Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, a disease that for years was considered to belong of the past, but tuberculosis is back causing over 2 million deaths per year. The infection can be dormant for decades and an active immune response can prevent the infection from progressing into active disease. However, the HIV/AIDS epidemic has caused an alarming rise in tuberculosis cases. The main infectious route for Mtb is through the airways into the lungs, where they encounter alveolar macrophages. Mtb are phagocytosed by these macrophages, but instead of being killing within the phagosome, Mtb modulates the cell to become a host in which the bacteria thrive. The lack of capacity to eradicate the infection stimulate cells of the immune system to gather around infected macrophages and form a granuloma that walls off the infection. Within this granuloma, Mtb can wait silently and later progress into active disease. However, only a fraction of exposed individuals develop disease, indicating that initial eradication of Mtb infections is possible. Such immediate response must be directed by the innate immunity comprised of phagocytes such as neutrophils (PMNs) and non-activated macrophages. Upon Mtb infection, macrophages become anergic and PMNs enter apoptosis. PMNs have a short lifespan and are cleared by neighbouring phagocytes, a mechanism described to resolve the inflammation and modulate tissue regeneration. We found that Mtb-induced apoptosis in PMNs was not dependent on phagocytosis of the bacteria, indicating that Mtb have the capacity to induce apoptosis in multiple PMNs. Complement-mediated phagocytosis induce survival signals such as Akt in PMNs, but despite this, complement-opsonized Mtb was able to override the anti-apoptotic activation in the cells. Since phagocytes clear apoptotic cells, we investigated how clearance of Mtb-induced apoptotic PMNs affected macrophages. We found that Mtb-induced apoptotic PMNs inflicted pro-inflammatory activation of the macrophages that cleared them. In addition, this activation was mediated by Hsp72 released from the Mtb-induced apoptotic PMNs. Furthermore, apoptotic PMNs can work in synergy with phagocytosed Mtb to activate macrophages and enhance intracellular killing of Mtb. Since dendritic cells are important for the regulation of immunity, we investigated whether Mtb-induced apoptotic PMNs affected the inflammatory response and maturation of dendritic cells. We found that Mtb-induced apoptotic PMNs trigger dendritic cells to enter a mature state able to activate naïve T-cell proliferation. We propose that infected apoptotic PMNs is a potent activator of the inflammatory response during infections. Taken together, PMNs not only kill their share of pathogens but also modulate other immune cells, thereby forming a link between the early innate and the adaptive immune response during microbial challenge with Mtb.

<p><p>Handhygien är en grundläggande princip som förebygger, kontrollerar och reducerar vårdrelaterade infektioner och är ensam den mest effektiva metoden för att bryta en smittspridning. Syfte: att belysa vårdpersonalens följsamhet till de basala rutinerna gällande handhygien. Metod: Författarna gjorde en litteratursökning och granskning av vetenskapliga artiklar inom området. Resultat: Vårdpersonalens följsamhet till handhygienrutinerna visade sig ligga mellan 16,5%-70% och var större efter än före patientvård. Det fanns faktorer som gynnade och minskade följsamheten. Diskussion: Författarna fann stöd i sin teori om att vårdpersonalen skyddar sig i första hand själv och utför därför handhygienen i större grad än efter patientkontakt. Då det visade sig att kunskap leder till ökad följsamhet ansåg författarna att en årlig genomgång av handhygienrutinerna är lika viktigt som att repetera hjärt- och lungräddning.</p></p>

<p>Campylobacter jejuni is the main cause of bacterial diarrhoeal illness in developed countries, with ~7000 cases being reported each year in Sweden. C. jejuni has received growing attention since it’s recognition as a human pathogen in the 1970s, but its epidemiology is complex and much still remains unknown. There are several potential reservoirs for C. jejuni, including environmental sources as water and soil, wild and domesticated animals, particularly poultry, but also other livestock and pets. In this study 348 Swedish C. jejuni isolates from the year 2000 from humans (n = 164) and broilers (n = 184) were characterized with multilocus sequence typing (MLST) with the aim of comparing the population structures and diversity of C. jejuni between isolates from the two hosts. MLST is a method for characterization of bacterial isolates that indexes the variation in DNA sequence of multiple protein encoding housekeeping genes. A secondary aim in this study was to compare populations of C. jejuni from 11 subgroups of isolates based on location of the sampling. The overlap between the populations was analyzed numerically based on genotypes detected and with analysis of phylogeny, gene flow and molecular variation. It was shown that the population structure of C. jejuni isolates from broilers and humans show a high degree of similarity, supporting broilers as an important source of human infection. However, even though the population structure of human and broiler C. jejuni were almost genetically indistinguishable other sources of C. jejuni infections in humans cannot be ruled out since the same genotypes can be found in other sources as well. Analysis of the 11 subgroups suggested that there may be a difference in populations infecting humans in different Swedish regions, and between populations of C. jejuni in broilers from different slaughterhouses. But this could be a result of chance since most of the subgroups were small. Future studies to improve the understanding of C. jejuni epidemiology, for which MLST has proven itself as a valid method, is important to develop control strategies to prevent infection with this common cause of diarrhoeal illness.</p>

<p>The third domain of life, Archaea is one of the three main evolutionary lineages together with the Bacteria and the Eukarya domains. The archaea are, despite their prokaryotic cell organisation, more closely related to eukaryotes than to bacteria in terms of the informational pathways (DNA replication, transcription and translation). Organisms from the archaeal hyperthermophilic genus <i>Sulfolobus</i> thrives in a hot (80°C), acidic (pH 2-4) and sulphur-rich environment.</p><p>In my thesis, I have used a variety of different approaches to study the <i>Sulfolobus</i> cell cycle. After dilution of a stationary phase cell culture with fresh medium, synchronous cell cycle progression was obtained. From the synchronised cell culture experiment we could conclude that the major cell cycle events (nucleoid segregation, cell division and chromosome replication) were tightly coupled to each other and to cellular mass increase. </p><p>Inhibitors of the elongation stage of chromosome replication, and of cell division, as well as drugs arresting the cell cycle in the post-replicative phase, were found in an in vivo screening of a range of antibiotics. The cell cycle was found to be regulated such that the previous cell cycle step had to be successfully accomplished before the next could initiate, except for DNA replication which could occur without an intervening cell division event.</p><p>The replication pattern of <i>Sulfolobus solfataricus</i> was analysed using a marker frequency assay. From the results, we were able to determine that a single origin is utilized in vivo, that the replication directionality is bidirectional, and also an approximate location of the replication origin within the genome.</p><p>Intracellular virus production in vivo of SIRV2 (<i>Sulfolobus islandicus</i> rod-shaped virus2) in <i>Sulfolobus islandicus</i> was also analysed. The effects on the host cell were determined, including loss of cell viability, inhibited initiation of replication at virus infection and DNA degradation and loss of cell integrity at the time of virus release. Also, for the first time intracellular virus DNA was visualized with flow cytometry.</p>

The third domain of life, Archaea is one of the three main evolutionary lineages together with the Bacteria and the Eukarya domains. The archaea are, despite their prokaryotic cell organisation, more closely related to eukaryotes than to bacteria in terms of the informational pathways (DNA replication, transcription and translation). Organisms from the archaeal hyperthermophilic genus Sulfolobus thrives in a hot (80°C), acidic (pH 2-4) and sulphur-rich environment. In my thesis, I have used a variety of different approaches to study the Sulfolobus cell cycle. After dilution of a stationary phase cell culture with fresh medium, synchronous cell cycle progression was obtained. From the synchronised cell culture experiment we could conclude that the major cell cycle events (nucleoid segregation, cell division and chromosome replication) were tightly coupled to each other and to cellular mass increase. Inhibitors of the elongation stage of chromosome replication, and of cell division, as well as drugs arresting the cell cycle in the post-replicative phase, were found in an in vivo screening of a range of antibiotics. The cell cycle was found to be regulated such that the previous cell cycle step had to be successfully accomplished before the next could initiate, except for DNA replication which could occur without an intervening cell division event. The replication pattern of Sulfolobus solfataricus was analysed using a marker frequency assay. From the results, we were able to determine that a single origin is utilized in vivo, that the replication directionality is bidirectional, and also an approximate location of the replication origin within the genome. Intracellular virus production in vivo of SIRV2 (Sulfolobus islandicus rod-shaped virus2) in Sulfolobus islandicus was also analysed. The effects on the host cell were determined, including loss of cell viability, inhibited initiation of replication at virus infection and DNA degradation and loss of cell integrity at the time of virus release. Also, for the first time intracellular virus DNA was visualized with flow cytometry.

Rift Valley Fever virus (RVFV) causes an infection with severe impact on animal and human health. The disease is endemic throughout almost the entire African continent and large regions of the Arabian Peninsula. During epidemics, high mortality is observed in animals, especially among cattle, goats, and sheep. In humans, the symptoms vary from a benign influenza-like disease to a life-threatening hemorrhagic fever. Due to the devastating effect on communities in endemic regions and the possibility of further spread of this virus, there is an imperative need to improve and develop control measurements against this emerging disease. Therefore, this thesis focuses on diagnostics and vaccines against RVFV. RVFV infection kinetics was studied in a mouse model system by detection and quantification of viral genomes, using a developed quantitative real-time PCR (QRT-PCR) method. This novel QRT-PCR method proved to be reliable and serves as a supplement to standard diagnostics, direct virus isolation and serological methods. High levels of viral RNA were found in blood and liver samples from experimentally infected mice during the first days post infection. Thereafter the levels declined rapidly and dropped below detection limit approximately seven days post infection. The QRT-PCR technique was also used in a study aimed to improve diagnosis of RVFV from field samples collected on filter strips. Today, the available RVFV vaccines are only approved for animal use and these vaccines have several shortcomings. Since RVFV is a highly pathogenic organism requiring bio-safety level 3 laboratories, two different none-replicating vaccine approaches have been applied and evaluated using a mouse model. A DNA based vaccine, administered via gene-gun, and the use of virus-like particles (VLP), by the intra-peritoneal route. RVFV specific and neutralising antibodies were raised with both vaccine approaches. However, VLP vaccination against Rift valley Fever proved to be more promising as a future vaccine, since higher titres of neutralising antibodies and improved survival rate were found upon a lethal RVFV challenge in mice. In conclusion, a sensitive and specific method for quantifying RVFV infection and a promising vaccine candidate against RVFV were developed.

At present, the human adenoviruses (Ads) comprise 51 members, which have been classified into six species (A to F). In general, adenovirus (Ad) tissue tropism or disease patterns vary according to species, although adenoviruses from different species can sometimes cause the same symptoms. The current interest in adenoviruses is partly due to the aim of using them as vectors for gene therapy. Hematopoietic cells are attractive targets for gene therapy and the transductions can be performed ex vivo. However, the most commonly used adenovirus vectors, based on Ad2 or Ad5, are inefficient in their transduction of hematopoietic cells since they attach poorly to these cells. Most Ads, including Ad2 and Ad5, appear to use the coxsackie-adenovirus receptor (CAR) (a component of tight junctions), for attachment to host cells. However, species B Ads do not bind to CAR and several studies have indicated that species B-based vectors would be more suitable for hematopoietic cells. Species B Ads can be further divided into species B1 and B2, which display different tissue tropisms. Species B1 Ads mostly cause acute respiratory infections whereas species B2 Ads have been associated with persistent infections of the kidney and urinary tract. One of the key determinants of tropism is believed to be the initial high-affinity attachment of the virion to host cell fiber receptors. By reciprocal blocking experiments and different ways of characterizing the species B attachment receptors, we have shown that the species B2 serotypes Ad11p and Ad35 and the species B1 serotypes Ad3p and Ad7p also differ in receptor usage. There are at least two different Ad species B receptors. Since one of these receptors appeared to be used by all four serotypes, we designated this receptor sBAR (species B adenovirus receptor). The other receptor appeared to be used exclusively by the two species B2 serotypes and was therefore designated sB2AR (species B2 adenovirus receptor). Binding to sBAR can be abolished by EDTA and restored with Mn2+ or Ca2+, whereas binding (of Ad11p and Ad35) to sB2AR is independent of divalent cations. Furthermore, sBAR appears to be trypsin sensitive whereas sB2AR is not. We also identified CD46 as a receptor for Ad11p. Even so, CD46 does not appear to be a functional receptor for Ad7p. Both Ad7p and Ad11p attached to CD46-transfected Chinese hamster ovary (CHO) cells more efficiently than to control CHO cells. However, only Ad11p (selectively) infected CD46-transfected CHO cells. Anti-CD46 antibodies inhibited Ad7p and Ad11p from binding to, and Ad11p from infecting, CD46-transfected CHO cells. However, in human cells, anti-CD46 antibodies had an inhibitory effect only on Ad11p binding (~30%) but did not affect Ad7p binding. In binding experiments with EDTA, divalent cations and pretrypsinized cells, Ad11p and Ad7p showed the same pattern in their binding to CHO-CD46 cells as in the previous study. Since Ad7p interacted almost as efficiently with control CHO cells as with CHO-CD46 cells after addition of Mn2+, it seems that Ad7p mainly addressed an endogenously expressed hamster receptor on CHO-CD46, the properties of which resemble sBAR. In addition, Ad3p and Ad7p attach poorly to PBMCs and CD46 is expressed on all nucleated cells. Thus, CD46 appears to correspond to sB2AR rather than to sBAR. With these differences in receptor usage in mind, we studied the binding and infectious capacity of these species B Ads in various hematopoietic cells. We found that all species B serotypes bound efficiently to CD34+ hematopoietic stem cells (HSCs) and also productively infected HSCs. However, only the sB2AR binding Ad serotypes Ad11p and Ad35 could attach primary PBMCs efficiently. Our results regarding the subsequent steps in infection of PBMCs suggest that both Ad11p and Ad35 enter PBMCs and deliver viral DNA to the nuclei of most PBMC cell types. However, productive infections were only clearly detected in stimulated T-cells (most frequently) and monocytes, whereas Ad infection seemed eclipsed in unstimulated lymphocytes. Replication of Ad DNA seemed seriously impaired in at least T-cells, indicating limited production of infectious particles in PBMCs. The capacity of species C Ads to establish persistent infections in lymphatic tissues has been described previously. These Ads also persistently infect various transformed hematopoietic cell lines in vitro. Our studies indicate that replication of the species B2 Ads is also restricted in cells of hematopoietic origin (both in primary and transformed cells). Taken together, the results indicate that species B2 Ads (as compared to other Ads) seem to enter and infect most hematopoietic cells efficiently, which is in line with the persistent nature of these Ads. They would presumably act as suitable vectors for efficient transduction of most cells of hematopoietic origin, as has already been shown for e.g. HSCs and dendritic cells. The finding that replication of Ads in T-cells appears to depend on the level of T-cell activation, strengthens the hypothesis that T-cells may serve as a reservoir for human Ads and raises possible safety issues for usage of species B-based vectors in hematopoietic cells.

A vast number of studies have been completed on the virulence determinants of Yersinia spp.; however, the focus of many of these studies has been on the virulence plasmid and the plasmid-encoded Type three secretion system. Nevertheless, many chromosomal genes whose products are directly involved in virulence have also been identified. Some of these critical virulence determinants are outer membrane proteins. Outer membrane proteins of Gram-negative bacteria often have important physiological roles; however, some have also been found to be important for pathogenesis. In this thesis, we investigated two Yersinia. pestis outer membrane proteins, Ail and OmpA, and their roles in virulence. We provide evidence that Y. pestis Ail is a highly expressed outer membrane protein that is absolutely essential for Y. pestis to resist the killing action of the complement system present in human blood and tissues, as well as the blood and tissues of other mammalian hosts. Furthermore, Ail was important for virulence in a Y. pestis-Canorhabditis elegans model of infection.The work in this thesis also provided the first evidence that another surface-exposed outer membrane protein, termed OmpA, is required for both Yersinia pseudotuberculosis and Y. pestis to survive and proliferate intracellularly in macrophages. Finally, we provide evidence that Y. pestis has a functional small RNA MicA that controls the expression of OmpA. This is the first demonstration of sRNA-mediated regulation of a Yersinia virulence factor. This work has paved the way for future studies on the role of outer membrane proteins in virulence, particularly the role of Ail and OmpA.

This thesis reports on cross-sectional surveys aimed at detecting and characterizing haemoprotozoan parasites infecting wild free-ranging non human primates (NHPs) in Kenya, East Africa. Blood samples from olive baboons (Papio cynocephalus anubis), vervet monkeys or African green monkeys (AGMs, Chlorocebus aethiops) and Syke's monkeys (Cercopithecus mitis) from five provinces of Kenya were analyzed. The haemoprotozoan parasites survey was performed with microscopic evaluation of blood smears, serological techniques and molecular tools. Blood specimens and serum samples from 121 NHPs were tested for the presence of Trypanosoma brucei (Study I). Indirect antibody enzyme-linked immunosorbent assay (Ab-ELISA) detected titers of anti-T. brucei antibodies in 19% (23/121) of the sera sampled. Subsequent field-oriented latex agglutination test (LAT) detected presence of T. brucei antigens in 16% (19/121) of the sera. However, there were no active infections detected on fixed blood smears, or wet blood films. Of the 378 NHPs sera samples tested for Leishmania major exposure using Ab-ELISA, 66% had detectable anti-L. major antibodies (study II). Western blot (WB) assay detected anti-L. major antibodies in sera from 46% (175/378) of the NHPs samples. Specific proliferation of peripheral blood mononuclear cells to L. major antigen was demonstrated in 23% (17/57) of AGMs samples. Haemoprotozoan parasites, Entopolypoides macaci and Hepatocystis kochi were detected by microscopic evaluation of Giemsa-stained blood smears from 179 NHPs (study III). The prevalence rate of E. macaci was 43% in African green monkeys, 35% in Syke’s monkeys and 33% in baboons. H. kochi infection rate was 18% in African green monkeys, 23% in baboons and 25% in Syke’s monkeys. Subsequent indirect immunofluorescent antibody test (IFAT) supported the morphologic appearance of E. macaci observed by microscopy. Molecular tools were used to detect and identify haemoprotozoan parasites in wild free-ranging NHPs (study IV). Nested polymerase chain reaction (PCR) targeting Babesia β-tubulin gene detected a 22% (27/125) B. microti infections in free-ranging NHPs in Kenya. PCR also detected 22% mixed infections by Hepatocystis and Entopolypoides, 12% Hepatocystis and Babesia and 7% Entopolypoides and Babesia (study V). Phylogenetic analysis inferred from mitochondrial cytochrome b (Cyt-b) gene confirmed the presence of Hepatocystis kochi whereas analysis of 18SS rRNA gene confirmed presence of two piroplasms, Babesia sp. and Entopolypoides macaci. In conclusion, epidemiological results from sero-prevalence studies provide strong circumstantial evidence that some species of Kenyan NHPs are naturally exposed to L. major and T. brucei infections and could be potential reservoir hosts for these haemoparasites. Molecular diagnosis revealed the occurrence of mixed parasite infections and confirmed the circulation of Babesia and Entopolypoides species in the same populations of Kenyan NHPs.

Drosophila melanogaster has been widely used as a model organism to study the immune responses against bacteria, fungi, parasites and viruses. Here, I present a D. melanogaster virus as a model to study persistent virus infections. I have discovered and characterized the Nora virus, a small picorna-like RNA virus able to persistently infect D. melanogaster. The Nora virus genome encodes four open reading frames; a feature not present in other picorna-like viruses. The Nora virus is not closely related to any other virus family, but rather is the first virus in a new family of picorna-like viruses. The major replicative proteins of this virus are encoded in the second open reading frame and the capsid proteins are encoded in the fourth open reading frame. The sequence of the capsid proteins are not obviously related to any other previously described protein. By looking at expressed sequence tags (EST) projects, we identified an EST sequence from the parasitic wasp Nasonia which appears to encode proteins that have sequence similarity to the Nora virus capsid proteins. I have shown that the Nora virus persists in the fly intestine however I did not observe serious pathological effects in the infected flies. The virus is shed through feces and the transmission occurs horizontally via the ingestion of virus-contaminated food. Moreover, I observed variability in the viral titers among single flies of the same infected stock. Some flies are able to clear the Nora virus but not others and this phenomenon seems to be titer-dependent. Surprisingly, none of the known Drosophila antiviral responses play a role against the Nora virus. In conclusion, my work shows that studying the Nora virus interaction with the Drosophila immune system can lead to new findings on viral persistence mechanisms of RNA viruses and of Drosophila viral innate immunity.