Browsing by Author "van Marle, Guido"
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- ItemOpen AccessAssessing the performance of a Loop Mediated Isothermal Amplification (LAMP) assay for the detection and subtyping of high-risk suptypes of Human Papilloma Virus (HPV) for Oropharyngeal Squamous Cell Carcinoma (OPSCC) without DNA purification(2018-02-08) Rohatensky, Mitchell G; Livingstone, Devon M; Mintchev, Paul; Barnes, Heather K; Nakoneshny, Steven C; Demetrick, Douglas J; Dort, Joseph C; van Marle, GuidoAbstract Background Oropharyngeal Squamous Cell Carcinoma (OPSCC) is increasing in incidence despite a decline in traditional risk factors. Human Papilloma Virus (HPV), specifically subtypes 16, 18, 31 and 35, has been implicated as the high-risk etiologic agent. HPV positive cancers have a significantly better prognosis than HPV negative cancers of comparable stage, and may benefit from different treatment regimens. Currently, HPV related carcinogenesis is established indirectly through Immunohistochemistry (IHC) staining for p16, a tumour suppressor gene, or polymerase chain reaction (PCR) that directly tests for HPV DNA in biopsied tissue. Loop mediated isothermal amplification (LAMP) is more accurate than IHC, more rapid than PCR and is significantly less costly. In previous work we showed that a subtype specific HPV LAMP assay performed similar to PCR on purified DNA. In this study we examined the performance of this LAMP assay without DNA purification. Methods We used LAMP assays using established primers for HPV 16 and 18, and new primers for HPV 31 and 35. LAMP reaction conditions were tested on serial dilutions of plasmid HPV DNA to confirm minimum viral copy number detection thresholds. LAMP was then performed directly on different human cell line samples without DNA purification. Results Our LAMP assays could detect 105, 103, 104, and 105 copies of plasmid DNA for HPV 16, 18, 31, and 35, respectively. All primer sets were subtype specific, with no cross-amplification. Our LAMP assays also reliably amplified subtype specific HPV DNA from samples without requiring DNA isolation and purification. Conclusions The high risk OPSCC HPV subtype specific LAMP primer sets demonstrated, excellent clinically relevant, minimum copy number detection thresholds with an easy readout system. Amplification directly from samples without purification illustrated the robust nature of the assay, and the primers used. This lends further support HPV type specific LAMP assays, and these specific primer sets and assays can be further developed to test for HPV in OPSCC in resource and lab limited settings, or even bedside testing.
- ItemOpen AccessAssessing the performance of a Loop Mediated Isothermal Amplification (LAMP) assay for the detection and subtyping of high-risk suptypes of Human Papilloma Virus (HPV) for Oropharyngeal Squamous Cell Carcinoma (OPSCC) without DNA purification(BioMed Central, 2018-02-08) Rohatensky, Mitchell G; Livingstone, Devon M; Mintchev, Paul; Barnes, Heather K; Nakoneshny, Steven C; Demetrick, Douglas J; Dort, Joseph C; van Marle, GuidoOropharyngeal Squamous Cell Carcinoma (OPSCC) is increasing in incidence despite a decline in traditional risk factors. Human Papilloma Virus (HPV), specifically subtypes 16, 18, 31 and 35, has been implicated as the high-risk etiologic agent. HPV positive cancers have a significantly better prognosis than HPV negative cancers of comparable stage, and may benefit from different treatment regimens. Currently, HPV related carcinogenesis is established indirectly through Immunohistochemistry (IHC) staining for p16, a tumour suppressor gene, or polymerase chain reaction (PCR) that directly tests for HPV DNA in biopsied tissue. Loop mediated isothermal amplification (LAMP) is more accurate than IHC, more rapid than PCR and is significantly less costly. In previous work we showed that a subtype specific HPV LAMP assay performed similar to PCR on purified DNA. In this study we examined the performance of this LAMP assay without DNA purification.
- ItemOpen AccessThe Development of Immunoreagents to Detect for a Cellular Immune Response(2019-08-20) Ma, Crystal Wai Yin; De Buck, Jeroen M.; Jenne, Craig N.; Santamaria, Pere; van Marle, GuidoCell mediated immunity (CMI) is the adaptive T-cell-mediated defense mechanism against pathogens. Oftentimes, by assessing lymphocyte counts and/or cytokine profiles, the presence of an infection can be detected. While current diagnostic tests (e.g. interferon gamma release assay, delayed type hypersensitivity test) exist, many are not widely adopted due to the requirement of specific equipment for quantification, time restraints and/or false negative results. As such, the discovery of new biomarkers that can accurately detect and quantify a T-cell mediated, antigen specific immune response would be very valuable. We aimed to explore cellular immune responses (CIRs) through the direct detection of antigen specific Major Histocompatibility Complex II - T Cell Receptor (MHC II-TCR) interactions. We anticipated to create bioreagents that could enumerate TCR:MHC interactions, thereby giving possible insights of a previously activated CMI by targeting effector T-cells. To do this, superantigens (SAgs) (here: TSST-1) were split and fused to a reporter protein (YFP), allowing for specific TCR-pMHC interactions on antigen presenting cells (APCs) to be detected through a fluorescent marker. In the presence of a cognate pMHCII:TCR interaction, our split superantigens would be able to bind and reassemble, thereby quantifying the interaction using fluorescence. To further detect for sensitized T-cells, we engineered peptide-recombinant T cell receptor ligands (pRTLs) to be displayed on the surface of fluorescent E. coli using a pAIDA1 expression system. This system is intended to mimic the expression of MHC II on APCs. Interactions between the T-cells and pRTLs on the surface the E. coli can therefore be visualized using microscopy followed by flow cytometry analysis, whereby clustering of GFP-bacteria around effector T-cells would arise as a result of successful peptide display. Our results indicated no binding occurred between the individual C-domain of TSST-1 and T-cells. It was also revealed that our designed pRTL that were expressed on the surface of E.coli were unable to bind to T-cells, possibly due to complications with expressing the pRTL using a bacterial surface expression vector. Here, we describe the methodology of our approach, including construct design, protein production and analysis. We also explore existing technologies,and suggest future approaches and considerations that can be made to supplement the current research we have performed in this project. In conclusion, our developed biomarkers did not successfully bind to T-cells as we had anticipated, however, we did develop valuable insight regarding the use of SAg and pRTLs are potential bioreagents for the detection of a cellular immune response.
- ItemOpen AccessEpitope Mapping of Bovine Viral Diarrhea Virus Antigens E1, E2, Erns, and NS3 using Phage Display and Peptide Scanning(2020-12-21) Bremner, William T. R.; van der Meer, Frank; Schryvers, Anthony Bernard; Coffin, Carla S.; van Marle, GuidoBovine viral diarrhea virus is a cattle pathogen with global distribution, and substantial economic impact. Control of viral transmission is challenging by the lifelong viral shedding by persistently infected (PI) animals, and the significant diversity of the virus. While surveillance and removal of PI animals is the primary focus of control programs, insight into the antigenicity of the virus could be valuable for developing broadly protective vaccines to reduce the spread of the virus within farms and in this way the incidence of persistently infected animals (PI’s). This study aims to characterise the viral proteins of BVDV which elicit specific antibody responses. These proteins are the surface glycoproteins E1, E2 and Erns, as well as the non-structural protein NS3. This work employs the techniques of phage display, and peptide scanning to identify epitope structures on these four antigens and puts them into the context of existing structural models. Linear epitopes of potential interest for future vaccine development were identified on each of the four antigens. While attempts at characterizing conformational epitopes highlighted shortcomings in the computational workflow for such efforts, valuable insight into the limitations and future directions are drawn.
- ItemOpen AccessExamining the immune response to infection in the context of Non-alcoholic fatty liver disease (NAFLD)(2022-02) Davis, Rachelle; Jenne, Craig; van Marle, Guido; Mahoney, DougNAFLD includes a spectrum of disease from minor steatosis to hepatic inflammation, and can develop into fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Due to the obesity epidemic, NAFLD is rapidly emerging as the most common chronic liver disease worldwide. Although the exact pathogenic progression of NAFLD is ambiguous, the evolution from steatosis to NAFLD is currently considered a multi-hit approach involving inflammation that may promote progression of the disease, although there is very little data regarding immune response to pathogens in the context of NAFLD. The hypothesis states the immune response to viral infection in the context of fatty liver is hyperinflammatory, resulting in accelerated and increased liver damage. Intravital microscopy (IVM) is a sophisticated imaging approach that allows us to see cellular interactions and behaviours in real-time in vivo. IVM of the fatty liver revealed anatomical changes to the liver after high fat diet (HFD) and a pro-inflammatory resident macrophage phenotype. After infection with S. aureus, HFD mice had decreased platelets and NETs in the liver, while plasma IL-17 was increased compared to control. However, NAFLD mice showed similar bacterial loads and alanine aminotransferase (ALT) levels compared to control. Infection with adenovirus resulted in a delayed GFP expression but increased liver damage as assessed by ALTs. Importantly, flow cytometric analysis showed exhausted T cells from NAFLD mice that did not activate as well as control T cells after stimulation, while IVM revealed no behavioural dysfunction of T cells from NAFLD mice post-viral infection. Overall, these results reveal small but important alterations in the immune response to both bacterial and viral infection in the context of NAFLD.
- ItemOpen AccessExploring the Effects of Inhaled Antibiotics on the Cystic Fibrosis Lung Microbiome and Pseudomonas aeruginosa Population Diversity and their Clinical Implications(2020-01-20) Heirali, Alya; Parkins, Michael D.; Storey, Douglas G.; van Marle, Guido; Surette, Michael G.The CF lung microbiome is composed of a diverse group of microorganisms. Where the constituents of the microbiome originate from remains poorly understood. The work presented herein shows that the home environment may serve as a reservoir for infection in patients with CF. Researchers have demonstrated that the CF lung microbial communities are dynamic and complex. As patients age and disease progression occurs the diversity of organisms colonizing the lower airways generally decreases and patients become dominated by organisms such as Pseudomonas aeruginosa. Several studies have attempted to increase our understanding of the shifts in the microbial communities prior to pulmonary exacerbations. However, there is a tremendous knowledge gap on how the microbiome changes through chronic suppressive inhaled antibiotics used by the majority of CF patients in Canada. Accordingly, we sought to investigate how inhaled aztreonam and tobramycin affect the CF lung microbiome and whether the microbiome can be used as a tool to predict patient response. We showed that the lung microbiome is relatively fixed in adults with CF despite potent inhaled antibacterial therapy. The relative abundance of Staphylococcus was associated with response in all three studies assessing the effects of inhaled antibiotics on the lung microbiome. Specifically, a higher abundance of Staphylococcus at baseline was associated with non-response to inhaled aztreonam and response to inhaled tobramycin – mirroring expected antibacterial activity and suggesting a potential biomarker for treatment response. Keywords: lung microbiome, cystic fibrosis, inhaled antibiotics, Staphylococcus, P. aeruginosa, biomarker
- ItemOpen AccessExploring the Impact of Bovine Leukemia Virus Proviral Load on Production, and its Potential Use for Control(2024-01-19) Shrestha, Sulav; van der Meer, Franciscus Johannes; Careem, Mohamed Faizal Abdul; Barkema, Herman W.; Orsel, Karsina; van Marle, GuidoThe main aim of this dissertation was to evaluate the efficacy of a bovine leukemia virus (BLV) control program by selective removal of high proviral load (HPL) BLV-infected subsets. Six chapters are included. 1) To be acquainted with the current understanding on BLV infection, transmission routes, diagnosis, control, and most importantly, BLV proviral load, a literature review was conducted. This review explored the applicability of BLV proviral load in disease diagnosis, BLV transmission risk assessment, and BLV control. 2) We implemented a cross-sectional study to evaluate the impact of BLV proviral load on milk production of dairy cows. Data obtained from nine dairy herds in Alberta, Canada demonstrated a significant reduction in milk, fat, and protein production of HPL cows when compared with the BLV-negative counterparts. 3) The effectiveness of HPL-cow focused BLV control program in reducing BLV prevalence and seroconversions within the herd was evaluated by conducting a 3-year study among ten dairy herds. The BLV prevalence decreased in four herds whereas the BLV incidence was reduced in nine herds, which supported the notion that removal of HPL cows can offer a feasible and economical option for BLV control. 4) A 1.5-year longitudinal study was designed by enrolling subset of cows from BLV-seropositive (further classified into various proviral load categories) and BLV-seronegative group to monitor the dynamics of various parameters such as BLV proviral load, lymphocyte, white blood cell (WBC) count, antibody titer, CD3+, CD4+, CD8+, CD21+, and WC1+ cell proportions. A relatively stable pattern of BLV proviral load, WBC, CD3+, and CD4+ cell proportion was observed, indicating frequent testing might not be required for these parameters in monitoring BLV infection. 5) A cross-sectional study was conducted to investigate the hematological and immunological impact of BLV infection which suggested a simpler categorization of HPL and LPL as an appropriate approach. Additionally, a lower proviral load cut-off was identified as an accurate threshold for identifying HPL cows. 6) Lastly, all the results and findings were thoroughly discussed, and future directions for using HPL-focused strategies as a potential tool for BLV control and management were elaborated.
- ItemOpen AccessHepatitis B Virus (HBV) Variants in Untreated and Tenofovir Treated Chronic Hepatitis B (CHB) Patients during Pregnancy and Post-Partum Follow-Up(PLoS One, 2015-10-16) Virine, Boris; Osiowy, Carla; Gao, Shan; Wang, Tong; Castillo, Eliana; Martin, Steven R.; Lee, Samuel S.; Simmonds, Kimberley; van Marle, Guido; Coffin, CarlaBACKGROUND: Chronic hepatitis B (CHB) is a dynamic disease that may be affected by immune changes in pregnancy. Guidelines suggest consideration of nucleos/tide analogs (NA), i.e., tenofovir, (TDF) in highly viremic mothers to reduce vertical transmission risk. HBV variability affects CHB outcome, but little is known about HBV genetic changes in pregnancy due to immune or NA selection. OBJECTIVES: To evaluate HBV diversity in NA treated or untreated pregnant vs. post-partum CHB carriers. STUDY DESIGN: In plasma collected from 21 mothers (7 matching pre/post-partum), HBV serological tests, genotype and viral load were assayed. The HBV pre-surface (S) /S overlapping polymerase (P) (N = 20), pre-core (C) /C (N = 11) and/or full genome PCR amplicons (N = 3) underwent clonal sequence analysis. RESULTS: The median age was 31 y, 71% Asian, 68% genotype B or C, 33% HBV eAg+, 5 received TDF (median HBV DNA 8.5 log IU/ml). In untreated mothers, median antepartum vs. post-partum ALT was 21 vs. 24 U/L and HBV DNA was 2.7 vs. 2.4 log(10) IU/ml. ALT and/or HBV DNA flares occurred during pregnant and/or post-partum period in 47% (10/21). Clonal sequencing antepartum showed the presence of minor "a determinant" and/or vaccine escape mutants (VEM) but drug resistant variants were infrequent. Analysis of pregnant vs. post-partum samples showed different HBV variants and viral diversity. CONCLUSIONS: Differences in immune and/or by NA selective pressures during pregnancy may affect HBV evolution during pregnancy. The presence of minor VEM warrant infant follow-up.
- ItemOpen AccessInfection of the Gut by HIV-1: The Pathogenic Role of the Nef Protein(2012-12-03) Grant, Tannika; van Marle, GuidoHuman Immunodeficiency virus type 1 (HIV-1) targets several cells in the immune system and induces their progressive decline and the Acquired Immunodeficiency Syndrome (AIDS). HIV produces viral proteins including Nef; an important multifunctional protein and viral pathogenic determinant. The gastrointestinal tract and impairment of its epithelial barrier has been implicated in HIV pathogenesis, thus an investigation of the contribution of HIV-1 Nef on intestinal barrier dysfunction was performed. An in vitro model system was established and then used to analyse the pathogenic role of Nef. Trans-epithelial electrical resistance, Real time (RT)-PCR and fluorescence microscopy methods were used. It was found that Nef expression in colonic Caco-2 cells, Jurkat and U937 cells; reduced expression of the Caspases, increased viability of cells, while decreasing expression of the tight junction proteins. Thus HIV-1 Nef protein may contribute to HIV pathogenesis by disrupting barrier integrity and cell death of colonic epithelial cells and monolayers.
- ItemOpen AccessLoop-Mediated Isothermal Amplification for Diagnosis of Major Infectious Diseases in Resource-Limited Settings(2015-07-09) Mintchev, Paul; van Marle, GuidoMajor Infectious Diseases (MIDs) continue to pose major global health-care challenges in 2015, particularly in resource-limited settings. Alongside the recent global Human Immu-nodeficieny Virus (HIV)/Acquired Immunodeficieny Syndrome (AIDS) pandemic, there has been a marked resurgence in fatalities related to Malaria and Leishmaniasis in Lower-to-Middle-Income Countries (LMICs). With the recent development of Loop-Mediated Isothermal Amplification (LAMP), researchers are hoping to bring rapid and inexpensive point-of-care diagnostics, based on nucleic acid amplification, to LMIC settings. We ex-plored the possibility of a portable, low-cost LAMP-based diagnostic device targeting HIV, Malaria, and Leishmaniasis, for use in resource-limited settings, in an idealized proof-of-concept study. Design and build an inexpensive functional prototype with the potential to quantify disease severity that demonstrates the feasibility of this diagnostic tool for the desired application was the primary outcome; however, much work remains to be done in order to optimize the method to a reliable and repeatable clinical tool.
- ItemOpen AccessNovel Approaches to Fight Prion Diseases(2020-04-29) Thapa, Simrika; Schaetzl, Hermann M.; Gilch, Sabine; Trang, Tuan; van Marle, Guido; Coffin, Carla S.; Telling, Glenn C.Prion diseases are fatal neurodegenerative disorders caused by PrPSc, the misfolded and infectious isoform of the cellular prion protein (PrPC). Currently, no preventive or therapeutic measures are available. In this work, we focused on therapeutic and prophylactic strategies against prion infections. In the therapeutic approach, we targeted cellular pathways and investigated the role of the quality control (QC) proteins, ERp57 and VIP36, on prion propagation. We found that the overexpression of ERp57 or VIP36 significantly reduced PrPSc levels in persistently prion-infected cells and decreased the susceptibility of uninfected cells to de novo prion infection. Moreover, lentiviral-mediated overexpression of ERp57 prolonged the survival of prion-infected mice. Mechanistically, we found that ERp57 overexpression reduced endoplasmic reticulum (ER) stress. To translate this proof-of-concept into potential drug therapy, we investigated the anti-prion effect of Sephin1, shown to prolong the phosphorylation of eIF2α and lower ER stress in the cells. In persistently prion-infected neuronal cells, we found that treatment with Sephin1 markedly reduced PrPSc levels. Moreover, Sephin1 reduced ER stress-induced PrP aggregates in cells and significantly extended the survival of prion-infected mice. These data provide the basis for targeting these cellular pathways as novel anti-prion therapy. In our prophylactic approach, we hypothesized that active vaccination is useful to contain chronic wasting disease (CWD), a contagious and expanding prion disease of cervids. Here, we vaccinated transgenic mice expressing elk prion protein with adjuvant CpG alone, or one of four recombinant PrP (rPrP) immunogens: deer dimer (Ddi), deer monomer (Dmo), mouse dimer (Mdi), and mouse monomer (Mmo). After challenging the animals with CWD prions intraperitoneally, we found that all vaccinated groups had longer survival times than the CpG control group. Interestingly, the Mmo-immunized group revealed that survival was extended by 60%. We also observed 28.4% and 24.1% prolongation in Dmo and Ddi groups, respectively. Our preliminary study in reindeer showed substantial humoral immune response induced by Mdi and Ddi, and the sera from the Ddi-vaccinated reindeer significantly reduced CWD prions in a cell culture model. Taken together, this study describes potential vaccine candidates against CWD. However, their protective effect in the natural cervid host needs further investigation.
- ItemOpen AccessPersistent Hepatitis B Virus in Hepatic and Extrahepatic Reservoirs in Hepatitis B Virus Related Oncogenesis(2020-05-04) Lau, Keith Chi Kei; Coffin, Carla S.; Burak, Kelly Warren; Mahoney, Douglas J.; van Marle, GuidoChronic infection by Hepatitis B virus (HBV) may result in hepatocellular carcinoma (HCC). Numerous viral factors are associated with oncogenesis and development of HCC, including integration, genetic variations in the X/basal core promoter/precore (X/BCP/PC) region, and occult infection. Although primarily hepatotropic, HBV is frequently found in circulating peripheral blood mononuclear cells (PBMCs) of the lymphoid system. Few studies have characterized this extrahepatic reservoir particularly in HBV-related HCC individuals. In this thesis, we hypothesized that pro-oncogenic HBV is present in both liver and extrahepatic reservoirs in chronic HBV (CHB) carriers with and without malignant disease. A highly sensitive molecular tool for accurate detection of low-level HBV and determination of genotype is described in this thesis. Subsequently, this technique was applied to plasma and PBMCs from post-liver transplant CHB carriers in combination with next generation sequencing to identify HCC-associated viral genotypes and genetic variants. The lymphoid reservoir in 32 CHB carriers with HCC and a representative patient with an extrahepatic lymphoid malignancy (i.e., dendritic cell sarcoma [DCS]), was evaluated for pro-oncogenic HBV. Interestingly, integrated virus was identified in genes implicated in cancer development in liver, PBMCs and DCS tumor. HCC-associated X/BCP/PC variants and genotypes were also present within lymphoid cells. Functional characterization of the most commonly detected variants (Guanine-1896-Adenine and Adenine-1762-Thymine/Guanine-1764-Adenine) were evaluated in vitro. Compared to wild-type, these mutants showed reduced viral HBeAg, modulated cytokine/chemokine expression, decreased APOBEC3G protein expression. The A1762T/G1764A mutant had more robust HBV replication than the G1896A variant. Overall, the findings in this thesis contributes to the literature on the epidemiological association of occult HBV, specific HBV SNPs, and integrated virus in hepatic and extrahepatic reservoirs. Persistent carcinogenic HBV poses continual risks of HCC recurrence and viral reactivation thus advocating for long-term HBV prophylaxis use post-transplant. Pro-oncogenic HBV within lymphoid cells may contribute towards oncogenesis of extrahepatic malignancies such as DCS. HCC-associated variants show functional differences in viral replicative fitness and host immune responses. Taken together, this thesis is significant for advancing the currently limited understanding of HBV molecular virology and pathogenesis of HBV-related carcinogenesis within the hepatic and extrahepatic reservoirs.
- ItemOpen AccessQuantification of HIV-1 Proviral DNA Forms in Gut Tissues Using Real-Time PCR(2015-09-29) Bahafzallah, Laila; van Marle, GuidoPrevious studies have shown that infection of gut tissues (esophagus-stomach-duodenum-colon) by HIV is compartmentalized with increased HIV-1 diversity and selection for drug resistance in the colon. We looked at the kinetics of HIV-1 in the gut tissues obtained from infected patients receiving ART (i.e AZT or ddI). We established real- time PCR protocols to detect HIV-1 proviral DNA forms. The total HIV-1 proviral DNA was the most frequent form found in the gut tissues. 2-LTR proviral DNA was also detected but in low levels which might indicate low viral replication. HIV-1 latent infection was also detected in the gut tissues as represented by the integrated proviral DNA levels. The levels detected of the proviral DNA forms varied between gut tissues, visits and patients, and no difference or correlation were found for patients receiving AZT or ddI.
- ItemOpen AccessA Serological Assessment and Development of a Microbiome and Virome Analysis in Beef Calves(2022-08) Louden, Julia; van der Meer, Frank; Sycuro, Laura; van Marle, Guido; Surewaard, BasBovine Respiratory Disease Complex (BRDC) is a multifactorial disease and the leading cause of morbidity and mortality on feedlots. In many instances, metaphylactic administration of antimicrobials is used to both treat and prevent BRDC. However, this practice is becoming less sustainable due to increasing antimicrobial resistance. Therefore, methods such as preconditioning are being explored to reduce BRDC incidence and the associated antimicrobial use. Preconditioning includes aspects such as fence line weaning and a strategic vaccination schedule to prepare cattle for life on the feedlot. The serological component of this study aimed to demonstrate a higher proportion of preconditioned (PC) animals would arrive on the feedlot with antibodies present than their auction derived (AD) and ranch-sourced (RS) counterparts. The vaccination protocol was evaluated by collecting serum samples from PC, AD, and RS calves upon arrival at the feedlot. Enzyme-linked immunosorbent assays were used to detect antibodies against viral BRDC-associated pathogens: Bovine Viral Diarrhoea Virus, Infectious Bovine Rhinotracheitis, Bovine Parainfluenza Virus Type 3, Bovine Respiratory Syncytial Virus, and Bovine Coronavirus (BoCoV). Significant differences were only found between PC and AD calves as well as PC and RS calves for BoCoV. A metatranscriptomics pilot study was also included in this work to determine optimal downstream processing of deep nasal swabs (DNS). Four PC calves that showed signs of BRDC during preconditioning were utilized in the pilot study. A total RNA isolation was performed on DNS before samples were divided in two, creating an “A” and “B” for each sample. “A” samples underwent rRNA depletion and poly-A tailed mRNA depletion. While “B” samples only had rRNA depleted. Results were interpreted through two bioinformatic alignment tools MetPhlAn3 and Kaiju. MetPhlAn3 identified a total of 20 species, while Kaiju identified over 5000. However, Kaiju also had a significantly high rate of false-positive when reporting positive controls; therefore, results from Kaiju must be interpreted with a high degree of caution. Data from both ELISA’s and the pilot study can provide insight for future directions for this project and, in doing so, support preconditioning as a viable method for the prevention of BRDC.
- ItemOpen AccessThe Development of a Self-Build Power Supply Unit to Assess the Challenges and Barriers Associated with Designing a Malaria Diagnostic Detection Device(2013-10-10) Prebeau-Menezes, Leif; van Marle, GuidoMalaria remains a serious public health problem across tropical and sub-tropical areas of Africa and around the globe. A potential solution lies in areas of biomedical research such as the development of a Malaria Diagnostic Detection Device (MDDD) and similar Point of Care (POC) devices. These diagnostic devices provide timely results leading to rapid clinical management decisions and patient care. Trials on some high technology POC devices have failed to deliver appropriate diagnostics in Africa due to the lack of availability of portable power utilities, specialized operational and maintenance training, and increasingly sophisticated supporting components, which were not available locally. Therefore, before major investments were made in developing the MDDD, a Self-Build Power Supply Unit (SBPSU) was constructed. It consisted of simple components that could be easily constructed, operated and maintained locally and economically in Ethiopia. The SBPSU replicates were tested and resulted in successfully running gel-electrophoresis units. The construction and replication of the SBPSU was also used to train research participants at the University of Gondar in Ethiopia. The outcome of this research provided an assessment of the requirements for developing the MDDD for use in Ethiopia. The lessons learned were incorporated into the design basis for the MDDD based on Loop mediated isothermal AMPlification (LAMP) assays for an accurate and rapid detection of malaria. It is expected that the implications of this work will foster future international biomedical research collaborations and provide insights for the next generation of POC infectious diseases detection devices.
- ItemOpen AccessUsing the social entrepreneurship approach to generate innovative and sustainable malaria diagnosis interventions in Tanzania: a case study(BioMed Central, 2010-02-03) Allen, Lisa K; Hetherington, Erin; Manyama, Mange; Hatfield, Jennifer M.; van Marle, Guido