimulates this effective immune response would be a valuable direction for future vaccine research. - PublicationAccès libreLight and electron microscopy studies of the midgut and salivary glands of second and third instars of the horse stomach bot, Gasterophilus intestinalis(2010)
; ; ; A morphological study of the midgut and salivary glands of second and third instars of Gasterophilus intestinalis (De Geer) (Diptera: Oestridae) was conducted by light, scanning and transmission electron microscopy. The midgut is anteriorly delimited by a proventriculus, without caeca, and is composed of posterior foregut and anterior midgut tissue from which a double-layered peritrophic matrix is produced. The midgut can be divided into anterior, median and posterior regions on the basis of the structural and physiological variations of the columnar cells which occur along its length. Two other types of cell were identified: regenerative cells scattered throughout the columnar cells, and, more rarely, endocrine cells of two structural types (closed and open). Different secretion mechanisms (merocrine, apocrine and microapocrine) occur along the midgut epithelium. Abundant microorganisms are observed in the endoperitrophic space of the anterior midgut. The origin and nature of these microorganisms remain unknown. No structural differences are observed between the second and third instar midguts. The salivary glands of G. intestinalis second and third instars consist of a pair of elongated tubular structures connected to efferent ducts which unite to form a single deferent duct linked dorsally to the pharynx. Several intermediate cells, without cuticle, make the junction with the salivary gland epithelium layer. Cytological characteristics of the gland epithelial cells demonstrate high cellular activity and some structural variations are noticed between the two larval stages. - PublicationAccès libreProtein expression profile of Gasterophilus intestinalis larvae causing horse gastric myiasis and characterization of horse immune reaction(2009)
; Background Little information is available on the immunological aspect of parasitic Gasterophilus intestinalis (Diptera, Oestridae) larvae causing horse gastric myiasis. The objectives of this research were to analyze the protein content of larval crude extracts of the migrating second and third larvae (L2 and L3) of G. intestinalis in order to characterize the immune response of horses.
Results The proteomic profile of L2 and L3, investigated by using one and two dimensional approaches, revealed a migration pattern specific to each larval stage. Furthermore, Western blots were performed with horse sera and with sera of Balb/c mice immunised with the larval crude extracts of L2 or L3, revealing a different immune reaction in naturally infected horses vs. artificially induced immune reaction in mice. The comparisons of the immunoblot profiles demonstrate that the stage L2 is more immunogenic than the stage L3 most likely as an effect of the highest enzymatic production of L2 while migrating through the host tissues. Fifteen proteins were identified by mass spectrometry.
Conclusion This work provides further information into the understanding of the interaction between G. intestinalis and their host and by contributing a novel scheme of the proteomic profile of the main larval stages. - PublicationAccès librePCR Detection and Serological Evidence of Granulocytic Ehrlichial Infection in Roe Deer (Capreolus capreolus) and Chamois (Rupicapra rupicapra)(2002)
; The role of wild mammals, such as roe deer (Capreolus capreolus) and chamois (Rupicapra rupicapra), in the epidemiology of granulocytic ehrlichiae in Switzerland was investigated. We tested blood samples for Ehrlichia phagocytophila genogroup 16S rRNA gene sequences by PCR and for immunoglobulin G antibodies against granulocytic ehrlichiae by indirect fluorescent-antibody assay (IFA). Overall means of 60.9% of 133 roe deer serum samples and 28.2% of 39 chamois serum samples were seroreactive by IFA. PCR results were positive for 18.4% of 103 roe deer serum samples as well. None of the 24 chamois blood samples tested were positive by PCR. Partial 16S rRNA gene and groESL heat shock operon sequences of three roe deer samples tested showed strong degrees of homology (99.7 and 98.6%, respectively) with the sequences of granulocytic ehrlichiae isolated from humans. These results confirm that chamois, and particularly roe deer, are commonly infected with granulocytic ehrlichiae and provide evidence that these wild mammals are potential reservoirs for granulocytic ehrlichiae in Switzerland. - PublicationAccès libre
- PublicationAccès libreDonnées nouvelles sur les Tiques Ixodides du canton du Tessin (Suisse) et sur la présence d'agents rickettsies dans leur hémolymphe(1982)
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