Testing and comparing the serum resistance of different strains and mutants of Mycoplasma agalactiae

Bachelorarbeit - Veterinärmedizinische Universität Wien - 2020Mycoplasma agalactiae ist ein wichtiger Erreger von kleinen Wiederkäuern, der chronische, schwer heilbare Infektionen verursacht. Diese sind hauptsächlich durch Mastitis, Arthritis und Keratokonjunktivitis gekennzeichnet. In einigen Fällen breitet sich der Erreger im gesamten Körper aus und kann auch zu Symptomen wie Vaginitis, Aborten und Totgeburten führen. Ein wichtiger Virulenzfaktor, welcher an der systemischen Ausbreitung vieler pathogener Bakterien beteiligt ist, ist die Serumresistenz. Dennoch ist nur wenig darüber bekannt, inwiefern M. agalactiae sich dem Komplementsystem entziehen oder es inaktivieren kann. Ziel dieses Projekts war es, die Serumresistenz verschiedener Stämme und Mutanten von M. agalactiae zu testen, da Unterschiede in diesen Merkmalen für die unterschiedliche Pathogenität verschiedener Stämme, Mutanten und Serovare verantwortlich sein könnten. Serumresistenztests wurden unter Verwendung von nicht sensibilisierten sowie M. agalactiae-sensibilisierten Schafseren aus vorangegangen experimentellen Infektionen mit den Wildtyp-Stämmen PG2 und GM139 sowie den phasen-fixierten Vpma Mutanten PLMU und PLMY durchgeführt. Vorläufige Daten aus ersten Experimenten zeigten signifikante Unterschiede in der Serumresistenz der vier verschiedenen Stämme und phasen-fixierten Mutanten. Darüber hinaus führte die Behandlung mit verschiedenen nicht-sensibilisierten sowie M. agalactiae-sensibilisierten Seren zu Unterschieden in der Serumresistenz der verschiedenen Stämme. Hierbei zeigte sich der internationale Referenzstamm PG2 sowohl für nicht-sensibilisierte als auch für sensibilisierte Seren signifikant anfälliger als der amerikanische Stamm GM139, welcher die höchste Anfälligkeit für das sensibilisierte Serum aufwies. Da PLMY und PLMU scheinbar gegensätzliche Eigenschaften in der Serumresistenz aufweisen, könnte die Vpma-Phasenvariation eine Rolle bei der Serumresistenz und damit der systemischen Ausbreitung der Erreger spielen. Weitere Experimente sollten durchgeführt werden, um diese Ergebnisse zu bestätigen und um weitere Erkenntnisse über die Rolle und die Mechanismen der Serumresistenz von M. agalactiae zu gewinnen. Zusätzlich zum experimentellen Teil befasst sich ein theoretischer Teil mit der Immunologie von Mykoplasmeninfektionen unter besonderer Berücksichtigung von M. agalactiae und der eng verwandten Art M. bovis.Bachelor thesis - University of Veterinary Medicine Vienna - 2020Mycoplasma agalactiae is an important pathogen of small ruminants, causing chronic, difficult-to-cure infections that are mainly characterized by mastitis, arthritis and keratoconjunctivitis. In some cases, the pathogen spreads throughout the whole body and can also lead to symptoms like vaginitis, still births and abortions. A key virulence trait involved in the systemic spread of many pathogenic bacteria is serum resistance, but only little is known about M. agalactiae’s abilities to resist, evade or inactivate the complement. The aim of this project was to test the serum resistance of different strains and mutants of M. agalactiae, as differences in these characteristics could be responsible for the differential pathogenicity of different strains, mutants and serovars. Serum bactericidal assays were performed with the wild-type pathogenic strains PG2 and GM139 as well as the representative phase-locked Vpma mutants PLMU and PLMY using non-sensitized and M. agalactiae-sensitized sheep sera from previous experimental sheep infections. Preliminary data revealed significant differences in the serum susceptibility of the four different strains and PLMs. Moreover, treatment with different non-sensitized, as well as M. agalactiae-sensitized sera also demonstrated differences in the serum resistance of the tested strains, with the PG2 strain being significantly more susceptible to both non-sensitized and sensitized sera than GM139, which showed the highest susceptibility to the sensitized serum. As PLMY and PLMU displayed contrary results in serum resistance assays, Vpma phase-variation might be playing a role in serum resistance, and thus the systemic spread of M. agalactiae. Further experiments should be conducted to confirm these results and to gain further insights in the role and mechanisms of serum resistance in different strains and mutants of M. agalactiae. In addition to the experimental part, a theoretical part deals with the immunology of Mycoplasma infections with special reference to M. agalactiae and M. bovis

Serum Resistance of Mycoplasma agalactiae Strains and Mutants Bearing Different Lipoprotein Profiles

In order to spread systemically, resistance against complement and other factors present in serum is an important trait in pathogenic bacteria. The variable proteins of Mycoplasma agalactiae (Vpmas) have been shown to affect differential adhesion, invasion and immune evasion, and undergo high-frequency phase-variation in expression. However, nothing is known about their involvement in M. agalactiae’s serum susceptibility. To evaluate this, the PG2 strain, the GM139 strain and the six Vpma phase-locked mutants (PLMs, PLMU to PLMZ) were tested for their ability to survive in the presence of non-sensitized and sensitized sheep serum, as well as guinea pig complement. Additionally, the reactivity of the sensitized sheep serum was analysed on the strains via western blotting. Overall data demonstrate PG2 strain to be more susceptible to sheep serum compared to the GM139 strain bearing a different Vpma profile. Significant differences were also observed between the different PLMs, with PLMU and PLMX showing the highest serum susceptibility in serum, while the other PLMs expressing longer Vpma proteins were more resistant. The results are in good correlation with previous studies where shorter lipoprotein variants contributed to a higher susceptibility to complement. Since none of the tested strains and PLMs were susceptible to non-sensitized sheep serum, antibodies seem to play an important role in serum killing

Novel zebrafish patient-derived tumor xenograft methodology for evaluating efficacy of immune-stimulating bcg therapy in urinary bladder cancer

BACKGROUND: Bacillus Calmette-Guérin (BCG) immunotherapy is the standard-of-care adjuvant therapy for non-muscle-invasive bladder cancer in patients at considerable risk of disease recurrence. Although its exact mechanism of action is unknown, BCG significantly reduces this risk in responding patients but is mainly associated with toxic side-effects in those facing treatment resistance. Methods that allow the identification of BCG responders are, therefore, urgently needed. METHODS: Fluorescently labelled UM-UC-3 cells and dissociated patient tumor samples were used to establish zebrafish tumor xenograft (ZTX) models. Changes in the relative primary tumor size and cell dissemination to the tail were evaluated via fluorescence microscopy at three days post-implantation. The data were compared to the treatment outcomes of the corresponding patients. Toxicity was evaluated based on gross morphological evaluation of the treated zebrafish larvae. RESULTS: BCG-induced toxicity was avoided by removing the water-soluble fraction of the BCG formulation prior to use. BCG treatment via co-injection with the tumor cells resulted in significant and dose-dependent primary tumor size regression. Heat-inactivation of BCG decreased this effect, while intravenous BCG injections were ineffective. ZTX models were successfully established for six of six patients based on TUR-B biopsies. In two of these models, significant tumor regression was observed, which, in both cases, corresponded to the treatment response in the patients. CONCLUSIONS: The observed BCG-related anti-tumor effect indicates that ZTX models might predict the BCG response and thereby improve treatment planning. More experiments and clinical studies are needed, however, to elucidate the BCG mechanism and estimate the predictive value