Sugar-rich larval diet promotes lower adult pathogen load and higher survival after infection in a polyphagous fly

Nutrition is a central factor influencing immunity and resistance to infection, but the extent to which nutrition during development affects adult responses to infections is poorly understood. Our study investigated how the nutritional composition of the larval diet affects the survival, pathogen load, and food intake of adult fruit flies, Bactrocera tryoni, after bacterial septic infection. We found a sex-specific effect of larval diet composition on survival post-infection: survival rate was higher and bacterial load was lower for infected females fed sugar-rich larval diet compared with females fed protein-rich larval diet, an effect that was absent in males. Both males and females were heavier when fed a balanced larval diet compared to protein- or sugar-rich diet, while body lipid reserves were higher in the sugar-rich larval diet compared with other diets. Body protein reserve was lower for sugar-rich larval diets compared to other diets in males, but not females. Both females and males shifted their nutrient intake to ingest a sugar-rich diet when infected compared with sham-infected flies without any effect of the larval diet, suggesting that sugar-rich diets can be beneficial to fight off bacterial infection. Overall, our findings show that nutrition during early life can shape individual fitness in adulthood.

The Fate of Foodborne Pathogens in Manure Treated Soil

The aim of this review was to provide an update on the complex relationship between manure application, altered pathogen levels and antibiotic resistance. This is necessary to protect health and improve the sustainability of this major farming practice in agricultural systems based on high levels of manure production. It is important to consider soil health in relation to environment and land management practices in the context of the soil microflora and the introduction of pathogens on the health of the soil microbiome. Viable pathogens in manure spread on agricultural land may be distributed by leaching, surface run-off, water source contamination and contaminated crop removal. Thus it is important to understand how multiple pathogens can persist in manures and on soil at farm-scale and how crops produced under these conditions could be a potential transfer route for zoonotic pathogens. The management of pathogen load within livestock manure is a potential mechanism for the reduction and prevention of outbreaks infection with Escherichia coli, Listeria Salmonella, and Campylobacter. The ability of Campylobacter, E. coli, Listeria and Salmonella to combat environmental stress coupled with their survival on food crops and vegetables post-harvest emphasizes the need for further study of these pathogens along with the emerging pathogen Providencia given its link to disease in the immunocompromised and its’ high levels of antibiotic resistance. The management of pathogen load within livestock manure has been widely recognized as a potential mechanism for the reduction and prevention of outbreaks infection but any studies undertaken should be considered as region specific due to the variable nature of the factors influencing pathogen content and survival in manures and soil. Mediocre soils that require nutrients could be one template for research on manure inputs and their influence on soil health and on pathogen survival on grassland and in food crops.

Detection of blueberry stunt phytoplasma in Eastern Canada using cpn60-based molecular diagnostic assays

Blueberry stunt phytoplasma (BBSP; ‘Candidatus Phytoplasma asteris’) is an insect-vectored plant pathogen that causes severe yield losses in blueberry (Vaccinium corymbosum), which is the most valuable fruit crop in Canada. Rapid, field-based diagnostic assays are desirable tools for the control of BBSP, as part of an integrated, proactive approach to production management termed biovigilance. We designed and validated a chaperonin-60 (cpn60)-targeted LAMP assay for detection of BBSP, providing a rapid, low cost, field-deployable diagnostic option. Our validation demonstrates that the assay is reproducible, with high analytical specificity and improved sensitivity when compared with 16S rRNA nested PCR. We applied the validated LAMP assay to nearly 2000 blueberry samples from Québec and Nova Scotia over three growing seasons (2016–2018). Our surveys revealed that BBSP is present in most sites across both provinces, though detection of the pathogen in individual plants varied in different tissues across sampling dates and across years, and evidence of spread between plants was limited. To quantify pathogen load in select plants, we designed additional qPCR and ddPCR assays, also based on cpn60. We found that pathogen load fluctuates in individual plants, both within and between growing seasons. Finally, we designed an interactive map to visualize the results of our surveys. These results provide a validated diagnostic assay that can be used as part of a biovigilance strategy for detecting and controlling infections caused by BBSP.

Hematopoietic Stem and Progenitor Cells Improve Survival from Sepsis By Boosting Immunomodulatory Cells

Sepsis is a dysregulated inflammatory syndrome that accounts for as many as 20% of deaths worldwide. Elevated production of pro-inflammatory cytokines during sepsis, such as IL-1, IL-6, interferons (IFNs), and tumor necrosis factor contribute to the development of fever, vasodilation, and multiorgan failure. Novel therapies to treat sepsis are urgently needed. Hematopoietic stem and progenitor cells (HSPC) are responsible for the day-to-day production of blood and immune cells. Recent work from our group and others indicates that during emergency hematopoiesis, inflammatory signals including cytokines, chemokines, and pathogen-derived molecules direct HSPCs to differentiate into effector immune cells. While these signals are essential for a proper immune response, excessive signaling in HSPCs can be detrimental and lead to their depletion. Thus, the interactions between HSPCs and their inflammatory environment may play a deterministic role in immune responses and sepsis. We used a mouse model of Group A Streptococcus (GAS) infection to examine the role of HSPCs in pathogenic infection and sepsis. GAS is a common pathogen that can cause a plethora of diseases from mild skin infections to life-threatening necrotizing fasciitis. We infected mice with 10 6 cfu GAS by intramuscular injection, which typically results in sepsis and death within 7 days, and examined the impact of this infection on peripheral blood (PB) and bone marrow (BM) populations. In just 24 hrs after GAS infection, BM myeloid and HSPC populations are significantly depleted, with myeloid cells being heavily trafficked into circulation following increased levels of monocyte chemoattractant protein-1 (MCP-1). Lineage tracing experiments using KRT18-CreERT2:Rosa26-lox-STOP-lox-TdTomato demonstrated that endogenous HSPCs differentiate toward the myeloid lineage after GAS infection. Based on these data, we hypothesized that the inflammatory environment of GAS infection drives rapid HSPC differentiation resulting in a depletion that could be rescued by the infusion of new HSPCs. To test this hypothesis, we infused GAS-infected mice with 10 4 naïve HSPCs (1.7x10 7 cells per m 2) and evaluated pathogen load and overall survival. This number of HSPCs infused is very low in comparison to the current granulocyte therapies that use ~10 10 cells per m 2 cells per infusion. BM and PB analysis showed that HSPC infusion restored HSPC levels and significantly increased myeloid progenitors and circulating myeloid cells. Strikingly, HSPC infusion in GAS-infected mice significantly increased survival, with 50-75% of mice surviving as opposed to 0-10% of controls. Despite the restoration of hematopoietic populations, surprisingly, GAS-infected mice infused with HSPCs did not show a reduction in pathogen load. Given that HSPC infusion significantly increased survival without impacting pathogen clearance, we sought to determine whether infused HSPCs served an immunomodulatory role. Analysis of BM and PB did not show any changes in lymphocyte populations, suggesting that Tregs and Bregs were not strongly affected. However, BM and PB MDSC populations were severely depleted during GAS sepsis, and HSPC infusion led to a dramatic restoration of these MDSC populations. Interestingly and in accordance with MDSC numbers, the overall cytokine levels of GAS-infected mice are lower after HSPC infusion. Notably, serum levels of cytokines known to drive the symptoms of sepsis, like TNF, IL-12, MIP-1a, IL-6, and IL-1b were dampened in HSPC-rescued mice. In conclusion, while HSPC infusion did not reduce bacterial load, it conferred a significant survival advantage to GAS-infected mice. Our data showing restoration of MDSCs and lower cytokine levels after HSPC infusion suggest that HSPC infusion supports the development of immunomodulatory cells that can prevent sepsis-related hyperinflammation and death. Current work is directed at defining specific HSPC subpopulations that mediate this effect. Importantly, the rescue potential of such low numbers of infused HSPCs highlights the feasibility of this technique and its potential applications. Overall, the information gained in this project may contribute to a new therapeutic strategy to use HSPCs to fight bacterial infections and sepsis where granulocyte infusions have so far produced only mixed results. Disclosures No relevant conflicts of interest to declare.

A novel wastewater-based epidemiology indexing method predicts SARS-CoV-2 disease prevalence across treatment facilities in metropolitan and regional populations

There is a need for wastewater based epidemiological (WBE) methods that integrate multiple, variously sized surveillance sites across geographic areas. We developed a novel indexing method, Melvin’s Index, that provides a normalized and standardized metric of wastewater pathogen load for qPCR assays that is resilient to surveillance site variation. To demonstrate the utility of Melvin’s Index, we used qRT-PCR to measure SARS-CoV-2 genomic RNA levels in influent wastewater from 19 municipal wastewater treatment facilities (WWTF’s) of varying sizes and served populations across the state of Minnesota during the Summer of 2020. SARS-CoV-2 RNA was detected at each WWTF during the 20-week sampling period at a mean concentration of 8.5 × 104 genome copies/L (range 3.2 × 102–1.2 × 109 genome copies/L). Lag analysis of trends in Melvin’s Index values and clinical COVID-19 cases showed that increases in indexed wastewater SARS-CoV-2 levels precede new clinical cases by 15–17 days at the statewide level and by up to 25 days at the regional/county level. Melvin’s Index is a reliable WBE method and can be applied to both WWTFs that serve a wide range of population sizes and to large regions that are served by multiple WWTFs.

Enhancers of host immune tolerance to bacterial infection discovered using linked computational and experimental approaches

Current therapeutic strategies against bacterial infections focus on reduction of pathogen load through antibiotics; however, stimulation of host tolerance to infection might offer an alternative approach. Here we used computational transcriptomics and a Xenopus embryo infection model to rapidly discover infection response pathways, identify potential tolerance inducer drugs, and validate their ability to induce broad tolerance. Xenopus embryos exhibit natural tolerance to A. baumanii, K. pneumoniae, S. aureus, and S. pneumoniae bacteria, whereas A. hydrophila and P. aeruginosa produce infection that leads to death. Transcriptional profiling led to definition of a 20-gene signature that allows for discrimination between tolerant and susceptible states, as well as identification of active and passive tolerance responses based on the degree of engagement of gene transcription modulation. Upregulation of metal ion transport and hypoxia pathways reminiscent of responses observed in primate and mouse infection models were identified as tolerance mediators, and drug screening in the susceptible A. hydrophila infection model confirmed that a metal chelator (deferoxamine) and HIF-1α agonist (1,4-DPCA) increase embryo survival despite high pathogen load. These data demonstrate the value of combining the Xenopus embryo infection model with multi-omics analyses for mechanistic discovery and drug repurposing to induce host tolerance to bacterial infection.

Landscape and tree community effects on the infestation of an endemic fungal pathogen in riparian forests were mediated by leaf traits

Background Plant pathogens are regarded as crucial agents shaping the dynamics of natural forest communities. Marssonina leaf spot of poplar is induced by an endemic pathogenic fungus Drepanopeziza populi, causing increased damage to riparian poplar stands in recent years. However, such endemic fungal diseases have received little attention at the landscape scale, despite the key role of landscape heterogeneity in the development and spread of emerging forest diseases. Moreover, most studies have insufficiently captured multiple ecological factors driving the infestation of an endemic pathogen acting at the landscape, community, and individual scales. Methods We measured pathogen load, disease prevalence, and disease severity of Marssonina leaf spot in poplars in riparian forests. We explored the direct and indirect effects of multiple ecological factors on pathogen infestation using a path analysis. Specifically, we first assessed the effects of landscape and community factors on leaf traits including leaf area, specific leaf area (SLA) and leaf dry matter content (LDMC), and then examined the role of these factors in shaping disease dynamics. Results Path analysis showed that landscape features had no direct impact on leaf traits and pathogen infestation, but directly affected tree community composition. Landscapes with higher forest cover resulted in higher host density and tree diversity. Host density was the most important factor of pathogen load, with higher host density resulting in more symptomatic leaves. Tree diversity had direct effects on disease prevalence, with poplars growing in mixed forest stands far less affected by pathogens than in pure stands. Moreover, disease prevalence was positively related to pathogen load. Tree diversity strongly reduced SLA, but increased LDMC. Higher SLA was found to increase pathogen load and disease severity, but higher LDMC was found to reduce both of them. Conclusions Our results show that the effects of landscape and tree community on Marssonina leaf spot disease are mediated by leaf traits. Disentangling the effects of biotic and abiotic factors affecting pathogen infestation contributes to reduce the overall impact of this disease, which can provide policy makers with sustainable management of endemic plant diseases in natural forests.

Far-UVC efficiently inactivates an airborne pathogen in a room-sized chamber

Many infectious diseases, including COVID-19, are transmitted by airborne pathogens. There is a need for effective environmental control measures which, ideally, are not reliant on human behaviour. One potential solution is Far-UVC which can efficiently inactivate pathogens, such as coronaviruses and influenza, in air. When appropriately filtered, and because of its limited penetration, there is evidence that Far-UVC does not induce acute reactions in the skin or eyes, nor delayed effects such as skin cancer. While there is laboratory evidence for far-UVC efficacy, there is limited evidence in full-sized rooms. In the first study of its type, we show that Far-UVC deployed in a room-sized chamber effectively inactivates aerosolised Staphylococcus aureus. At a room ventilation rate of 3 air changes per hour (ACH), with 5 filtered sources the steady-state pathogen load was reduced by 92.1% providing an additional 35 equivalent air changes (eACH). This reduction was achieved using Far-UVC intensities consistent with current regulatory limits. Far-UVC is likely to be more effective against common airborne viruses, including SARS-CoV-2, and should thus be an effective and “hands-off” technology to reduce airborne disease transmission. The findings provide room-scale data to support the design and development of safe and effective Far-UVC systems.

Mosquitoes (Diptera: Culicidae) in the Dark—Highlighting the Importance of Genetically Identifying Mosquito Populations in Subterranean Environments of Central Europe

The common house mosquito, Culex pipiens s. l. is part of the morphologically hardly or non-distinguishable Culex pipiens complex. Upcoming molecular methods allowed us to identify members of mosquito populations that are characterized by differences in behavior, physiology, host and habitat preferences and thereof resulting in varying pathogen load and vector potential to deal with. In the last years, urban and surrounding periurban areas were of special interest due to the higher transmission risk of pathogens of medical and veterinary importance. Recently, surveys of underground habitats were performed to fully evaluate the spatial distribution of rare members of the Cx. pipiens complex in Europe. Subterranean environments and their contribution to mosquito-borne pathogen transmission are virtually unknown. Herein, we review the underground community structures of this species complex in Europe, add new data to Germany and provide the first reports of the Cx. pipiens complex and usually rarely found mosquito taxa in underground areas of Luxembourg. Furthermore, we report the first finding of Culiseta glaphyroptera in Luxembourg. Our results highlight the need for molecular specimen identifications to correctly and most comprehensively characterize subterranean mosquito community structures.

Longitudinal monitoring of individual infection progression in Drosophila melanogaster

The innate immune system is critical for host survival of infection. Infection models in organisms like Drosophila melanogaster are key for understanding evolution and dynamics of innate immunity. However, current toolsets for fly infection studies are limited in their ability to resolve changes in pathogen load on the hours time-scale, along with stochastic responses to infection in individuals. Here we report a novel bioluminescent imaging strategy enabling non-invasive characterization of pathogen load over time. We demonstrate that photon flux from autobioluminescent reporter bacteria can be used to estimate pathogen count. Escherichia coli expressing the ilux operon were imaged in whole, living flies at relevant concentrations for immune study. Because animal sacrifice was not necessary to estimate pathogen load, stochastic responses to infection were characterized in individuals for the first time. The high temporal resolution of bioluminescence imaging also enabled visualization of the fine dynamics of microbial clearance on the hours time-scale. Overall, this non-invasive imaging strategy provides a simple and scalable platform to observe changes in pathogen load in vivo over time.