Toxic benthic cyanobacteria (Microcoleus autumnalis): Genetic structure and ecological effects

<p>Toxic, benthic cyanobacterial proliferations occur in rivers globally and are increasing in both frequency and severity. More than 100 dog deaths have been reported in New Zealand since 2005, and dogs have died due to cyanotoxins from benthic cyanobacteria in California and France in recent years. In New Zealand, toxic, benthic proliferations are typically dominated by the genus Microcoleus, which produces the potent neurotoxins called anatoxins. Investigations of the drivers of Microcoleus proliferations have been carried out, revealing important relationships between growth, nutrients and flow. Despite increased attention over the past 15 years, significant knowledge gaps remain regarding regulation of anatoxin concentrations in mats and the ecological impacts of toxic Microcoleus-dominated proliferations in aquatic ecosystems. In this study, I aimed to address these crucial knowledge gaps. Concentrations of anatoxins in Microcoleus-dominated mats are highly variable, ranging from 0 μg g⁻¹ to over 2000 μg g⁻¹ dry weight. Like other cyanobacteria, both toxic and non-toxic strains coexist, and it is possible that the relative abundance of these strains is related to the observed variability in anatoxin concentrations. I developed a quantitative PCR assay specific to the Microcoleus anaC gene and combined this with a 16S rRNA quantitative PCR specific to cyanobacteria. This assay enabled the relative abundance of toxic and non-toxic strains to be quantified for the first time in environmental samples of Microcoleus-dominated mats. The quantitative PCR assay was applied to 122 environmental samples. Toxic cells made up 0% to 30.3% of the samples and significant differences were detectable among rivers, with wide variation evident both within and among rivers. Anatoxin contents in samples were significantly correlated with the proportion of toxic cells, suggesting that variation in anatoxin concentrations are related primarily to the dominance of toxic strains. A field study was conducted in California to identify cyanotoxin producers, investigate the utility of the primers from the quantitative PCR developed in the previous chapter in an international setting and determine the extent of spatial variability in anatoxin concentrations and quotas. Samples were collected from the Eel and Russian rivers in northern California and gene screening, followed by sequencing, was conducted to identify likely cyanotoxin producers. Cyanotoxin concentrations were measured using liquid chromatography tandem-mass spectrometry and the anatoxin quotas were determined using droplet digital PCR. Cyanotoxin producers were identified as Microcoleus producing anatoxins and Nodularia producing nodularin. Anatoxin concentrations were highly variable and differed significantly among sites; however, this variability did not extend to anatoxin quotas, indicating that the abundance of toxic cells also drives anatoxin content in this system. The anatoxin congener dihydroanatoxin-a comprised a significant proportion of the total anatoxins (38%–71%), indicating that this congener should be included in monitoring. Mats dominated by the green alga Cladophora glomerata contained both anatoxins and nodularin; thus, they may pose an exposure risk to cyanotoxins. Microcoleus-dominated mats grow in waters with moderate concentrations of dissolved inorganic nitrogen and low concentrations of dissolved reactive phosphorus. Acquisition of phosphorus from organic sources, in addition to within-mat nutrient cycling, may explain how Microcoleus can obtain high biomass in low nutrient environments. In chapter 4, I compare the alkaline phosphatase activity of four toxic and four non-toxic strains under four phosphorus regimes to identify the potential for Microcoleus to use organic phosphorus as a nutrient source. Toxic strains exhibited greater alkaline phosphatase activity than non-toxic strains; however, considerable variability was evident among strains with the same toxigenicity. Environmental mat samples were collected from sites on an upstream-downstream gradient and across a diel cycle, and alkaline phosphatase activity measured. There was a significant difference in alkaline phosphatase activity between the most upstream and downstream site. No diel changes in alkaline phosphatase activity were apparent. The presence of alkaline phosphatase activity suggests that organic phosphorus sources may be accessible to Microcoleus. To date, management of, and research on, toxic, benthic cyanobacterial proliferations has been focused largely on identifying drivers of growth and toxin production. As a result, there is a significant lack of information on the effects of Microcoleus-dominated proliferations on the wider ecosystem. In chapter 5, an ecotoxicological study was undertaken on Deleatidium spp. larvae using purified anatoxins to address this knowledge gap. Deleatidium spp. larvae were exposed to a range of doses of anatoxin-a, dihydroanatoxin-a, or a mixture of homoanatoxin-a and dihydrohomoanatoxin-a. No significant mortality was observed for any dose or toxin congener. Larvae exposed to high doses (300 μg L⁻¹ to 600 μg L⁻¹) of dihydroanatoxin-a had measurable concentrations of the toxin in their tissues 24-hours post-exposure. The lack of mortality observed, combined with detectable anatoxins in tissues post-exposure, is indicative of a potential pathway for anatoxin transfer or bioaccumulation, and warrants further investigation. Overall, this thesis has addressed a number of critical knowledge gaps in our understanding of Microcoleus. Continued effort is needed to broaden our understanding of the physiological and ecological role of anatoxins to improve risk assessments and ultimately inform better management of Microcoleus proliferations.</p>

Toxic benthic cyanobacteria (Microcoleus autumnalis): Genetic structure and ecological effects

<p>Toxic, benthic cyanobacterial proliferations occur in rivers globally and are increasing in both frequency and severity. More than 100 dog deaths have been reported in New Zealand since 2005, and dogs have died due to cyanotoxins from benthic cyanobacteria in California and France in recent years. In New Zealand, toxic, benthic proliferations are typically dominated by the genus Microcoleus, which produces the potent neurotoxins called anatoxins. Investigations of the drivers of Microcoleus proliferations have been carried out, revealing important relationships between growth, nutrients and flow. Despite increased attention over the past 15 years, significant knowledge gaps remain regarding regulation of anatoxin concentrations in mats and the ecological impacts of toxic Microcoleus-dominated proliferations in aquatic ecosystems. In this study, I aimed to address these crucial knowledge gaps. Concentrations of anatoxins in Microcoleus-dominated mats are highly variable, ranging from 0 μg g⁻¹ to over 2000 μg g⁻¹ dry weight. Like other cyanobacteria, both toxic and non-toxic strains coexist, and it is possible that the relative abundance of these strains is related to the observed variability in anatoxin concentrations. I developed a quantitative PCR assay specific to the Microcoleus anaC gene and combined this with a 16S rRNA quantitative PCR specific to cyanobacteria. This assay enabled the relative abundance of toxic and non-toxic strains to be quantified for the first time in environmental samples of Microcoleus-dominated mats. The quantitative PCR assay was applied to 122 environmental samples. Toxic cells made up 0% to 30.3% of the samples and significant differences were detectable among rivers, with wide variation evident both within and among rivers. Anatoxin contents in samples were significantly correlated with the proportion of toxic cells, suggesting that variation in anatoxin concentrations are related primarily to the dominance of toxic strains. A field study was conducted in California to identify cyanotoxin producers, investigate the utility of the primers from the quantitative PCR developed in the previous chapter in an international setting and determine the extent of spatial variability in anatoxin concentrations and quotas. Samples were collected from the Eel and Russian rivers in northern California and gene screening, followed by sequencing, was conducted to identify likely cyanotoxin producers. Cyanotoxin concentrations were measured using liquid chromatography tandem-mass spectrometry and the anatoxin quotas were determined using droplet digital PCR. Cyanotoxin producers were identified as Microcoleus producing anatoxins and Nodularia producing nodularin. Anatoxin concentrations were highly variable and differed significantly among sites; however, this variability did not extend to anatoxin quotas, indicating that the abundance of toxic cells also drives anatoxin content in this system. The anatoxin congener dihydroanatoxin-a comprised a significant proportion of the total anatoxins (38%–71%), indicating that this congener should be included in monitoring. Mats dominated by the green alga Cladophora glomerata contained both anatoxins and nodularin; thus, they may pose an exposure risk to cyanotoxins. Microcoleus-dominated mats grow in waters with moderate concentrations of dissolved inorganic nitrogen and low concentrations of dissolved reactive phosphorus. Acquisition of phosphorus from organic sources, in addition to within-mat nutrient cycling, may explain how Microcoleus can obtain high biomass in low nutrient environments. In chapter 4, I compare the alkaline phosphatase activity of four toxic and four non-toxic strains under four phosphorus regimes to identify the potential for Microcoleus to use organic phosphorus as a nutrient source. Toxic strains exhibited greater alkaline phosphatase activity than non-toxic strains; however, considerable variability was evident among strains with the same toxigenicity. Environmental mat samples were collected from sites on an upstream-downstream gradient and across a diel cycle, and alkaline phosphatase activity measured. There was a significant difference in alkaline phosphatase activity between the most upstream and downstream site. No diel changes in alkaline phosphatase activity were apparent. The presence of alkaline phosphatase activity suggests that organic phosphorus sources may be accessible to Microcoleus. To date, management of, and research on, toxic, benthic cyanobacterial proliferations has been focused largely on identifying drivers of growth and toxin production. As a result, there is a significant lack of information on the effects of Microcoleus-dominated proliferations on the wider ecosystem. In chapter 5, an ecotoxicological study was undertaken on Deleatidium spp. larvae using purified anatoxins to address this knowledge gap. Deleatidium spp. larvae were exposed to a range of doses of anatoxin-a, dihydroanatoxin-a, or a mixture of homoanatoxin-a and dihydrohomoanatoxin-a. No significant mortality was observed for any dose or toxin congener. Larvae exposed to high doses (300 μg L⁻¹ to 600 μg L⁻¹) of dihydroanatoxin-a had measurable concentrations of the toxin in their tissues 24-hours post-exposure. The lack of mortality observed, combined with detectable anatoxins in tissues post-exposure, is indicative of a potential pathway for anatoxin transfer or bioaccumulation, and warrants further investigation. Overall, this thesis has addressed a number of critical knowledge gaps in our understanding of Microcoleus. Continued effort is needed to broaden our understanding of the physiological and ecological role of anatoxins to improve risk assessments and ultimately inform better management of Microcoleus proliferations.</p>

Pan-Piscine Orthoreovirus (PRV) Detection Using Reverse Transcription Quantitative PCR

Piscine orthoreovirus (PRV) infects farmed and wild salmon and trout species in North America, South America, Europe, and East Asia. PRV groups into three distinct genotypes (PRV-1, PRV-2, and PRV-3) that can vary in distribution, host specificity, and/or disease potential. Detection of the virus is currently restricted to genotype specific assays such that surveillance programs require the use of three assays to ensure universal detection of PRV. Consequently, herein, we developed, optimized, and validated a real-time reverse transcription quantitative PCR assay (RT-qPCR) that can detect all known PRV genotypes with high sensitivity and specificity. Targeting a conserved region at the 5′ terminus of the M2 segment, the pan-PRV assay reliably detected all PRV genotypes with as few as five copies of RNA. The assay exclusively amplifies PRV and does not cross-react with other salmonid viruses or salmonid host genomes and can be performed as either a one- or two-step RT-qPCR. The assay is highly reproducible and robust, showing 100% agreement in test results from an inter-laboratory comparison between two laboratories in two countries. Overall, as the assay provides a single test to achieve highly sensitive pan-specific PRV detection, it is suitable for research, diagnostic, and surveillance purposes.

124. Establishment of a Post-Influenza Aspergillosis Model in Corticosteroid-Immunosuppressed Mice

Background Post-influenza aspergillosis (PIA) is a feared complication in patients with severe influenza, especially those receiving corticosteroids. However, validated murine models of PIA in a background of corticosteroid immunosuppression are lacking, compounding efforts to better characterize the immunopathology and treatment of this emerging entity. Methods 8-week-old female BALB/c mice were infected with ~5% of the lethal dose of a mouse-adapted influenza A/Hong Kong/1968 (H3N2) strain (flu), delivered by aerosolization, versus control (aerosolized saline). Mice then received two intraperitoneal injections of 10 mg cortisone acetate (CA) or mock injections on days 5 and 8 after flu infection. On day 9, mice were intranasally challenged with 50,000 A. fumigatus AF-293 conidia or mock-infected with saline. Survival was monitored until day 16 and infection severity was scored using the modified murine sepsis score (MSS, 0 = healthy to 3 = moribund). Pulmonary fungal burden was determined by an 18S quantitative PCR assay on day 16 or upon death. 15-16 mice per group were assessed across 3 independent experiments. Results Flu infection alone caused modest early morbidity, followed by full recovery of the mice until day 16. Treatment with CA after flu infection led to 12% mortality and increased morbidity that persisted until day 16 (median MSS = 0.8). Similarly, mice infected with AF after CA treatment had 12% mortality and a median MSS of 0.7. Combination of all 3 challenges (flu, CA, and AF) led to 40% mortality and severe morbidity in surviving mice (median MSS = 2.7). Likewise, prior flu infection of CA-treated, AF-infected mice increased the pulmonary fungal burden from 27k to 80k median conidial equivalents. In contrast, mice not receiving CA treatment showed consistently low morbidity (median day-16 MSS = 0.5) and minimal fungal burden after AF challenge, regardless of prior flu infection. Conclusion We have established a model of PIA in CA-immunosuppressed mice that underscores the detrimental effect of corticosteroid therapy on the outcomes of PIA. In the future, we will employ this model to study the impact of various pharmacological interventions on the natural history of PIA in the background of corticosteroid immunosuppression. Disclosures Dimitrios P. Kontoyiannis, MD, Astellas (Consultant)Cidara Therapeutics (Advisor or Review Panel member)Gilead Sciences (Consultant, Grant/Research Support, Other Financial or Material Support, Honoraria)

SPECIFIC DETECTION OF SARS-COV-2 VARIANTS B.1.1.7 (ALPHA) AND B.1.617.2 (DELTA) USING A ONE-STEP QUANTITATIVE PCR ASSAY

In this report, we describe the development of an RT-qPCR assay, termed Alpha Delta assay, which can detect SARS-COV-2 (SC-2) and distinguish between the Alpha (B.1.1.7) and Delta (B.1.617.2) variants. The Alpha- and Delta-specific reactions in the assay target mutations that are strongly linked to the target variant. The Alpha reaction targets the D3L substitution in N gene, and the Delta reaction targets the spike gene 156-158 mutations. Additionally, we describe a second Delta-specific assay that we use as a confirmatory test for the Alpha Delta assay that targets the 119-120 deletion in the Orf8 gene. Both reactions have similar sensitivities of 15-25 copies per reaction, similar to the sensitivity of commercial SC-2 detection tests. The Alpha Delta assay and the Orf8-119del assay were successfully used to classify clinical samples that were subsequently analyzed by whole genome sequencing. Lastly, the capability of the Alpha Delta assay and Orf8-119del assay to identify correctly the presence of Delta RNA in wastewater samples was demonstrated. This study provides a rapid, sensitive and cost-effective tool for detecting and classifying two worldwide dominant SC-2 variants. It also highlights the importance of a timely diagnostic response to the emergence of new SC-2 variants with significant consequences on global health.

RPS27A is a Poor Prognostic Predictor for Hepatocellular Carcinoma via Interacting with METTL3-Mediated RNA Modifications

Background: Increasing evidence has pointed to the involvement of RNA modifications in the pathogenesis of human cancers. However, they are rarely studied in hepatocellular carcinoma (HCC). Method: We summarized multiple types of RNA modification-related genes (RMRGs) from public references, and identified differentially expressed RMRGs (DEGs) between HCC tissues and matched normal samples, where their genetic variation were then investigated. The potential hub genes in the protein-protein interaction (PPI) network constructed by co-expression genes of RMRGs were recognized and verified in METTL3-knockdown HCC cell lines by quantitative PCR assay.Results: Seventy-six RMRGs, including six writers, seven readers, and seven erasers, were collected, of which 34 were identified and validated as DEGs. YTHDC2 exhibited the highest mutation rate, while ADAT2 showed widespread deletions. High correlations were observed between the expressions of 34 RMRGs. The PPI network constructed by 1080 co-expression DEGs related to RNA regulations consisted of 513 nodes and 11557 edges, with RPS27A presented the most directed edges and maximum closeness centrality. Patients with high expression of RPS27A showed worse overall survival (P < 0.01) and disease-free survival (P = 0.019). Moreover, RPS27A was found upregulated on high-risk metastatic and recurrent HCC tissues. Quantitative PCR assay indicated that RPS27A was significantly decreased in cancer cell lines when METTL3 was knocked down. Conclusions: Remarkable differences were observed for RNA modifications between HCC and normal samples, and RPS27A could be a poor prognostic predictor for HCC via interacting with METTL3-mediated RNA modifications.

Influence of Environmental Factors on Occurrence of Cyanobacteria and Abundance of Saxitoxin-Producing Cyanobacteria in a Subtropical Drinking Water Reservoir in Brazil

Blooms of cyanobacteria are frequent in Brazilian water reservoirs used for drinking water. The warning for the presence of potential toxin-producing cyanobacteria is typically based on time-consuming microscopy, rather than specific molecular detection of toxic genes in cyanobacteria. In this study, we developed a quantitative PCR assay for the detection of cyanobacteria producing the neurotoxin saxitoxin (STX). The assay targets the sxtA gene in the sxt gene cluster. Potential and dominant STX-producers in the Itupararanga reservoir were the genera Raphidiopsis, Aphanizomenon and Geitlerinema. Numbers of the sxtA gene varied from 6.76 × 103 to 7.33 × 105 cells mL−1 and correlated positively with SXT concentrations in the water. Concentrations of STX and the sxtA gene also correlated positively with TN:TP ratio and pH, but correlated negatively with inorganic nutrients and turbidity, confirming that regulation of the SXT production was impacted by environmental variables. In contrast, the occurrence of another cyanotoxin, microcystin, did not correlate with any environmental variables. The developed qPCR assay was found to be a rapid and robust approach for the specific quantification of potential STX-producing cyanobacteria and should be considered in future investigations on toxic cyanobacteria to provide an early warning of potential toxin episodes.