Pfister, Kurt

2015, Lis, Katarzyna, Pfister, Kurt

Anaplasma marginale is a tick-borne pathogen that affects ruminants worldwide, causing a disease called anaplasmosis. The disease is endemic in tropical and subtropical regions of the New World, Europe, Africa, Asia and Australia where it causes large economic losses in the cattle industry.
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.

2015, Kenneil, Rachel, Pfister, Kurt

Le rickettsies Anaplasma marginale provoque l'anaplasmose bovine, une maladie hémolytique qui est un grave problème pour l'industrie bovine dans les régions tropicales et subtropicales du monde entier. L’A. centrale est une espèce moins pathogène qui est étroitement liée à l’A. marginale et elle est utilisée comme vaccin vivant contre l'anaplasmose. Malheureusement, le vaccin A. centrale ne peut être produit qu’à partir du sang de bovins infectés, qui risquent d’être contaminés par d'autres agents pathogènes. Ce projet a comparé le vaccin vivant A. centrale avec A. marginale UFMG1, une souche brésilienne faiblement pathogène qui a été proposée comme un vaccin vivant issu de culture cellulaire et potentiellement plus sûr. Les veaux ont été infectés par UFMG1 ou A. centrale, puis inoculés avec la souche israélienne pathogène A. marginale Gonen.
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.