Viruses cause 60% of human infections and are probably the most common cause of infectious disease acquired indoors. Rapid spread of viral illness in indoor establishments facilitates disease morbidity and mortality. The goal of this dissertation is to clarify the role of fomites in the viral infection cycle. Research methods include investigation of published literature, and the use of polymerase chain reaction (PCR) for viral detection. The Appendix A study reviewed published literature to assess the significance of fomites in the transmission of ten common respiratory and enteric viruses (rhinovirus, respiratory syncytial virus (RSV), influenza A, parainfluenza 1 (HPIV1), coronavirus, rotavirus, calicivirus, hepatitis A virus (HAV), astrovirus and adenovirus). Results suggest that fomites play an important role in the transmission of common viral pathogens, and the use of disinfectants may limit the spread of viral disease. The Appendix B study examined PCR primer detection limits by determining the time length viruses can be isolated on fomites. Results indicated that poliovirus 1 and hepatitis A virus could be detected for up to 60 days. Parainfluenza 1 virus isolation yielded detection at 30 days and 50 days. Norovirus isolation yielded detection at 20 days and 30 days. Influenza virus isolation results were inconsistent, yielding no initial detection and detection up to 20 days. Appendix C assessed the occurrence of human parainfluenza 1 virus (HPIV1) on surfaces in office settings. HPIV1 was detected on 37% of fomites. HPIV1 was detected most on desktops (47%), and least on light switches (19%). Study results indicated a statistically significant difference between positive fomites in different buildings (Chi-square p < 0.011), and between building cubicles and conference room fomites (Chi-square p < 0.011). Appendix D evaluated the prevalence of influenza A virus on surfaces in day care and home settings. Influenza A was isolated on 23% of fall day care fomites and 53% of spring day care fomites. Influenza was isolated on 59% of home fomites sampled during March, and no influenza was detected on home fomites sampled during the summer. Overall, Influenza A virus was isolated on over 50% of fomites in homes and day care centers.

Background: Real-time quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR) is recently used for characterization and expression analysis of miRNAs. The data from such experiments need effective analysis methods to produce reliable and high-quality data. For the miRNA prostate cancer qRT-PCR data used in this study, standard housekeeping normalization method fails due to non-stability of endogenous controls used. Therefore, identifying appropriate normalization method(s) for data analysis based on other data driven principles is an important aspect of this study. Results: In this study, different normalization methods were tested, which are available in the R packages Affy and qpcrNorm for normalization of the raw data. These methods reduce the technical variation and represent robust alternatives to the standard housekeeping normalization method. The performance of different normalization methods was evaluated statistically and compared against each other as well as with the standard housekeeping normalization method. The results suggest that qpcrNorm Quantile normalization method performs best for all methods tested. Conclusions: The qpcrNorm Quantile normalization method outperforms the other normalization methods and standard housekeeping normalization method, thus proving the hypothesis of the study. The data driven methods used in this study can be applied as standard procedures in cases where endogenous controls are not stable.

Shigatoxin-producing Escherichia coli (STEC) can cause infections in humans which can beserious and sometimes fatal. There is a great need for methods that are able to detect differentserogroups of STEC. In this project, conventional and real-time PCR assays for detection ofSTEC O104:H4 and O121, as recommended by the European Union Reference Laboratory(EU-RL) for STEC, were validated. The specificity, limit of detection, repeatability,efficiency and robustness were determined for three real-time PCR assays. The validationshowed that the real-time PCR reactions were specific and sensitive although some additionaltests are required.

The purpose of this study was first to describe the clinical findings, case management and short-term outcome of horses with septic synovial structures over the last 25 years, and to identify risk factors and treatment modalities associated with specific short-term outcomes. Secondly, we wanted to evaluate a broad range real time polymerase chain reaction (RT PCR) assay for the diagnosis of septic synovitis from synovial fluid (SF) samples of horses, and compare its performance to currently available diagnostic methods. For the retrospective study, 367 cases met the inclusion criteria. Lavage of the synovial structure and endoscopic surgery were associated with an increased likelihood of discharge from hospital, however, none of the local antimicrobial delivery modalities were associated with a significant change in outcome. No significant improvement in hospital outcome of horses with septic synovitis was identified over the past 25 years. For the RT PCR study, 48 SF samples from horses with suspected septic synovitis were included, and RT PCR and microbial culture was performed on all samples. One additional RT PCR assay was performed on samples with discordant results or identification of dissimilar organisms. Thirty-eight percent of SF samples had positive culture results, and 42% of SF samples had positive RT PCR results. Sensitivity and specificity for the RT PCR assay relative to agreement of observers on the likelihood of infection were 87% and 72%, respectively, whereas for culture they were 56% and 86%, respectively (P=0.001). The combination of culture and RT PCR assay resulted in sensitivity and specificity of 85% and 81%, respectively. The broad range RT PCR assay was more sensitive than culture for identification of horses with septic synovitis. Further refinement of the RT PCR assay technique may facilitate use in a clinical setting. / Equine Guelph, University of Guelph

A PCR Differentiation Method, a hybrid of the US EPA and MOE methods, for quantifying human infectious C. parvum/C. hominis as a group and non-human infectious C. andersoni/C. muris was developed. Primers and probe sets targeting the hsp70 gene were designed for C. andersoni/C. muris; those for C. parvum/C. hominis were obtained from the MOE method. Results showed that C, andersoni/C. muris primers were specific for C. andersoni/C. muris oocysts, while those for C. parvum/hominis primers detected C. parvum/hominis and C. meleagridis. All primers were then used to quantify oocysts from urban and agricultural environmental water samples in Kitchener/Waterloo. Human infectious Giardia lamblia was also incorporated into this study. C. parvum/C. hominis and Giardia lamblia were detected at urban and agricultural areas, whereas C. andersoni/C. muris was only detected at agricultural sites. The PCR Differentiation Method is a reliable method for quantifying Cryptosporidium and Giardia lamblia in environmental water samples. / Best in Science Program of the Ontario Ministry of the Environment, Natural Sciences and Engineering Council (NSERC) of Canada Discovery Grants

Neotyphodium endophytes (Claviceptaceae) are asexual filamentous fungi found living between the cells of many cool season forage grasses including tall fescue, meadow fescue and perennial ryegrass. They produce a range of alkaloids, including ergovaline and lolitrem B, which have been shown to be directly associated with the livestock disorders fescue toxicosis and ryegrass staggers syndrome, while others, including peramine and the lolines, have been linked to increased insect and drought resistance of the grass host. In the past decade, the Neotyphodium strains AR1, MaxQ and MaxP were selected because they did not produce the alkaloids associated with livestock disorders. Subsequently, artificial associations were established between them and commercial forage grass cultivars. The slow growth rate of Neotyphodium endophytes in vitro and the low success rate of the present methods for establishing artificial associations between endophytes and grass hosts are limiting the rate at which new novel endophytes can be incorporated into plant breeding programs and eventually commercialised. In this thesis, the type and concentration of the growth medium was shown to affect radial growth rate, colony appearance and mycelial morphology of three strains of Neotyphodium endophytes. The floret inoculation of meadow fescue with the U2 strain of N. uncinatum using several techniques involving liquid culture was attempted but was unsuccessful in creating any artificial associations. Neotyphodium endophytes are unstable in stored seed. In New Zealand, it is critical that pastures are infected with protective Neotyphodium endophytes to ensure that they will not be destroyed by exotic pests. The present methods for determining the percentage of viable endophyte infection of a seed lot are too slow for efficient use in the commercial seed industry. In this thesis, primers specific to the â-tubulin gene of N. coenophialum, N. lolii and N. uncinatum were designed and successfully used to detect these species in planta. However, using these primers to develop a method to accurately determine the viable endophyte infection rate of a seed lot using RT-qPCR was unsuccessful.

Enteric adenoviruses (EAd) types 40 and 41 (Ad40 and Ad41) representing subgenus F, are primary pathogens of children being second only to rotaviruses as the most important cause of infantile diarrhea. The EAds differ from all other adenoviruses in their inability to grow in most conventional established cell lines and have been suggested to be deficient in some early gene functions since they could be complemented by Ad 5 early regions EIA and E1B. In order to search for differences that could explain its characteristic growth restriction, the early regions EIA and E1B of Ad41 (strain D389) were sequenced, analysed and compared with the corresponding regions of Adl2, Ad7, Ad2, and Ad4. As revealed by the analysis of Ad2, three major mRNAs of 9S, 12S and 13S are generated from region EIA. The EIA region of Ad41 encodes two mRNAs corresponding to the 12S and 13S mRNAs. Only the 13S mRNA is transcribed at detectable levels. This mRNA can be translated into a 251 aa putative protein that contains the three highly conserved domains found in all other human adenoviruses and shown to be responsible for many important regulatory functions during infection. The E1B region of Ad41 encodes three transcripts that correspond to 22S, 14S and 9S mRNA of Ad2. No equivalent to the 13S mRNA of Ad2 E1B is found. In addition the Ad41 14S mRNA exhibits an additional exon of 23 bp created by a donor and an acceptor splice sites not desribed for other adenovirus E1B sequences. Due to their growth restriction in conventional cultures, rapid diagnostic procedures developed for the enteric adenovirus infections have mainly been aimed at the detection of viral antigens or nucleic acids. This thesis also describes several procedures developed for the general detection of adenoviruses and specific detection of the enteric types in stools specimens. General and specific hybridization assays were developed by use of two BamHI clones obtained from the EIA region of Ad41. One- and two-step PCR procedures were also developed for the general detection of adenoviruses using primers corresponding to highly conserved sequences within the hexon gene. Subgenus F specific one- and two-step PCRs were developed by using primers located in the Ad41 E1B region. The one-step PCR systems were tested and validated against isolation in tissue culture, DNA restriction enzyme analysis and a commercial latex agglutination test in the study of 60 specimens obtained from children with rotavirus negative diarrhea. The asymptomatic fecal excretion of adenoviruses was evaluated by two-step PCR amplifications on samples from 50 healthy children, 50 healthy adults, and 50 adults suffering from diarrhea. Finally, a simplified procedure for detection, discrimination and typing of EAd was also designed by combining the one-step PCR amplification of the hexon region with the restriction of the 300 bp product. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1992</p> / digitalisering@umu

Human papillomavirus (HPV) infections of the genital tract are now recognized to be among the most prevalent sexually transmitted diseases and also a contributing factor to some cancers of the lower genital tract of women and men. Presence of HPV in a clinical specimen is confined to detection of the HPV genome by DNA hybridization techniques. In this thesis, the commonly used DNA hybridization techniques Southern blot and filter in situ hybridization (FISH), were first used for detection of genital HPV infection. In order to increase and simplify the detection of HPV in clinical specimens a more sensitive technique, the polymerase chain reaction (PCR) was subsequently utilized. For type-specific amplificaiton of HPV 6, 16, 18 and 33 by PCR, oligonucleotide primers located in the E6 and E7 regions of the HPV genome were selected. They were found to specifically amplify the four types. To be able to amplify a broad spectrum of genital HPV types, general primers located in the E7 and El region of the HPV genome, were designed and evaluated. They were found to amplify a wide range of genital HPV types. To further increase the sensitivity and specificity, a two-step PCR using general primers, was assembled and evaluated against a one-step PCR on cervical scrapes from young women in a population-based study. The two-step PCR increased the sensitivity about three-fold compared to the one-step PCR. By Southern blot and FISH, 46% of women with abnormal Papanicolaou (Pap) smears were shown to carry HPV DNA. Of the women analysed by Southern blot, 39 % harboured HPV DNA and 25 % proved HPV 16 positive. Of the samples analysed with FISH, 27 % contained HPV DNA, compared to 11 % of samples from a group of reference women with normal cytology. With the Southern blot technique, HPV DNA was detected in 66% of women with cervical intraepithelial neoplasia grade III (CIN III) lesions. Fifty-four percent of the women with CIN III lesions were positive for HPV 16 DNA. By type-specific PCR, 12 out of 13 women with cervical squamous carcinoma were shown to carry HPV 16 and/or 18. Among women with adenosquamous carcinoma of the cervix, HPV 18 was the most prevalent type (26%) but HPV 16 was also found in a proportion of the women(15 %). Nine of 13 premenopausal cases with cervical adenocarcinoma were HPV positive compared to only 2 of 13 postmenopausal cases (p&lt; 0.015). HPV 16 DNA was detected in 48%of women with cervical intraepithelial neoplasia (CIN), by the use of type-specific PCR. Three different groups of women with normal cytology were studied. Among women attending a family planning clinic in Kenya, 19% were shown to carry HPV virus, by the use of general primers. HPV 16 was found in 5.2% of these women and HPV 18 in 3.9%. In anothergroup of women, attending the gynecological department in Umeå, HPV 16 DNA was detected in 21 % by type-specific PCR. However, if consideration was taken to the medical status of the women, only 10% of women without any medical history were HPV 16 DNA positive, versus 54% of women with diseases and women with a relative progesterone dominance. Finally, by use of a two-step PCR using general primers, 20% of young women from Umeå taking part in a population-based study were demonstrated to carry HPV DNA. The most prevalent types were HPV 6 (2.0%) and HPV 16(2.7%). Among the women in this study with normal cytology, 19%were HPV positive. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1991, härtill 9 uppsatser.</p> / digitalisering@umu

Genetically modified (GM) crops attracted interest globally when use of these crops resulted in significant increases in yield and production. These increases were due to protection of crops from pests, weeds and diseases. However, evolution of resistance by pests threatens the continued efficacy of GM crops. One such example is the resistance to Cry1Ac toxin in Helicoverpa armigera (Lepidoptera: Noctuidae). Resistance in this pest was due to a mutation in the aminopeptidase N1 (APN) Cry receptor gene, encoding the receptor for Cry1Ac. Laboratory studies have indicated that species in families Noctuidae, Pyralidae and Plutellidae can develop resistance to Bttoxins. To date, field-evolved resistance has only been reported in Busseola fusca (Fuller) (Lepidoptera: Noctuidae) in South Africa, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) in the south-eastern United States, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) in Puerto Rico, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae) in India, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) in northern China and Plutella xylostella (Linnaeus) (Lepidoptera: Plutellidae) in The Philippines and Hawaii. Resistance development in lepidopteran species is thus a common phenomenon. The stem borer B. fusca is a major insect pest to Bt-maize in the Vaalharts irrigation scheme (South Africa). The first official report of B. fusca resistance to Cry1Ab toxin was recorded in 2007, although farmers observed increased damage to Bt-maize from stem borers as early as 2004. A second report of resistance in an area nearby followed in 2009. No study has yet been done to determine the molecular mechanism of B. fusca resistance to Cry1Ab. As mentioned, a mutation in the APN receptor gene is responsible for H. armigera resistance to Cry1Ac. Although B. fusca has developed resistance to the B. thuringiensis Cry1Ab toxin, the binding-patterns and -sites of Cry1Ac and Cry1Ab are similar. Thus a similar mutation may be responsible for B. fusca resistance to Cry1Ab. Aminopeptidase, cadherin and alkaline phosphatase are the major Cry toxin receptors that have been identified in lepidopteran species. The present study was concerned with the investigation of mutations in these receptor genes. However, in order to study mutations, sequence data of receptor genes are essential. Degenerate primers were designed based on conserved regions observed in multiple protein sequence alignments of aminopeptidase N (isogenes 1 to 6), cadherin and alkaline phosphatase of several lepidopteran species. Primers were degenerate to take into consideration the variant regions in receptor gene sequences among lepidopteran species. These primers were used to amplify genomic DNA (gDNA) from susceptible and resistant larvae by using PCR. Sequences of PCR amplicons were determined through Sanger sequencing reactions and subjected to BLAST searches. Results of the BLAST searches showed some similarities to the respective receptor genes. These sequences were also used in phylogenetic analysis. This analysis intended to determine the phylogenetic relationship of the respective receptor genes between B. fusca and other lepidopteran species. Mutations could not be identified in the present study, due to a lack in receptor gene sequence data for B. fusca. Thus a goal of the present study was to generate sequence data for B. fusca. In addition to the proposed objectives, cytochrome b gene sequences of B. fusca were used to determine the phylogenetic relationship between B. fusca and other lepidopteran species. Genome sequencing of B. fusca is recommended, as this will provide a platform for genomic, transcriptomic and proteomic studies on this species. These studies will provide much needed information, which can be used to formulate strategies to prevent resistance development in and spread of resistance to other B. fusca populations in sub-Saharan Africa. / Thesis (MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013.

Genetically modified (GM) crops attracted interest globally when use of these crops resulted in significant increases in yield and production. These increases were due to protection of crops from pests, weeds and diseases. However, evolution of resistance by pests threatens the continued efficacy of GM crops. One such example is the resistance to Cry1Ac toxin in Helicoverpa armigera (Lepidoptera: Noctuidae). Resistance in this pest was due to a mutation in the aminopeptidase N1 (APN) Cry receptor gene, encoding the receptor for Cry1Ac. Laboratory studies have indicated that species in families Noctuidae, Pyralidae and Plutellidae can develop resistance to Bttoxins. To date, field-evolved resistance has only been reported in Busseola fusca (Fuller) (Lepidoptera: Noctuidae) in South Africa, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) in the south-eastern United States, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) in Puerto Rico, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae) in India, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) in northern China and Plutella xylostella (Linnaeus) (Lepidoptera: Plutellidae) in The Philippines and Hawaii. Resistance development in lepidopteran species is thus a common phenomenon. The stem borer B. fusca is a major insect pest to Bt-maize in the Vaalharts irrigation scheme (South Africa). The first official report of B. fusca resistance to Cry1Ab toxin was recorded in 2007, although farmers observed increased damage to Bt-maize from stem borers as early as 2004. A second report of resistance in an area nearby followed in 2009. No study has yet been done to determine the molecular mechanism of B. fusca resistance to Cry1Ab. As mentioned, a mutation in the APN receptor gene is responsible for H. armigera resistance to Cry1Ac. Although B. fusca has developed resistance to the B. thuringiensis Cry1Ab toxin, the binding-patterns and -sites of Cry1Ac and Cry1Ab are similar. Thus a similar mutation may be responsible for B. fusca resistance to Cry1Ab. Aminopeptidase, cadherin and alkaline phosphatase are the major Cry toxin receptors that have been identified in lepidopteran species. The present study was concerned with the investigation of mutations in these receptor genes. However, in order to study mutations, sequence data of receptor genes are essential. Degenerate primers were designed based on conserved regions observed in multiple protein sequence alignments of aminopeptidase N (isogenes 1 to 6), cadherin and alkaline phosphatase of several lepidopteran species. Primers were degenerate to take into consideration the variant regions in receptor gene sequences among lepidopteran species. These primers were used to amplify genomic DNA (gDNA) from susceptible and resistant larvae by using PCR. Sequences of PCR amplicons were determined through Sanger sequencing reactions and subjected to BLAST searches. Results of the BLAST searches showed some similarities to the respective receptor genes. These sequences were also used in phylogenetic analysis. This analysis intended to determine the phylogenetic relationship of the respective receptor genes between B. fusca and other lepidopteran species. Mutations could not be identified in the present study, due to a lack in receptor gene sequence data for B. fusca. Thus a goal of the present study was to generate sequence data for B. fusca. In addition to the proposed objectives, cytochrome b gene sequences of B. fusca were used to determine the phylogenetic relationship between B. fusca and other lepidopteran species. Genome sequencing of B. fusca is recommended, as this will provide a platform for genomic, transcriptomic and proteomic studies on this species. These studies will provide much needed information, which can be used to formulate strategies to prevent resistance development in and spread of resistance to other B. fusca populations in sub-Saharan Africa. / Thesis (MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013.