Browsing by Author "Gilleard, John Stuart"

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    Cathelicidin contributes to prompt protective inflammation against Toxoplasma gondii
    (2019-07-10) Tan, Yi Lin; Cobo, Eduardo R.; Kastelic, John P.; Gilleard, John Stuart; Finney, Constance A. M.
    Toxoplasma gondii is an intracellular parasite infecting all warm-blooded animals, including humans. Although infected immunocompetent individuals are usually asymptomatic, in immunocompromised individuals, T. gondii can affect the central nervous system and may cause congenital toxoplasmosis and death. Cathelicidins are short cationic peptides secreted by leukocytes and epithelial cells with antimicrobial and immunomodulatory activities. It has been proposed that cathelicidin might be a key defense against intracellular pathogens. For instance, human cathelicidin (LL-37), either endogenous or synthetic exogenous, reduced survival of intracellular Mycobacterium tuberculosis in macrophages. However, the role of cathelicidin in toxoplasmosis has been barely investigated. The objective was to elucidate the contributions of cathelicidin during acute systemic and long-term infection with T. gondii. In an acute generalized model of toxoplasmosis, C57BL/6 cathelicidin-deficient (Camp-/-) and wild type (Camp+/+) mice were challenged with luciferase-green fluorescence protein tagged T. gondii (high virulence Type 1 RH, 1 x 105, intra-peritoneal). Although all mice had succumbed by day 5 post infection, Camp-/- mice failed to initiate pro-inflammatory responses in vital organs (ileum, colon, liver, spleen and brain; p<0.05) at early infection (1 d). Consequently, more parasite load was detected in those vital organs (p<0.05). In long-term toxoplasmosis, we determined that Camp-/- mice were more susceptible to oral challenge with T. gondii cysts (low-virulence Type 2 ME 49); all Camp-/- infected mice died by day 12 post infection (p<0.05), whereas Camp+/+ infected and PBS treated mice survived throughout the 14 d study. These Camp-/- mice had more severe hepatitis, with evident liver necrosis and increased inflammatory cytokines, Ifn-γ and Tnf-α gene expression, than Camp+/+ mice (p<0.05). In Camp-/- infected mice, there was pronounced inflammation and Ifn-γ gene synthesis in their spleen (p<0.05) and in their ileum, more severe epithelial disruption, with fewer goblet cells. In in vitro studies, human macrophages (THP-1) infected by T. gondii (ME 49) expressed elevated endogenous gene transcriptional cathelicidin (p<0.05) together with induced gene transcriptional expression of IFN-γ and TNF-α (p<0.05). Camp-/- bone marrow derived macrophages (BMMs) challenged with T. gondii (RH or ME 49) secreted more Tnf-α (p<0.05) compared to infected Camp+/+ BMMs. The T. gondii burdens (both RH and ME 49 strains) were similar between Camp+/+ and Camp-/- BMMs. However, when THP-1 cells macrophages were pre-stimulated with recombinant human LL-37 cathelicidin (2 uM), T. gondii burden was reduced (p<0.05). In summary, this study identified the critical role of cathelicidin in initiating host immune responses, promptly during toxoplasmosis. In the absence of cathelicidin, inflammatory responses in vital organs (liver and spleen) were exaggerated at later infection (3 and 14 d) and they were detrimental to the host. Immunomodulatory roles of cathelicidin were manifested in infected macrophages, where the peptide supressed exaggerated inflammatory responses and aided macrophage killing capabilities. Cathelicidin secreted by macrophages during T. gondii infection has a vital role in host-pathogen balance, controlling parasite burden and preventing overwhelming inflammatory responses.
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    Investigating the genetic basis of ivermectin resistance in Haemonchus contortus
    (2018-04-30) Rezansoff, Andrew Mischa; Gilleard, John Stuart; Hansen, David; Rogers, Sean M.; Mains, Paul E.; Wasmuth, James D.; Geary, Tim C.
    Parasitic nematodes have a major impact on human and animal health and their control is threatened by the emergence of resistance to the anthelmintic drugs on which control depends. Although resistance to ivermectin is widespread in many livestock parasites, the genetic mechanisms remain elusive. In this thesis, a number of approaches are undertaken to investigate the genetics of ivermectin resistance in Haemonchus contortus, an important parasitic nematode research model. A transcriptomic comparison of the susceptible MHco3(ISE) and two ivermectin resistant strains, MHco4(WRS) and MHco10(CAVR), was undertaken to investigate if differences in gene expression revealed insights into ivermectin resistance mechanisms. Although this approach did not reveal clear ivermectin resistance gene candidates, the results have some important implications for RNAseq analysis in genetically diverse organisms. First, extremely high levels of genetic diversity in H. contortus had a major impact on RNAseq analysis and needed to be accounted for reliable identification of differentially expressed genes. Second, there were a remarkably large number of genes differentially expressed between the three H. contortus strains. In a novel genetic crossing approach, two independent serially backcrossed H. contortus populations were utilized in which ivermectin resistance loci had been introgressed into the MHco3(ISE) genome. Markers for six leading candidate ivermectin resistance genes from the literature were examined and none were found to show evidence of introgression in the backcross populations. A seventh marker, Hcms8a20 showed clear evidence of introgression and was later confirmed by collaborators using a whole genome sequencing approach to show its location in a 11.2 Mb introgressed region. A set of 25 in vivo or in vitro phenotyped ivermectin resistant H. contortus field populations were then used to investigate evidence of selection on the Hcms8a20 marker and four other loci distributed across the 11.2 Mb introgressed region. A deep amplicon sequencing approach revealed that, of the loci tested, only Hcms8a20 showed consistent evidence of purifying selection in the ivermectin resistant field populations. Overall, the results provide strong evidence that a major ivermectin resistance locus is located within a few megabases of the Hcms8a20 locus in many different ivermectin resistant H. contortus field populations.
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    Novel split trehalase-based biosensors for the detection of biomarkers of infectious diseases
    (2019-05-15) Drikic, Marija; De Buck, Jeroen M.; Gilleard, John Stuart; Dong, Tao G.; Turner, Raymond Joseph; Savchenko, Alexei; Campbell, Robert D.
    Infectious diseases remain a serious public health challenge in the field of human and veterinary medicine. For instance, infectious diseases were among the leading causes of mortality worldwide in 2016 according to the WHO. In veterinary medicine, infectious diseases have a significant negative impact on animal productivity and welfare leading to decreased profits and increased health-associated costs. Furthermore, the WHO estimates that 61% of diseases affecting the human population are zoonotic. Therefore, programs to control and eradicate these diseases are of crucial importance. Correct diagnosis, supported by robust diagnostic tests, is an essential first step in achieving these goals. An ideal diagnostic test needs to be accurate and robust while being easily accessible and widely-available. Although different studies over the years have aimed to develop such a diagnostic test, there have not been many that have managed to enter into clinical practice. The most successful class of biosensors currently on the market is the glucometer used to monitor blood glucose. Glucometer's sensitivity and specificity, as well as its performance in clinical samples, have been mainly perfected. Measuring devices have been miniaturized and their production costs optimized. In this thesis, we developed and characterized a novel biosensor able to detect antibodies and other infectious disease markers. The biosensor is based on the protein complementation principle and uses an E. coli glycolytic enzyme, trehalase (TreA), as a reporter. Trehalase converts trehalose to glucose, which can then be detected by a glucometer. TreA was split into two non-functional fragments, and each fragment was fused to the sensing element that was specific for the targeted analyte. In the presence of the analyte, the sensing elements bind to it and induce the dimerization of the TreA which then becomes active. In the second chapter, we demonstrated that conditional complementation of the trehalase fragments leading to trehalose hydrolysis and glucose production could be used to detect antibodies, bacterial cells, small molecule, protein-protein interaction, and protein aggregation. We also demonstrated the retention of activity of split TreA in undiluted clinical samples like blood or milk. In the third chapter, we attempted to increase the sensitivity and shorten the time for signal detection of the TreA biosensor by introducing split inteins. We placed the inteins within the previously developed biosensor, but we were not successful in achieving analyte-mediated dimerization of the two biosensor components. In the fourth chapter, we applied this TreA biosensor to develop and validate a new diagnostic tool for the quantification of the total amount of immunoglobulins in bovine colostrum and serum (named STIGA). We demonstrated that STIGA performs more efficiently in quantifying total immunoglobulins in colostrum and calf serum than other diagnostic tools (Colostrometer and Brix) that are used on a farm. Therefore, it is a suitable detection assay to establish colostrum quality and calf immune status in the field. We also proposed a modified format of STIGA for this on-farm application and demonstrated that its performance remained high. In the fifth chapter, we explored the applicability of a modified STIGA with the anticipation to detect immunoglobulins in different animal species. We analyzed 29 animal species and proved that the same detection protocol could be applied with success to the majority of animal species. In conclusion, we engineered a trehalase-based biosensor that requires minimal sample preparation and can be integrated with existing glucometers or sensors, which offers a versatile and convenient method for point-of-care applications.