Einleitung Streptococcus (S.) suis verursacht bei Schweinen unter anderem Meningitis, Arthritis, Serositis und Endokarditis und ist eine der größten Herausforderungen für die Schweineindustrie. Von 29 beschriebenen Serotypen sind die Serotypen 2, 7 und 9 unter invasiven Isolaten weltweit besonders prävalent, vor allem in Europa. Bis heute gibt es keinen zugelassenen Impfstoff zur Prävention von S. suis-Erkrankungen in Europa, daher ist im Feld die Anwendung stallspezifischer Impfstoffe verbreitet. Diese bieten jedoch höchstens homologen Schutz und ihre Wirkung kann durch prädisponierende Faktoren wie eine Infektion mit dem porcine reproductive and respiratory syndrome Virus (PRRSV) beeinträchtigt werden. Daher hat sich die Forschung auf Antigene fokussiert, die potentiell heterologen Schutz vermitteln. Zielstellung Ziele der Studie waren die Charakterisierung vernachlässigter invasiver S. suis Pathotypen der wichtigen Serotypen 7 und 9 und die Etablierung neuer Infektionsmodelle im Schwein. Des Weiteren sollte die immunogene und protektive Wirkung des Immunoglobulin (Ig) M-degradierenden Enzyms von S. suis, IdeSsuis, im Serotyp 9 Infektionsversuch untersucht werden. Material und Methoden In dieser Arbeit wurden in vitro Versuche und experimentelle Infektionen im Schwein durchgeführt. Dazu gehörte die Geno- und Phänotypisierung von 22 S. suis Serotyp 7 Stämmen und vier Serotyp 9 Stämmen. Die Genotypisierung erfolgte mittels multiplex (MP) Polymerase-Kettenreaktion (PCR), einer PCR zur Differenzierung verschiedener Genvarianten des muramidase-release protein (MRP) sowie mittels multilocus sequence typing (MLST). Zur Phänotypisierung der S. suis Stämme wurden bactericidal assays eingesetzt, die als Bakteriämiemodell fungierten. Auf diese Weise konnten die Empfänglichkeit gegenüber S. suis Stämmen sowie deren Virulenz beurteilt werden. Durch Zugabe von rekombinantem (r) IdeSsuis wurde die Rolle adaptiven IgMs in der Begrenzung der Bakteriämie untersucht. Anhand von Western Blot Analysen erfolgte die Untersuchung der Expression und Funktionalität von IdeSsuis sowie die Expression von MRP in S. suis Serotyp 7 Stämmen. Enzyme-linked immunosorbent assays (ELISA) kamen zum Einsatz, um die Entwicklung von IgM und IgG Spiegeln in Ferkeln im zeitlichen Verlauf und die IgG Spiegel nach rIdeSsuis Immunisierung zu messen. Durch rIdeSsuis Immunisierung induzierte Antigen-spezifische T-Helferzellen (Th-Zellen) wurden mithilfe der Durchflusszytometrie untersucht. Schließlich erfolgte die Durchführung zweier S. suis Serotyp 7 Etablierungsversuche mit je 18 bzw. 5 Ferkeln sowie zwei S. suis Serotyp 9 Impf- und Infektionsversuche mit je 18 Ferkeln. Sektionsproben wurden histologisch untersucht. In einem Fall wurde eine Endokarditis mittels fluorescence in situ hybridization (FISH) charakterisiert. Ergebnisse Die meisten untersuchten Serotyp 7 Stämme gehörten dem Sequenztyp (ST) 29 an, einem emerging pathotype in Europa. Trotz der engen phylogenetischen Verwandtschaft, war mrp in den Stämmen sehr variabel. Phänotypisch bildeten alle Stämme gleichermaßen eine kleine MRP Variante, MRPs. Für vier ausgewählte Serotyp 7 Stämme wurde die Expression von IdeSsuis gezeigt, jedoch mit Unterschieden in Größe und Funktionalität. Bactericidal assays dieser vier Stämme zeigten starke Proliferation im Blut von Absatzferkeln, aber Abtöten im Blut von Läuferschweinen aus zwei Herden mit unterschiedlichem S. suis Status. Dieses Überlebensmuster unterschied sich deutlich von dem eines Serotyp 9 Stammes. Durch Zugabe von rIdeSsuis, konnte gezeigt werden, dass das Abtöten der Serotyp 7 Stämme im Blut von Läuferschweinen des infizierten Bestandes IgM-vermittelt war. Unabhängig von der Herkunft entwickelten sich die IgM Spiegel in den Ferkeln im zeitlichen Verlauf fast synchron. Schließlich konnte die Virulenz eines Serotyp 7 Stammes in einem intravenösen Infektionsversuch mit 5 Ferkeln gezeigt werden. Alle Tiere entwickelten schwere Symptome einer S. suis Erkrankung. In einem Impf- und Infektionsversuch mit einem hoch virulenten Serotyp 9 Stamm wurde die immunogene und protektive Wirkung einer rIdeSsuis Immunisierung untersucht. Neun Ferkel wurden mit rIdeSsuis oder einem Placebo prime-boost-boost immunisiert und zwei Wochen später infiziert. Neunzig Prozent der Placebotiere entwickelten schwere Symptome einer S. suis Erkrankung und starben oder mussten aus Tierschutzgründen euthanasiert werden. Alle geimpften Tiere überlebten den Versuch, fielen jedoch mit Fieber und Lahmheiten auf. Es konnte also gezeigt werden, dass eine rIdeSsuis Immunisierung vor Mortalität, nicht aber Morbidität durch den Infektionsstamm schützt. Alle immunisierten Ferkel serokonvertierten und Antigen-spezifische Th-Zellen wurden nachgewiesen. Weder die IgG Antwort noch die Th-Zell Antwort wurde jedoch durch die Infektion verstärkt. In einem weiteren Impf- und Infektionsversuch mit einem anderen Serotyp 9 Stamm fiel ein Tier am 11. Tag nach der Infektion mit Zeichen einer akuten Leptomeningitis auf, nachdem es zuvor klinisch völlig unauffällig war. In der Sektion des Tieres wurde eine Endokarditis der Mitralklappe diagnostiziert, die mit Biofilm-Bildung assoziiert war, was mithilfe von Histologie und FISH gezeigt werden konnte. Zusätzlich wurde eine fibrinopurulente Leptomeningitis diagnostiziert. Das Ferkel hatte Antikörper gegen rIdeSsuis und tötete den Infektionsstamm ex vivo im Blut ab. Schlussfolgerungen In dieser Arbeit habe ich zwei wichtige S. suis Pathotypen charakterisiert: S. suis Serotyp 7 Stämme des ST29 und einen hoch virulenten Serotyp 9 Stamm vom ST94. Die Serotyp 7 Stämme stellten einen besonderen Pathotypen dar, bei dessen Bekämpfung IgM eine wichtige Rolle spielt. Bei einer Serotyp 9 Infektion konnten trotz des Abtötens der Bakterien im Blut und opsonisierender Antikörper Biofilmbildung und eine folgende akute Leptomeningitis nicht verhindert werden. Letztlich wurde Schutz vor einem hoch virulenten Serotyp 9 Stamm durch rIdeSsuis Immunisierung und damit zum ersten Mal für ein Antigen Protektion vor den wichtigen Serotypen 2 und 9 aufgezeigt. Dies ist ein vielversprechendes Ergebnis hinsichtlich der Entwicklung eines Impfstoffes gegen S. suis. / Introduction Streptococcus (S.) suis causes meningitis, arthritis, serositis and endocarditis and is one of the biggest challenges for the swine industry. Of 29 described serotypes, the serotypes 2, 7 and 9 are highly prevalent amongst invasive S. suis isolates worldwide, especially in European countries. To date, no commercially produced vaccine is available in Europe for prevention of S. suis disease, thus the use of autogenous vaccines in the field is common. However, bacterins may at most confer homologous protection and their efficacy may be influenced by predisposing factors such as an infection with the porcine reproductive and respiratory syndrome virus (PRRSV). Research has therefore focused on subunit vaccines with the potential to elicit cross-protection against various serotypes. Aim of the study The objective of this study was to characterize neglected invasive S. suis pathotypes of the important serotypes 7 and 9 and to establish infection models in the main host of S. suis, the pig. A further aim was to investigate immunogenicities and protective efficacies of the immunoglobulin (Ig) M-degrading enzyme of S. suis, IdeSsuis, in a serotype 9 challenge experiment. Materials and methods In vitro experiments and experimental challenges in swine were conducted as part of this thesis. This included the geno- and phenotyping of 22 S. suis serotype 7 strains and four serotype 9 strains. Genotyping was conducted using a multiplex (MP) polymerase chain reaction (PCR), a PCR for differentiation of variants of the muramidase-released protein (MRP) gene and multilocus sequence typing (MLST). For phenotyping of the S. suis strains, bactericidal assays were carried out which served as a model for bacteraemia. This way, susceptibility to S. suis strains and virulence of different strains was assessed. Further, through addition of recombinant (r) IdeSsuis, the role of adaptive IgM in limiting bacteraemia was elucidated. Western blot analyses were conducted to investigate expression and functionality of IdeSsuis as well as expression of MRP in S. suis serotype 7 strains. Enzyme-linked immunosorbent assays (ELISA) were used to determine the development of IgM and IgG levels in piglets over time and to assess IgG levels following rIdeSsuis immunization. Antigen-reactive T-helper (h) cells induced by rIdeSsuis immunization were investigated using flow cytometry. Finally, two experiments to establish a serotype 7 infection model with 18 and 5 piglets each and two vaccination and challenge experiments using different S. suis serotype 9 strains (n=18 piglets/ experiment) were conducted. Samples of dissected animals were examined histologically. In one case, an endocarditis was analysed using fluorescence in situ hybridization (FISH). Results Most of the investigated serotype 7 strains belonged to sequence type (ST) 29 which was thus shown to be an emerging pathotype in Europe. Despite the close phylogenetic relation of the strains, mrp was highly variable. Phenotypically, all strains expressed a small variant of MRP, MRPs. Four selected serotype 7 strains were shown to express IdeSsuis with differences in size and functionality. Bactericidal assays of these four strains revealed high proliferation in blood of weaning piglets but killing in blood of growing piglets of two herds which differed in their S. suis infection status. This survival pattern was distinct from a serotype 9 strain. Addition of rIdeSsuis revealed that killing of the serotype 7 strains in blood of growing piglets of the infected herd was IgM-mediated. Independent of the originating herd, IgM levels of the piglets rose almost synchronous over time. Finally, the virulence of a serotype 7 strain was proven in an intravenous challenge experiment with five pigs which all developed severe clinical signs of S. suis disease. In a vaccination and challenge experiment using a highly virulent serotype 9 strain, immunogenicities and protective efficacies of rIdeSsuis immunization were investigated. Nine piglets were prime-boost-boost vaccinated with rIdeSsuis or placebo-treated and challenged two weeks later. Ninety per cent of the placebo-treated piglets developed severe clinical signs of S. suis disease and died or had to be euthanized due to animal welfare reasons. All vaccinated piglets survived the experiment, however elevated body temperatures and lameness were also noted in this group. Accordingly, rIdeSsuis vaccination protected from mortality but not morbidity caused by the challenge strain. Seroconversion of the immunized piglets and antigen-reactive Th cells were detected. Neither the IgG response nor the Th cell response was boosted through the challenge. In a further vaccination and challenge experiment with a different serotype 9 strain, one animal was clinically unobtrusive following infection and then developed an acute leptomeningitis on the 11th day post infection and had to be euthanized. Dissection of the animal revealed an endocarditis on the mitral valve which was proven to be associated with biofilm formation by histology and FISH. In addition, a fibrinosuppurative leptomeningitis was diagnosed. The piglet had specific antibodies against rIdeSsuis and mediated killing of the challenge strain in a bactericidal assay. Conclusions In this thesis, I characterized two important pathotypes: S. suis serotype 7 strains of ST29 and a highly virulent serotype 9 strain of ST94. The serotype 7 strains represent a distinct pathotype and IgM plays a significant role in their control. Following a serotype 9 infection, biofilm formation and a subsequent acute leptomeningitis could not be prevented despite blood bactericidal activity and opsonizing antibodies. Finally, protection against challenge with a highly virulent serotype 9 strain through rIdeSsuis immunization was demonstrated. Thereby, for the first time an antigen was shown to confer cross-protection against the important serotypes 2 and 9, which is highly encouraging regarding the development of a vaccine against S. suis.

Florida's mild climate supports year round enzootic transmission of arthropod-borne (arbo) viruses, such as St. Louis Encephalitis virus (SLEV), West Nile virus (WNV), and Eastern Equine Encephalomyelitis virus (EEEV). First isolated in 1960 from two Florida blue jays, Highlands J virus (HJV) is endemic to the state and vectored by the same mosquitoes as EEEV (Henderson et al, 1962). EEEV and HJV are both alphaviruses, but HJV is not pathogenic to humans, occasionally causes encephalitis in horses, and is a recognized pathogen in some bird species (turkeys, emus, etc) (Cilnis et al, 1996). The Florida Sentinel Chicken Arboviral Surveillance Program, established in 1978, utilizes sentinel chickens to detect arboviral activity throughout the state. Current serologic antibody detection methods include the hemagglutination inhibition (HAI), IgM antibody capture enzyme-linked immunosorbent (MAC-ELISA), and serum neutralization plaque reduction (PRNT) assays (Blackmore et al, 2003). In 2003, sentinel chickens detected significantly greater alphavirus activity than seen in the previous 15 years (Stark & Kazanis, 2003). This increase raised concerns that bridging into the human population had become a serious threat as well as an important issue for veterinary health. The objective of this study was to determine if cross-reactions with Highlands J virus were impacting the serologic diagnostic tests routinely performed for identification of EEEV. For 2003, the HAI test detected 476 alphavirus positive sentinels. We tested 316 of these chickens in the PRNT, which identified 176 EEEV positive sentinels and 75 HJV positive sentinels. Our results indicate that Highlands J virus is extensively cross-reactive in the HAI test and that the MAC-ELISA is more specific for the detection of antibodies solely to EEEV. We demonstrated that EEEV antibody titers in the HAI test were positively correlated to antibody titers in the PRNT assay. Analysis of alphaviral activity by county indicates widespread transmission of HJV across the northern and panhandle regions of the state; however EEEV activity was greater than HJV activity in all but four counties. Consequently, distinguishing between the two agents can reduce the expenditure of resources on unnecessary vector control and medical alerts to protect the public health from Highlands J virus.

<p><I> Trypanosoma congolense </i> is a single-cell blood parasite and an important pathogen causing African trypanosomiasis, also called ngana, in livestock. Ngana in cattle is a chronic disease associated with anemia, cachexia and increased susceptibility to secondary infections. Infection of mice can be used as an experimental model to study the host-parasite relationship. As determined by their survival time, BALB/c mice are highly susceptible to <i> T. congolense </i> infection, whereas C57BL/6 mice are relatively resistant. The surfaces of African trypanosomes are covered with a layer of a single species of glycoprotein, called variant surface glycoprotein (VSG). Production of antibodies to the VSG of African trypanosomes is one of the major immune responses leading to control of parasitemia. The reaction of antibodies with VSG of trypanosomes, for presently unknown reasons, predominantly activates the alternative complement pathway rather than the classical pathway of complement. IgM antibodies are the first and predominant class of anti-trypanosomal antibodies in infected animals. Antibody-mediated phagocytosis of <i> T. congolense </i> by macrophages is considered a major mechanism of control of parasitemia, besides antibody/complement-mediated lysis and cytotoxic effect by macrophage-derived nitric oxide (NO). The receptor(s) on macrophages that recognizes IgM antibody-coated trypanosomes and enables their phagocytosis is unknown. Interaction of antibodies with the VSG of trypanosomes not only causes phagocytosis of trypanosomes by macrophages, but also leads to the release of sVSG from the trypanosomes. sVSG has been found to modulate various functions of the host: induction of polyclonal B cell activation and modulation of macrophage functions, such as the induction of TNF-á synthesis and the inhibition of IFN-ã-induced nitric oxide production. The objectives of this thesis are:</p> <p> 1) to test whether CR3 (Mac-1; CD11b/18) is involved in IgM anti-VSG-mediated phagocytosis of <i> T. congolense </i> by macrophages </p> <p> 2) to test the effects of anti-VSG antibody and complement on the release of soluble VSG from <i> T. congolense </i> </p> <p>1) When the trypanosomes were incubated with IgM anti-VSG antibody and fresh mouse serum, fragments of complement component C3 were found to be deposited onto <i> Trypanosoma congolense </i>. Thus, it was assessed whether complement receptor CR3 (CD11b/CD18; receptor for iC3b) might be involved in IgM anti-VSG mediated phagocytosis of <i> T. congolense </i>. In the presence of fresh mouse serum, there was significantly and markedly less phagocytosis of IgM-opsonized <i> T. congolense </i> by CD11b-deficient macrophages compared to phagocytosis by normal macrophages (78% fewer <i> T. congolense </i> were ingested per macrophage). There also was significantly less TNF-á (38% less), but significantly more NO (63% more) secreted by CD11b-deficient macrophages that had engulfed trypanosomes than by equally treated normal macrophages. It was concluded that CR3 is the major, but not the only, receptor involved in IgM anti-VSG-mediated phagocytosis of <i> T. congolense </i> by macrophages. It was further concluded that signaling via CR3, associated with IgM anti-VSG-mediated phagocytosis of <i> T. congolense </i>, either directly or indirectly, enhances synthesis of disease-producing TNF-á and inhibits the synthesis of parasite-controlling NO.</p> <p> 2) This investigation revealed that there was more sVSG released from <i> T. congolense </i> by interaction with IgM anti-VSG than by interaction with equal amounts of IgG2a anti-VSG. The release of sVSG occurred in an antibody dose-dependent pattern. It was also found that IgM anti-VSG, after interacting with the surface of <i> T. congolense </i>, formed soluble immune complexes with released sVSG. The results also showed that antibody-induced release of sVSG can occur without complement, but is enhanced by complement. It was further tested whether fresh sera from either relatively resistant C57BL/6 mice or highly susceptible BALB/c mice, which differ in their complement cascade, had different effects on the release of sVSG from <i> T. congolense </i>. The results showed that antibody-induced shedding of sVSG was higher in the presence of fresh C57BL/6 serum than in the presence of fresh BALB/c serum. All these data suggest that the concentration of anti-VSG antibody, antibody class and source of complement can affect the release of sVSG from <i> T. congolense </i></p>.

<p><I> Trypanosoma congolense </i> is a single-cell blood parasite and an important pathogen causing African trypanosomiasis, also called ngana, in livestock. Ngana in cattle is a chronic disease associated with anemia, cachexia and increased susceptibility to secondary infections. Infection of mice can be used as an experimental model to study the host-parasite relationship. As determined by their survival time, BALB/c mice are highly susceptible to <i> T. congolense </i> infection, whereas C57BL/6 mice are relatively resistant. The surfaces of African trypanosomes are covered with a layer of a single species of glycoprotein, called variant surface glycoprotein (VSG). Production of antibodies to the VSG of African trypanosomes is one of the major immune responses leading to control of parasitemia. The reaction of antibodies with VSG of trypanosomes, for presently unknown reasons, predominantly activates the alternative complement pathway rather than the classical pathway of complement. IgM antibodies are the first and predominant class of anti-trypanosomal antibodies in infected animals. Antibody-mediated phagocytosis of <i> T. congolense </i> by macrophages is considered a major mechanism of control of parasitemia, besides antibody/complement-mediated lysis and cytotoxic effect by macrophage-derived nitric oxide (NO). The receptor(s) on macrophages that recognizes IgM antibody-coated trypanosomes and enables their phagocytosis is unknown. Interaction of antibodies with the VSG of trypanosomes not only causes phagocytosis of trypanosomes by macrophages, but also leads to the release of sVSG from the trypanosomes. sVSG has been found to modulate various functions of the host: induction of polyclonal B cell activation and modulation of macrophage functions, such as the induction of TNF-á synthesis and the inhibition of IFN-ã-induced nitric oxide production. The objectives of this thesis are:</p> <p> 1) to test whether CR3 (Mac-1; CD11b/18) is involved in IgM anti-VSG-mediated phagocytosis of <i> T. congolense </i> by macrophages </p> <p> 2) to test the effects of anti-VSG antibody and complement on the release of soluble VSG from <i> T. congolense </i> </p> <p>1) When the trypanosomes were incubated with IgM anti-VSG antibody and fresh mouse serum, fragments of complement component C3 were found to be deposited onto <i> Trypanosoma congolense </i>. Thus, it was assessed whether complement receptor CR3 (CD11b/CD18; receptor for iC3b) might be involved in IgM anti-VSG mediated phagocytosis of <i> T. congolense </i>. In the presence of fresh mouse serum, there was significantly and markedly less phagocytosis of IgM-opsonized <i> T. congolense </i> by CD11b-deficient macrophages compared to phagocytosis by normal macrophages (78% fewer <i> T. congolense </i> were ingested per macrophage). There also was significantly less TNF-á (38% less), but significantly more NO (63% more) secreted by CD11b-deficient macrophages that had engulfed trypanosomes than by equally treated normal macrophages. It was concluded that CR3 is the major, but not the only, receptor involved in IgM anti-VSG-mediated phagocytosis of <i> T. congolense </i> by macrophages. It was further concluded that signaling via CR3, associated with IgM anti-VSG-mediated phagocytosis of <i> T. congolense </i>, either directly or indirectly, enhances synthesis of disease-producing TNF-á and inhibits the synthesis of parasite-controlling NO.</p> <p> 2) This investigation revealed that there was more sVSG released from <i> T. congolense </i> by interaction with IgM anti-VSG than by interaction with equal amounts of IgG2a anti-VSG. The release of sVSG occurred in an antibody dose-dependent pattern. It was also found that IgM anti-VSG, after interacting with the surface of <i> T. congolense </i>, formed soluble immune complexes with released sVSG. The results also showed that antibody-induced release of sVSG can occur without complement, but is enhanced by complement. It was further tested whether fresh sera from either relatively resistant C57BL/6 mice or highly susceptible BALB/c mice, which differ in their complement cascade, had different effects on the release of sVSG from <i> T. congolense </i>. The results showed that antibody-induced shedding of sVSG was higher in the presence of fresh C57BL/6 serum than in the presence of fresh BALB/c serum. All these data suggest that the concentration of anti-VSG antibody, antibody class and source of complement can affect the release of sVSG from <i> T. congolense </i></p>.

The Neisseria genus includes pathogens, Neisseria gonorrhoeae (Ngo) and Neisseria meningitidis, as well as commensals. Ngo, the cause of gonorrhea, induces massive inflammation but a surprising lack of adaptive immune responses. We have observed that Ngo can inhibit both T cell activation and dendritic cell maturation through interaction with the host expressed co-inhibitory receptor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). Therefore, I wondered whether B cells may also be affected in this manner. Herein, I examine primary human B cell responses to infection with Ngo, as well as the other Neisseria species. B cells infected with Ngo show no sign of inhibition, regardless of their ability to bind CEACAM1, instead responding to gonococci with robust activation and proliferation. There are distinct subsets of B cells found in the periphery and, intriguingly, the IgM memory B cell subset expand and produce polyreactive IgM in response to goncoccal infection. These cells are innate in function, producing low affinity, polyclonal IgM that is protective against bacterial and fungal dissemination. This effect was broadly specific for Neisseria sp., as B cell infection with all commensal Neisseria species examined induced innate B cell responses. Curiously, meningococcal strains avoid inducing the innate B cell responses, making it enticing to hypothesize that its avoidance of such an ancient immune response may contribute to its ability to cause disease in humans. Finally, I tested whether gonococcal Opa protein binding to CEACAM1 affects primary human B cell activation, and show that no inhibition was observed. This absence of co-inhibitory function of neisserial-bound CEACAM1 may reflect inherent differences between distinctive cell types. Combined, the results in this thesis contribute new insight regarding the poorly characterized human IgM memory B cells, as well as to the function of CEACAM1 in lymphocytes.

The Neisseria genus includes pathogens, Neisseria gonorrhoeae (Ngo) and Neisseria meningitidis, as well as commensals. Ngo, the cause of gonorrhea, induces massive inflammation but a surprising lack of adaptive immune responses. We have observed that Ngo can inhibit both T cell activation and dendritic cell maturation through interaction with the host expressed co-inhibitory receptor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). Therefore, I wondered whether B cells may also be affected in this manner. Herein, I examine primary human B cell responses to infection with Ngo, as well as the other Neisseria species. B cells infected with Ngo show no sign of inhibition, regardless of their ability to bind CEACAM1, instead responding to gonococci with robust activation and proliferation. There are distinct subsets of B cells found in the periphery and, intriguingly, the IgM memory B cell subset expand and produce polyreactive IgM in response to goncoccal infection. These cells are innate in function, producing low affinity, polyclonal IgM that is protective against bacterial and fungal dissemination. This effect was broadly specific for Neisseria sp., as B cell infection with all commensal Neisseria species examined induced innate B cell responses. Curiously, meningococcal strains avoid inducing the innate B cell responses, making it enticing to hypothesize that its avoidance of such an ancient immune response may contribute to its ability to cause disease in humans. Finally, I tested whether gonococcal Opa protein binding to CEACAM1 affects primary human B cell activation, and show that no inhibition was observed. This absence of co-inhibitory function of neisserial-bound CEACAM1 may reflect inherent differences between distinctive cell types. Combined, the results in this thesis contribute new insight regarding the poorly characterized human IgM memory B cells, as well as to the function of CEACAM1 in lymphocytes.

Gene expression can be regulated at multiple steps once transcription is initiated. I have studied two different gene models, the α-Fetoprotein (AFP) and the immunoglobulin heavy chain (IgM) genes, to better understand post-transcriptional gene regulation mechanisms. The AFP gene is highly expressed during fetal liver development and dramatically repressed after birth. There is a mouse strain-specific difference between adult levels of AFP, with BALB/cJ mice expressing 10 to 20-fold higher levels compared to other mouse strains. BALB/cJ mice express low levels of Zhx2 and thus incompletely repress AFP. Despite differences in steady state AFP mRNA levels in the adult liver between Balb/cJ and wild-type mice, transcription rates across this gene were similar, indicating a post-transcriptional regulatory mechanism. I found accumulated unspliced RNA across multiple AFP introns in wild-type mice where mature AFP mRNA levels are low, suggesting overall AFP splicing is inefficient in the presence of Zhx2. The IgM gene is alternative processed to produce two mRNA isoforms through a competition between cleavage/polyadenylation (μspA) and splicing reactions and the pA/splice RNA expression ratio increases during B cell maturation. Cotranscriptional cleavage (CoTC) events, driven by specific cis-acting elements, are required downstream of some poly(A) signals to terminate transcription. In some cases, a pause site can produce similar effect. I explored whether there is a CoTC-like element within the IgM gene that may contribute to developmental changes in the mRNA ratio. In both a B cell and plasma cell line there was a gradual decrease in transcripts downstream from the μspA signal, suggesting that there is not evidence for a CoTC element within the IgM gene. To examine the effect a CoTC element would have on the competition between the splice and μspA reactions, we inserted the CoTC sequence of the β-globin gene into different locations downstream of the μspA signal. While the β-globin CoTC element caused cotranscriptional cleavage in all locations, it only affected the μspA/splice ratio when located close to the μspA site. This suggests there is a position effect of the inserted CoTC element on the competing polyadenylation and splicing reactions within the IgM transcripts.

OBJECTIVES: Measles and rubella are nationally notifiable, vaccine-preventable diseases targeted for elimination by the Pan American Health Organization (PAHO). To support national and international elimination efforts, surveillance optimization is important to ensure rapid case detection, document endemic transmission interruption, identify susceptible populations and inform immunization strategies. While current national surveillance captures confirmed-case data, its performance cannot be assessed using PAHO-recommended surveillance indicators as suspect-case investigation data are required for their estimation. In Canada, the investigation of clinically-suspect measles-like illness (MLI) is highly dependent on laboratory evidence, providing an opportunity to use laboratory data to estimate MLI investigation rates. The Measles and Rubella Surveillance (MARS) pilot project was developed to address existing surveillance challenges with the central hypothesis that (I) ‘it is feasible to develop and implement a real‐time, web‐based measles and rubella surveillance system in the Canadian setting’, and the following sub‐hypotheses: (II) ‘implementation of real‐time surveillance in MARS pilot provinces will result in increased timeliness of national measles and rubella surveillance when compared with established confirmed-case surveillance’, and (III) ‘it is possible to use augmented laboratory data to estimate the performance of national measles and rubella surveillance using adapted PAHO indicators’. METHODS: A MARS application was designed to support centralized real-time measles/ rubella investigation reporting and alerting with integration of non-nominal laboratory and epidemiological data, then developed and piloted using the web-based Canadian Network for Public Health Intelligence platform in British Columbia, Alberta and Newfoundland from June/2011-May/2012. Pre- and post-pilot laboratory surveys were conducted to retrospectively assess national surveillance performance in ‘outbreak’ and ‘non-outbreak’ settings during the 2005‐2011 and pilot years using various surveillance indicators and attributes. Measles IgM serology testing was used as a laboratory-based proxy for MLI investigation to support indicator estimation. RESULTS: Real-time, integrated surveillance was successfully implemented in MARS pilot provinces as modeled within the context of established reporting roles, and surveillance indicators and attributes were estimated using augmented laboratory data. MARS surveillance was more timely than confirmed-case surveillance, and real-time MARS reports exceeded all laboratory-related PAHO targets evaluated: 100% met ‘sample collection’ and ‘receipt’ timelines, and 91.7% met ‘result' timelines (Targets:≥80%); 99.8% of all MLI investigations were discarded (Target:≥95%). A national ‘non-outbreak’ baseline rate of 14 MLI investigations/100 000 population was estimated, whereas MARS pilot sites averaged 22 MLI investigations/100 000 population during the pilot year. While ‘non-outbreak’ investigation rates varied between provinces, all annual provincial and national rates estimated for the 2005‐2011 and MARS pilot years exceeded the PAHO investigation target of ≥2 suspected cases/100 000 population in settings attempting elimination. CONCLUSIONS: The MARS model supported more timely and integrated national measles and rubella surveillance, and enabled indicator‐based performance assessment. Results underscore the importance of laboratory data when evaluating and documenting surveillance performance to support elimination efforts. Consideration should be given to national MARS implementation and its use as a model adaptable to the case-based surveillance of other nationally notifiable diseases.

This thesis focuses on humoral immune response of specific hosts to antigens of various developmental stages of bird schistosomes T. regenti and T. szidati, and follows up on previous research of antibody response in non-specific hosts (mouse, human). Sera of experimentally infected and hunted-down wild ducks were examined using the ELISA and western blot methods. The sera samples were taken in predefined intervals. Results of the ELISA analysis show the process of humoral immune response after infection by bird schistosomes. The level of specific antibodies IgY against homogenate of T. regenti cercariae increased significantly 20 d.p.i. in ducks infected by T. regenti. Such reaction wasn't observed in ducks infected by T. szidati. Slight changes in level of specific antibodies IgM against T. szidati cercariae homogenate were observed 10 d.p.i. only in fully immunocompetent ducks and in reinfected ducks. Examination of hunted-down wild ducks didn't prove infection by bird schisosomes; this conclusion was confirmed by results of the ELISA analysis. IgY antibodies from ducks infected by T. regenti demonstrated strong reactions with 2 antigens in ranges 49-47 kDa and 47-45 kDa. Other reactions, which were recognized, have not been observed in all specimen. An Western blott with homogenate from 7 days...

The aim of the thesis was to develop an immunoassay for the detection of dangerous bacterial pathogen, methicillin-resistant Staphylococcus aureus (MRSA). MRSA infections cause global problems in health facilities which provide acute and follow-up care in inpatient and outpatient parts. That is why early and rapid diagnosis and targeted thereapies are very important for the subjects. E. coli was purified using the QIAquick PCR kit Purificatin isolated bacterial constructs, which were subsequently purified and dialyzed. The recombinant protein PBP2a in different concentrations was applied to a nitrocellulose membrane in the form of lines. Furthermore was performed optimized blot-line method for the detection of specific antibodies against the recombinant antigen PBP2a in the classes IgG, IgA and IgM. Several different concentrations of the conjugate Goat Anti-human IgG-AP, Goat Anti-human IgA-AP or Goat anti-Human IgM-AP were used for the detection. The color intensity of each line of the strip was evaluated with Immunoblot software. The measured values were used to determine diagnostic sensitivity, specificity and overall diagnostic match. Further testing of precision was carried out under repeatability conditions (intra-assay) and reproducibility (inter-assay). Precision of the method was expressed by coefficient of variation. As the most suitable for the manufacture of a IgG kit was determined the concentration of the antigen PBP2a from 0.30 mg/ml to 0.45 mg/ml and the concentration of IgG conjugate from 1:1500 to 1:1800. For class IgA as the most appropriate antigen was determined concentration PBP2a from 0.40 mg/ml to 0.52 mg/ml and conjugate concentrations of IgA from 1:500 to 1:1000. The coefficient of variation under repeatability conditions for the entire range of the IgG class is 10.09 %, and for IgA is 8.91 %. Variation coefficient reproducibility conditions for the entire range of the IgG class is 9.23 % and for IgA is 9.60 %. Precision of the method under conditions of repeatability and reproducibility for classes IgG and IgA meets the criteria for the manufacture of a diagnostic kit. Titration results showed that particular batch of cards made of nitrocellulose membrane coated with antigen PBP2a must always be verified on the panel of reference samples and the values of concentrations (for both antigen and conjugate) should be set according to the needs, but differently for the class of immunoglobulins IgG and IgA.