<p>The adenoviruses are nonenveloped double stranded DNA viruses, which cause enteric dysentary and respiratory infection. Adenovirus has become a focus of the water treatment community because of its apparent resistance to ultraviolet disinfection; it is the basis for stringent new EPA regulations regarding all viruses in both surface and ground waters. Most of the work done so far, however, has involved the use of monochromatic (254 nm) low pressure (LP) UV sources and subsequent assay of viral infectivity in cell culture models. LP UV lamps primarily damage DNA, while polychromatic UV sources may damage other parts of the virus as well. Recent research has shown that these newer, polychromatic UV sources--such as medium pressure (MP) UV--are more effective than monochromatic LP UV for disinfection of adenovirus. The objectives of this work were to study adenoviral response to UV using both LP and MP UV as well as using both standard cell culture infectivity assays and more direct methods of assessment based on molecular biology. These include quantitative long PCR for assessment of DNA damage and SDS-PAGE for assessment of protein damage; transmission electron microscopy was used to examine the structure of UV treated viral particles. This work was only the second significant study to show the response of adenoviruses to medium pressure UV and the first to thoroughly examine the response of adenoviruses to both LP and MP UV using cell culture-independent methods. Results confirm that adenovirus is sensitive to MP UV when assayed in cell culture; they show that LP and MP UV are equally effective at inducing damage to the adenoviral genome and that MP UV is more effective than LP UV at damaging the viral proteins. This work helps deepen our understanding of UV disinfection of adenovirus.</p> / Dissertation

Drug resistance is a major obstacle to management and control of malaria and currently progressing at a rapid rate across Africa. This thesis has examined factors influencing evolution of resistant P. falciparum at two sites in Africa, including parasite migration, cross mating and fitness cost of resistance. In Asar village, eastern Sudan, the frequencies of drug sensitive and resistant parasites were monitored throughout the dry season in the absence of anti-malarial drug usage to examine whether persistence of resistant parasites is reduced in the absence of drug pressure. Two cohorts of P. falciparum infected patients were treated with chloroquine in the transmission season (Oct-Dec), and followed monthly in the dry season into the next transmission season. A large proportion of the cohort maintained sub-patent asymptomatic P. falciparum infections throughout the entire study period. Alleles of the chloroquine resistance transporter (Pfcrt) and multi-drug resistance protein (Pfmdr1) were examined. Mutant alleles of Pfcrt reached fixation following CQ treatment and remained high in the transmission season. However, at the start of the dry season, wild type alleles of both genes started to emerge and increased significantly in frequency as the season progressed. The mutant Pfcrt haplotype was invariably CVIET, indicating migration of CQ resistant parasites into an area; otherwise the CVMNK haplotype is normal. In addition, microsatellite haplotypes of dihydrofolate reductase (dhfr) gene and dihydropteroate synthase (dhps) genes, which control the parasite response to pyrimethamine and sulfadoxine respectively, were characterized. One major dhfr haplotype with double dhfr mutations and two major mutant dhps haplotypes were seen in eastern Sudan. These haplotypes are distinct from those prevailing in other African countries, suggesting the likely local origin of dhfr and dhps haplotypes conferring drug resistance. Transmission capacities of different P. falciparum clones within a single infection in The Gambia have a high ability to produce gametocytes and infect Anopheles mosquitoes even when they exist at levels not detectable by microscopy and PCR. These findings emphasize the crucial role of gametocyte complexity and infectivity in generating the remarkable diversity of P. falciparum genotypes seen in infected people. Parasites with different resistant dihydrofolate reductase (dhfr) haplotypes have the ability to infect Anopheles mosquitoes following drug treatment, and cross-mating between parasites with different dhfr haplotypes was detected. Our results showed that the major dhfr haplotype in the Gambia is similar to the common one seen in other African countries, suggesting that parasite migration plays a major role in spread of resistance. Indeed, the dominant resistant haplotype seen in infected patients was readily transmitted to infect mosquitoes.

Universiẗat, Diss., 2005--Jena.

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Emergence of avian origin and equine origin canine influenza viruses (CIVs) in Asia and the United States brings important concerns. Humans are in closer and more frequent contact with dogs than other common hosts of influenza. Thus, CIV is a potential threat to human health. However, little is known about the determinants of CIV host tropism or the transmissibility of CIVs to humans. An amino acid change (W222L) was implicated in modifying hemagglutinin receptor binding by CIV. This was tested using reverse genetics, glycan microarray and virus histochemistry. Glycan microarray demonstrated that avian-origin CIV (H3N2-222W) bind predominantly to alpha-2, 3 linked glycans. Virus histochemistry indicated that rH3N2-222L had higher binding affinity with epithelial cilia of canine tracheal tissue and weaker binding with avian tracheal tissue. Ferret infection demonstrated that the avian-origin H3N2 CIV could cause infection and limited to rhinitis, suggesting that CIV could infect humans.

The potential of increasing disease problems through the use of recycled irrigation water in horticultural operations is a serious concern, yet basic research on waterborne plant pathogens in Virginia is lacking. In this work seasonal fluctuations and locations of Pythiaceae in a recycled water irrigation system at a container nursery were determined. <i>Pythium</i> spp. were recovered more frequently and in greater numbers than <i>Phytophthora</i> spp. Species of <i>Phytophthora</i> recovered in filtering assays were identified as <i>P. capsici, P. citricola, P. citrophthora, P. cryptogea, P. drechsleri,</i> and <i>P. nicotianae. P. cryptogea</i> and <i>P. drechsleri</i> were the only <i>Phytophthora</i> spp. recovered from baits placed on the surface of the irrigation reservoir, whereas a greater diversity of species was recovered from baits placed at depths. Hymexazol-amended medium was found to have limitations in recovery of <i>Phytophthora</i> spp. In pathogenicity tests, <i>P. cactorum, P. capsici, P. citrophthora,</i> and <i>P. nicotianae</i> caused significant mortality of <i>Salvia officinalis</i> and <i>P. cactorum</i> showed limited pathogenicity on <i>Gerbera jamesonii</i>. Asymptomatic (aboveground) plants were found to harbor inoculum long after <i>Phytophthora</i>-inoculation. Fresh weight analyses of roots and shoots of asymptomatic plants demonstrated that <i>Phytophthora</i> inoculation may either reduce or stimulate plant shoot growth, but little effect is apparent on roots. Irrigation with naturally infested irrigation water reduced plant growth. This research provides data for prioritizing development of detection technology and management practices for plant pathogens in irrigation water. The results may also lead to improvements in conventional water assay protocols for plant pathogens. / Master of Science

Amylopectin granules in Apicomplexan protozoa are hypothesized to be used as an energy source to aid the parasites in surviving in the environment allow latent stages to excyst and release infective stages, allow them to be mobile, invade host cells, and to continue their life cycle. The objective of this project was to determine if parasite glycolytic enzymes: alpha-amylase, amyloglucosidase, enolase, lactate dehydrogenase, and phosphorylase could be used to decrease amylopectin stores and subsequently infectivity of Cryptosporidium parvum oocysts/sporozoites in both fresh oocysts and stored oocysts. In addition, glycolytic enzymes and substrates: glucose, glucose-1-phosphate, and glycogen synthase were investigated to determine if they can be used to increase amylopectin stores and thus increase infectivity to aid in detection/storage of oocysts. Oocysts of Cryptosporidium parvum were suspended in 1mg/ml glycolytic enzymes or substrates (except glucose - 0.05M and glycogen synthase - 1U/ml) and electroporated. Oocysts were incubated at 37&#176;C for one hour to allow treatments to react with amylopectin followed by incubation on HCT-8 cells for 24 hours for infection. Real-time PCR and immunohistochemistry were performed to determine the effect of the enzymes on infectivity. An amylopectin assay and excystation assay was performed to determine if the enzymes degraded amylopectin and if decreased amylopectin reduced excystation. Alpha amylase and amyloglucosidase had the greatest impact on reducing both amylopectin and infectivity of fresh oocysts with reductions of 99.5% and 99.1% in infective oocysts, respectively (P<0.05). These results suggest that amylopectin may be an important factor in infection, although further research is needed. In stored oocysts, enzymes significantly reduced amylopectin content but not infectivity. In fresh oocysts, amylopectin content was correlated to excystation and infectivity with a decrease in amylopectin correlating to decreased excystation and infectivity. In contrast, there was no direct correlation for stored oocysts. When glucose, glucose-1-phosphate, or glycogen synthase was used to increase infectivity, results show that glycogen synthase had little effect, but glucose and glucose-1-phosphate significantly increased amylopectin content, excystation, and infectivity. In conclusion, amylopectin may be an important polysaccharide store of Cryptosporidium parasites to cause infection by allowing excystation of the oocysts to release infective sporozoites. / Ph. D.

Transmissible spongiform encephalopathies (TSEs) are a group of fatal, neurodegenerative diseases that can affect both humans and animals. TSEs can be sporadic, familial, or acquired diseases. The prion hypothesis states that a misfolded form of the host glycoprotein, PrPC, acts as the infectious agent in TSE disease. The misfolded form, PrPSc, is increased in β-sheet content, detergent insoluble and partially resistant to proteinase K (PK) digestion. Based on the prion hypothesis, most current post-mortem diagnostic tests rely on the presence of PrPSc as indicative of TSE disease. However, recently experimental cases of TSE disease have been identified where no PrPSc deposition is evident. One example of this is a murine transgenic model of Gerstmann Sträussler Scheinker (GSS) disease. GSS is a familial TSE disease, caused by a number of different mutations in human PrP including a point mutation from proline to leucine at residue 102. A murine model of GSS disease, produced through gene-targeting, contains the same point mutation at the equivalent residue, 101, in murine PrP. These mice do not develop spontaneous disease during their lifespan, but when inoculated intra-cerebrally with either human P102L GSS (101LL/GSS) or hamster 263K scrapie (101LL/263K); develop a clinical disease and vacuolar TSE-related pathology. Upon biochemical and immunohistochemical analysis, the brain tissues of these clinically ill mice contain little or no detectable PrPSc. However titration experiments have previously shown infectivity titres of 107-109IU/g of brain tissue. Standard PK digestion (at 37°C), NaPTA precipitation and isolation of PrPSc through detergent insolubility and differential centrifugation all confirmed the observation of little or no detectable PK-resistant PrP (PrP-res) in the 101LL/GSS and 101LL/263K brain tissues, despite the high levels of TSE infectivity. The presence of PrPSc and/or TSE infectivity in the spleen during disease pathogenesis is dependent upon TSE agent strain and host species. Previous studies utilising wild-type mice infected with ME7, have shown that the levels of infectivity observed in spleen tissue are 2- 3log10 lower than those observed in the brain tissue of the same mice. However, experiments conducted as part of this thesis showed that sub-passage of both the brain and spleen tissue from clinically ill 101LL/GSS and 101LL/263K mice into 101LL mice by intra-cerebral inoculation result in short incubation periods, indicating that infectivity levels were similarly high in both tissues. Biochemical analysis of the primary spleen tissue identified the presence of PrP-res, albeit at lower levels than those observed in wild-type spleens infected with a standard laboratory TSE strain, ME7 or 79A. However, the presence of PrP-res indicates that the spleen has a role in disease pathogenesis, which will require further investigation. Additionally, the spleen tissue maintains the discrepancy between PrP-res and TSE infectivity that is observed in the brain tissue of these models and further questions the prion hypothesis. As little or no PrP-res was detectable in the brain tissues of 101LL/GSS and 101LL/263K mice by standard biochemical and immunohistochemical techniques, it was hypothesised that an in vitro amplification technique, protein misfolding cyclic amplification (PMCA) could amplify PrPSc to detectable levels. A series of optimisation experiments were performed to produce a reliable positive control for amplification of mouse PrPSc from a standard laboratory mouse TSE strain, 79A or ME7, with a normal wild-type mouse brain homogenate substrate. While a wide range of technical and experimental conditions were investigated, consistent and reproducible amplification of mouse PrPSc was not achieved and therefore amplification of PrPSc from 101LL/GSS and 101LL/263K tissues could not be performed as interpretation of results would be complicated without the presence of a positive control. Previous research has shown that while other commercial assays, e.g. TeSeE (BioRad), identified tissues from these models as borderline positive or negative for TSE disease, one TSE diagnostic assay, the IDEXX HerdChek kit, that utilises the Seprion ligand, identified both the brain and spleen tissue from 101LL/GSS and 101LL/263K clinical mice as positive for TSE disease. In order to identify if TSE infectivity is associated with the target of the Seprion ligand, brain tissue homogenates from 101LL/GSS, 101LL/263K and a positive control wild-type/79A homogenate were depleted of the Seprion ligand target utilising a PAD-beads kit (Microsens Biotechnologies), which incorporates the Seprion ligand as the capture agent, in combination with magnetic beads. Upon inoculation, a single depletion of the homogenates produced no significant reduction in incubation period to clinical disease in either the depleted homogenates or the wash buffers produced, in comparison to a non-depleted brain homogenate. This result indicates that a single depletion with the Seprion ligand, did not remove enough of the aggregated protein to significantly alter the level of infectivity in the depleted homogenate and that any infectious agent, which was initially bound to the Seprion ligand due to non-specific interactions, was then released during the wash steps of the procedure. Proteomic differences between all components produced during a single depletion of an infected brain homogenate, wild-type/79A, or a normal uninfected brain homogenate were assessed to potentially identify the target of the Seprion ligand. In conclusion, these murine models of TSE disease, 101LL/GSS and 101LL/263K, which contain both high infectivity levels with little or no PrP-res in the brain tissue and similar high levels of infectivity with low levels of PrP-res in the spleen, questions the accepted correlation between levels of infectivity and PrP-res or PrPSc as proposed by the prion hypothesis. It is hypothesised that either an alternative form of PrP, which has not yet been identified is the infectious agent in these disease models, or that the TSE infectious agent is a component which associates with PrPSc rather than being PrPSc itself. The eventual identification of the infectious agent present in these unusual disease models will increase our understanding of these diseases, potentially offer improved diagnostics for infectivity, and perhaps identify novel therapeutic targets.

Bakgrund: Trots tydliga föreskrifter om basala hygienrutiner följs dessa inte alltid av personal på vårdavdelningar. Studier visar att orsaken till detta beror på olika faktorer. Syfte: Att undersöka kunskapen om, samt följsamheten till Socialstyrelsens föreskrifter om basala hygienrutiner hos undersköterskor, sjuksköterskor och läkare på ett universitetssjukhus i Mellansverige. Metod: En kvantitativ studie med deskriptiv och jämförande design. Datainsamlingen skedde med hjälp av ett observationsprotokoll samt en enkätundersökning. Antal medverkande i observationsmomentet var 93 deltagare respektive 81 i enkätundersökningen.  Data analyserades med ANOVA samt deskriptivt statistik. Resultat: Samtliga professioner hade relativt god kunskap gällande kunskapsfrågorna samt ansåg att kunskap är viktigt. Bristande kunskaper sågs gällande handdesinfektion samt regler för naglar och armbandsur. Gällande arbetskläder och håruppsättning var följsamheten god bland samtliga professioner. Gällande följsamheten i patientnära kontakt var följsamheten relativt god men skillnader inom olika områden kunde ses. Tidspress och placering av hygienprodukter visades påverka hur följsamheten sköts. Slutsats: Studien visar att följsamheten till de basala hygienrutinerna i helhet är relativt god bland professionerna. Dock ses brister vilket tyder på att kunskapen är otillräcklig. Ökad kunskap och förbättrad följsamhet kan leda till kortare vårdtider, minskat antal vårdrelaterade infektioner samt en ökad patientsäkerhet. / Background: Despite clear basic hygiene rules, the adherence is poor among healthcare workers. Previous studies have shown that this depends on several factors. Aim: To examine certified nursing assistants´, registered nurses´ and doctors´ knowledge and adherence of the basic hygiene rules at a teaching hospital in central Sweden. Method: A quantitative study with descriptive and comparative design was chosen. The data was collected using an observation protocol and a questionnaire. Number of participants in the observation was 93 persons and 81 persons in the questionnaire. The data was analyzed using ANOVA and descriptive statistics. Results: All participants had relatively good knowledge regarding the basic hygiene rules and they found the subject important. Lack of knowledge was seen regarding the use of hand disinfectants and in the use of wristwatches and rules for nails. Regarding rules for clothes and hair, adherence was good to the basic hygiene rules among all participants. In contact with the patient, adherence was relatively good. Time pressure and placement of hygiene products have been shown to affect compliance. Conclusion: The overall result shows that adherence to the basic hygiene rules is relatively high among all professions. Faults were seen due to lack of knowledge. Increased knowledge and improved adherence to the basic hygiene rules can result in shorter hospitalization, decreased nosocomial infections and increased patient safety.