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Lyophilization maximises vaccines’ stability, shelf life; maintains chemical or biological functions; enables easier transportation and storage compared to a cold chain. Success of vesicular stomatitis virus (VSV) based vaccines has been highlighted by its increased adoption by pharmaceutical companies and most importantly, the recently approved vaccine for high consequence Ebola virus (EBOV). Lujo virus (LUJV) and Lassa virus (LASV), both arenaviruses, are still without licensed vaccines, however, researchers are exploring VSV-vectored vaccines. Many of these highly pathogenic viruses are endemic in tropical regions like Africa where slight breach in cold chain may affect stability of vaccines. It was established that slight changes in cold chain conditions do not affect efficacy of VSV-based vaccines, however, it is imperative to optimise thermostability of VSV-based vaccines using lyophilization. The primary hypothesis of this study is that lyophilized VSV-LASV and VSV-EBOV vaccines exposed to sub-optimal temperature will retain infectivity in cell culture and protect against LASV challenge in inbred Hartley guinea pigs. Different combinations of commercially available stabilisers were used to assess the thermostability of VSV-based vaccines at 4, 21 and 37 °C of storage temperatures. Lyophilized VSV-LASV and VSV-EBOV were recoverable in Vero cells, the recovery titer showed an inverse relationship with exposed temperature and duration of storage of 1, 7, 35 and 90 days. Furthermore, lyophilized VSV-LASV affords 100% protection and sterilising immunity against LASV infection in inbred Hartley guinea pigs.

Blastomycosis is a pulmonary disease caused by Blastomyces dermatitidis, a dimorphic fungus endemic to Manitoba and northwestern Ontario. Immunosuppression is a major risk factor affecting disease susceptibility, yet host immunity is not well understood. Genetic immunodeficiencies can also influence disease, with variants in IL6, GATA2, and VDBP shown to influence susceptibility. However, additional genetic factors in disease susceptibility and severity remain undetected. Our study seeks to establish what is known about susceptibility to blastomycosis and explore genetic risk factors in a case-control cohort. We conducted a scoping review to establish current knowledge on blastomycosis immunity and a literature review to identify candidate genes influencing susceptibility to fungal and mycobacterial infections. Exomes from 18 blastomycosis cases and 9 controls were sequenced, variants were identified, and filtered according to best practices. We performed candidate gene prioritization and variant aggregation to identify genetic associations and explored the full exome dataset. The scoping review included 58 articles on susceptibility to blastomycosis. TNF-a, GM-CSF, CD4+ deficiency, and the IL-12-IFN-y pathway had the most evidence as susceptibility factors. The literature review identified 86 candidate genes relevant to fungal and mycobacterial infections. One hundred and three genetic variants in 42 candidate genes were identified in the exome dataset. No variants associated with susceptibility were identified in a single-variant analysis although two non-synonymous variants in TYK2 were enriched among cases suggesting a possible role in susceptibility. Gene-based association analysis found TLR1 and GATA2 enriched in controls (p = 0.024 and 0.051, respectively) suggesting a possible protective effect, although GATA2 has previously been associated with blastomycosis susceptibility. Gene cluster analysis identified genetic variants in genes of chromatin remodeling

Recent mumps outbreaks have been observed in vaccinated young adults due to MuV genotype G, whereas the current vaccine is a mixture of two genotype A strains. These outbreaks could be attributed to waning vaccine immunity or antigenic differences between the vaccine and circulating MuV, HN, and F glycoproteins. These glycoproteins are essential targets for the immune system, and antigenic variations may reduce the recognition of mumps antibodies, rendering the population susceptible to MuV. We established stable cell lines expressing the MuV glycoproteins to study cross-reactivity between genotype A and genotype G. Cross-reactivity of antibodies between genotypes was assessed by IFA analysis using patient sera from vaccinated individuals without genotype G infection (vaccinated-only), unvaccinated individuals with a history of genotype G infection (infected-only), and vaccinated individuals with genotype G infection (vaccinated-infected). A limiting dilution assay was used to determine antibody titer, defined as the reciprocal of the last serum dilution at which fluorescence could be detected, and the geometric mean titer (GMT) was calculated. Titer ratios were calculated by dividing the genotype A titer by the genotype G titer, and the arithmetic mean was then calculated. Titer ratios showed that the vaccinated-only individuals had a higher titer for genotype A than for genotype G. In contrast, the infected-only individuals had a lower titer for genotype A compared to genotype G. Notably, no difference in titer ratio was observed for individuals vaccinated and infected with mumps. Additionally, a comparison of GMT in different groups showed that vaccinated-only individuals had lower titers for genotype G than infected-only and vaccinated-infected individuals. These results show a differential antibody response to vaccine vs. wild-type strains of MuV, which may explain the increased susceptibility of mumps in vaccinated young adults. However, there is no correlatio

Acinetobacter baumannii is a Gram-negative opportunistic pathogen that has a high incidence of infection among individuals with compromised immune systems. Currently, 80% of all hospital-acquired A. baumannii infections are multidrug-resistant to at least three different families of antibiotics, including last-line treatments. Therefore, there is an urgent need for effective therapeutic strategies which requires a thorough understanding of bacterial mechanisms of resistance. Infections caused by A. baumannii are difficult to eradicate, not only due to its extensive antibiotic resistance, but also due to its ability to adapt and persist against harsh environmental and host stressors; a prominent one being oxidative stress via the generation of reactive oxygen species (e.g., H2O2). A. baumannii is known to employ protective enzymes such as catalases to degrade hydrogen peroxide to water and oxygen. Mutants with single- and double-deletions of the catalase genes katG and katE, and their respective complements, were subjected to phenotypic assays and omics analysis to help us elucidate the overall contribution of this oxidative stress response in A. baumannii. The catalase mutants exhibited reduced fitness in rich medium, which was exacerbated when supplemented with H2O2. They also displayed differential antibiotic susceptibilities that were presumed to be mediated by the resistance-nodulation-division (RND) efflux pumps. Further assessment of overall efflux activity and expression profile of the RND efflux pump-encoding genes inferred alternative antibiotic resistance mechanism(s), most likely secondary to oxidative stress. Moreover, as H2O2 is generated by the innate immune cells as a pathogen containment and clearing strategy, we were prompted to examine bacterial virulence with a macrophage survivability assay. While the absence of catalases KatG and KatE sensitized A. baumannii to oxidative stress, their involvement in the virulence of this bacterium proved to be c

Acinetobacter spp. are a group of Gram-negative bacteria found in a variety of different environments such as hospitals, food, soil and water sources. In fact, investigation of Acinetobacter spp. has been declared a One Health crisis. highlighting that not just clinical, but also animal and environmental samples are important to our understanding and resolution of these infections. Many strains of the most notorious member, Acinetobacter baumannii are multi-drug resistant (MDR), and virulent, causing infections in humans including soft tissue and wound infections, ventilator-associated pneumonia and even bacteremia. Various antibiotic resistance genes (ARGs) are common in Acinetobacter spp. In Chapter 3, the diversity of Acinetobacter spp. is highlighted with the discovery of novel sequence types and therefore novel clonal complexes from non-clinical sources. We found that the most common ARGs are efflux pumps and the presence of ARGs did not necessarily determine antibiotic susceptibility. There was also no correlation between virulence and geographic origin or isolation source. In Chapter 4, we explore clinical MICs of colistin resistance observed in clinical isolates of Acinetobacter courvalinii and Acinetobacter pittii, and in non-clinical isolates of Acinetobacter seifertii. Again, with Chapter 3 as a basis, Chapter 5 describes the discovery of an ADC-5 cephalosporinase encoding gene from a tank milk isolate of A. baumannii, the first to be observed in a non-clinical isolate. Efflux pumps were the most commonly detected ARG and this was studied in more detail in Chapter 6. Determinants of substrate selectivity using molecular genetic techniques. Finally, because of the classification of A. baumannii as a One Health challenge. understanding the impact of a wide-spread environmental non-antibiotic compound, SA, and its effects on antibiotic susceptibility and virulence are explored in Chapter 7. Studying the impact of SA on ARGs and virulence genes (VGs) using a

With the widespread use of antiretroviral therapy, HIV drug resistance (HIVDR) rates are increasing globally. Many reported cases of HIV are found in areas without readily accessible testing centres capable of processing blood samples. Blood samples received from these areas can become degraded due to storage conditions during transportation to another facility or country capable of testing. Degraded samples present challenges for HIVDR testing, due to compromised viral RNA integrity interfering with RNA amplification and sequencing. Similarly, archival samples and dried blood spots (DBS) can also pose challenges. DBS samples are a highly accessible and cost-effective sample collection method used for HIVDR testing in resource-limited settings within Canada and across the world; however, they can be difficult to process through traditional methods due to susceptibility to degradation and low input volumes. To address this, we have developed a multiplex, amplicon-based, direct-to-sequencing HIVDR testing protocol compatible with degraded samples. This approach uses a hemi-nested approach with primer pools spanning the entire pol region of the HIV-1 genome. Protocol primers also incorporated Illumina P5/P7 adaptors, single index barcodes, and unique molecular identifiers (UMIs or Primer ID). This approach allow direct sequencing of amplicons, decreasing overall cost, mitigating PCR biases, and increasing clinical throughput. Here, we show the utility of this approach, successfully targeting the entire pol gene region of the HIV-1 subtype B genome in degraded and DBS samples. Preliminary work suggests the protocol may be adaptable for other major subtypes circulating in Canada as data shows the primer pools can amplify other major HIV-1 subtypes, albeit this may require further optimization. This protocol provides a potential option for NGS-based HIV drug-resistance testing from degraded samples and phylogenetic studies, while also building towards the ultimate goal of

The loss of effective therapeutic options due to the increase instances of antimicrobial resistant bacterial infections is of great concern to the World Health Organization (WHO). The WHO released a list to prioritize efforts in fighting this problem and placed carbapenem-resistant Acinetobacter baumannii as the top priority in need of development of new therapeutic options. A. baumannii is a Gram-negative opportunistic pathogen that can survive within clinical environments. A lot is known about the ability of this species to resist and tolerate the effects of antimicrobials. It possesses three efflux pumps of the resistance-nodulation-division (RND) efflux family, AdeAB(C), AdeFGH, and AdeIJK, that when overexpressed are responsible for reducing the susceptibility of A. baumannii to several antibiotic classes. These RND efflux pumps, and AdeFGH in particular, were the focus of my study. In this study I have investigated regulation and physiological function of AdeFGH, which is the least well studied RND efflux pump of the three. I have shown that AdeFGH expression has profound effects on the motility and biofilm formation of A. baumannii and that this is most likely due to increased efflux of acyl homoserine lactones. The effect of AdeFGH on motility and biofilm formation appears to be AdeL-independent. Interestingly, the overexpression of AdeL led to increased virulence in Galleria mellonella. I have discovered that the expression of adeFGH is induced by nitrosative stress and regulated by not only its cognate regulator, AdeL, but also by the global regulator, CsrA. I have investigated the role of CsrA in the physiology of A. baumannii via transcriptomics and phenotypic microarray. Additional investigations into AdeIJK showed it was crucial for A. baumannii virulence in G. mellonella and increased expression lead to increased tolerance to osmotic stress. All of this combined furthers our understanding of A. baumannii physiology and identifies several new potential

Members of the Ophiostomatales are of economic concern as many are blue-stain fungi including some plant pathogens. The taxonomy of these fungi has been challenging due to the convergent evolution of traits associated with insect dispersal. This project involved comparative sequence analysis of fungal mitochondrial genomes belonging to Ophiostomatales (phylum Ascomycota), which are also rich reservoirs of mobile genetic elements. The work is divided into two chapters. The first chapter is focused on three members of Leptographium sensu lato with inflated mitogenomes owing to abundant introns, including complex intron arrangements. Phylogeny based on mitogenome-derived protein sequences and nuclear markers helped in confirming the separation of Leptographium and Grosmannia species. The second chapter investigates the mitogenomes of nine strains of Ophiostoma ips with a varied global distribution which were sequenced and compared with other members of the Ophiostomatales. A mitogenome intron landscape demonstrated the distribution of the mobile genetic elements and provided insight into the evolutionary dynamics of introns among members of this group of fungi. Examples of complex or nested introns composed of two or three intron modules have been observed in some species, and RNA-Seq analysis suggests possible splicing pathways with regard to resolving complex introns in Ophiostoma ips. Mitochondrial DNA and RNA data for several members of the Ophiostomatales provide the basis for further studies relating to alternative splicing, evolutionary intron dynamics, and taxonomic studies.

Anaerobic digestion (AD) has shown the potential to reduce the abundance of antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs) in animal manures. It stands as a promising option to reduce the risk of the spread of antimicrobial resistance (AMR) due to livestock production and manure applications. However, the underlying mechanisms driving these changes still need to be fully understood. This multidisciplinary study aimed to utilize metagenomics to investigate the molecular and microbial mechanisms associated with the evolution of ARGs and MGEs during the anaerobic digestion (AD) of bovine dairy manure. The research focused on three main aspects: 1) examining the long-term effects of mesophilic (MAD) and thermophilic (TAD) anaerobic digestion on the entire set of ARGs (resistome) and MGEs (mobilome) in manure; 2) comparing the impact of alternative manure treatments, such as storage and solid-liquid separation, on resistomes and mobilomes; 3) identifying microbial groups potentially associated with ARGs and MGEs and microbial shifts potentially driving changes in resistomes and mobilomes. A meta-analysis conducted early in this research informed the primary focus of this research. Two anaerobic digesters operating at mesophilic (36 °C) and the other at thermophilic (55 °C) temperatures were set up, operated, monitored, and studied for 4 and 2 years, respectively. Metagenomics analyses were used to evaluate resistomes, mobilomes, and microbiomes in the mesophilic and thermophilic digesters operating under steady state and in the bovine manure used as substrate. The results indicated that MAD and TAD lowered ARG levels in fresh cattle manure by over 50% and MGEs by over 65%. Surprisingly, TAD did not outperform MAD at reducing ARGs and MGEs. Co-occurrence analysis indicated a strong association between microbial groups from the phyla Bacillota (e.g., Jeotgalicoccus, Streptococcus, Enterococcus), Actinomycetota (e.g., Brevibacterium, Rhodococcus)

Sexually transmitted blood-borne infections (STBBIs) are a group of infectious diseases that spread through sexual contact or blood-to-blood contact. STBBIs are of international concern due to their severe impact on quality of life and morbidity. Surveillance strategies are imperfect, in part due to the frequency of asymptomatic infection. Wastewater-based testing (WBT), the analysis of sewage influent for a pathogen, is one possible solution to supplement current clinical surveillance approaches. WBT has proven to be an effective strategy for monitoring the spread of other pathogens, including SARS-CoV-2 and its variants by allowing for the community-level detection of the infection markers and can act as an early indicator of transmission that precedes clinical indicators. In this work, detectability in wastewater was investigated for six pathogens: Neisseria gonorrhoeae, Chlamydia trachomatis, Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Human papillomavirus (HPV) types 16 and 18, and Human Immunodeficiency virus (HIV). Neither HIV nor HCV were detected in this study in any Canadian wastewater samples. N. gonorrhoeae, C. trachomatis, HBV, and HPV-16/18 were detected in the solids fraction of wastewater. The methodology was optimized by evaluating multiple extraction kits and input volumes, resulting in a final protocol utilizing 100 mL of wastewater influent extracted with the MagAttract PowerMicrobbiome DNA/RNA Kit. This protocol was employed to survey nine to eleven Canadian correctional facilities and one urban wastewater treatment plant over two six-week testing periods for N. gonorrhoeae, C. trachomatis, HPV-16/18, and HBV. Methods were also developed to evaluate circulating HPV genotypes using Illumina high-throughput sequencing technology. Using direct-to-sequencing primers conjugated to the GP5+/GP6+ primers, two urban wastewater treatment plants, two town sites, and three correctional facilities were surveyed. Ultimately, we detected 26 distinct HPV