Prokaryotic microbial ecology as an ecosurveillance tool for eukaryotic pathogen colonisation: Meiothermus and Naegleria fowleri.

Naegleria fowleri has been detected in drinking water distribution systems (DWDS) in Australia, Pakistan and the United States and is the causative agent of the highly fatal disease primary amoebic meningoencephalitis. Previous small scale field studies have shown that Meiothermus may be a potential biomarker for N. fowleri. However, correlations between predictive biomarkers in small sample sizes often breakdown when applied to larger more representative datasets. This study represents one of the largest and most rigorous temporal investigations of Naegleria fowleri colonisation in an operational DWDS in the world and measured the association of Meiothermus and N. fowleri over a significantly larger space and time in the DWDS. A total of 232 samples were collected from five sites over three-years (2016-2018), which contained 29 positive N. fowleri samples. Two specific operational taxonomic units assigned to M. chliarophilus and M. hypogaeus, were significantly associated with N. fowleri presence. Furthermore, inoculation experiments demonstrated that Meiothermus was required to support N. fowleri growth in field-collected biofilms. This validates Meiothermus as prospective biological tool to aid in the identification and surveillance of N. fowleri colonisable sites.

Microbial source tracking of fecal pollution to coral reef lagoons of Norfolk Island, Australia.

Fecal pollution contributes to global degradation of water quality and requires identification of the source(s) for predicting human health risk, tracking disease, and developing management strategies. While fecal indicator bacteria are commonly used to detect fecal pollution, they cannot identify sources. Novel approaches, such as microbial source tracking (MST), can be applied to evaluate the origin of fecal pollution. This study examined fecal pollution in the coral reef lagoons of Norfolk Island, Australia where reef health decline has been related to nutrient input. The primary objective of this study was to evaluate the host sensitivity and specificity of two human wastewater-associated marker genes (Bacteroides HF183 (HF183) and cross-assembly phage (crAssphage)) and four animal feces associated marker genes targeting avian, ruminant, dog, and pig (Helicobacter-associated GFD (GFD), Bacteroides BacR (BacR), Bacteroides DogBact (DogBact), and Bacteroides Pig-2-Bac (Pig-2-Bac)) in wastewater and animal fecal samples collected from Norfolk Island. The prevalence and concentrations of these marker genes along with enterococci genetic marker (ENT 23S rRNA) of general fecal pollution and human adenovirus (HAdV), which is considered predominantly a pathogen but also a human-wastewater associated marker gene, were determined in surface, ground, and marine water resources. A secondary objective of this study was to assess the sources and pathways of fecal pollution to a sensitive marine environment under rainfall events. HF183, crAssphage, HAdV, and BacR demonstrated absolute host sensitivity values of 1.00, while GFD and Pig-2-Bac had host sensitivity values of 0.60, and 0.20, respectively. Host specificity values were > 0.94 for all marker genes. Human and animal (avian, ruminant, dog) fecal sources were present in the coral reef lagoons and surface water whereas groundwater was polluted by human wastewater markers. This study provides understanding of fecal pollution in water resources on Norfolk Island, Australia after precipitation events. The results may aid in effective water quality management, mitigating potential adverse effects on both human and environmental health.

Gut Microbial Perturbation and Host Response Induce Redox Pathway Upregulation along the Gut-Liver Axis during Giardiasis in C57BL/6J Mouse Model.

Apicomplexan infections, such as giardiasis and cryptosporidiosis, negatively impact a considerable proportion of human and commercial livestock populations. Despite this, the molecular mechanisms of disease, particularly the effect on the body beyond the gastrointestinal tract, are still poorly understood. To highlight host-parasite-microbiome biochemical interactions, we utilised integrated metabolomics-16S rRNA genomics and metabolomics-proteomics approaches in a C57BL/6J mouse model of giardiasis and compared these to Cryptosporidium and uropathogenic Escherichia coli (UPEC) infections. Comprehensive samples (faeces, blood, liver, and luminal contents from duodenum, jejunum, ileum, caecum and colon) were collected 10 days post infection and subjected to proteome and metabolome analysis by liquid and gas chromatography-mass spectrometry, respectively. Microbial populations in faeces and luminal washes were examined using 16S rRNA metagenomics. Proteome-metabolome analyses indicated that 12 and 16 key pathways were significantly altered in the gut and liver, respectively, during giardiasis with respect to other infections. Energy pathways including glycolysis and supporting pathways of glyoxylate and dicarboxylate metabolism, and the redox pathway of glutathione metabolism, were upregulated in small intestinal luminal contents and the liver during giardiasis. Metabolomics-16S rRNA genetics integration indicated that populations of three bacterial families-Autopobiaceae (Up), Desulfovibrionaceae (Up), and Akkermanasiaceae (Down)-were most significantly affected across the gut during giardiasis, causing upregulated glycolysis and short-chained fatty acid (SCFA) metabolism. In particular, the perturbed Akkermanasiaceae population seemed to cause oxidative stress responses along the gut-liver axis. Overall, the systems biology approach applied in this study highlighted that the effects of host-parasite-microbiome biochemical interactions extended beyond the gut ecosystem to the gut-liver axis. These findings form the first steps in a comprehensive comparison to ascertain the major molecular and biochemical contributors of host-parasite interactions and contribute towards the development of biomarker discovery and precision health solutions for apicomplexan infections.

Occurrence of multiple respiratory viruses in wastewater in Queensland, Australia: Potential for community disease surveillance.

The early warning and tracking of COVID-19 prevalence in the community provided by wastewater surveillance has highlighted its potential for much broader viral disease surveillance. In this proof-of-concept study, 46 wastewater samples from four wastewater treatment plants (WWTPs) in Queensland, Australia, were analyzed for the presence and abundance of 13 respiratory viruses, and the results were compared with reported clinical cases. The viruses were concentrated using the adsorption-extraction (AE) method, and extracted nucleic acids were analyzed using qPCR and RT-qPCR. Among the viruses tested, bocavirus (BoV), parechovirus (PeV), rhinovirus A (RhV A) and rhinovirus B (RhV B) were detected in all wastewater samples. All the tested viruses except influenza B virus (IBV) were detected in wastewater sample from at least one WWTP. BoV was detected with the greatest concentration (4.96-7.22 log10 GC/L), followed by Epstein-Barr virus (EBV) (4.08-6.46 log10 GC/L), RhV A (3.95-5.63 log10 GC/L), RhV B (3.74-5.61 log10 GC/L), and PeV (3.17-5.32 log10 GC/L). Influenza viruses and respiratory syncytial virus (RSV) are notifiable conditions in Queensland, allowing the gene copy (GC) concentrations to be compared with reported clinical cases. Significant correlations (ρ = 0.60, p < 0.01 for IAV and ρ = 0.53, p < 0.01 for RSV) were observed when pooled wastewater influenza A virus (IAV) and RSV log10 GC/L concentrations were compared to log10 clinical cases among the four WWTP catchments. The positive predictive value for the presence of IAV and RSV in wastewater was 97 % for both IAV and RSV clinical cases within the four WWTP catchments. The overall accuracy of wastewater analysis for predicting clinical cases of IAV and RSV was 97 and 90 %, respectively. This paper lends credibility to the application of wastewater surveillance to monitor respiratory viruses of various genomic characteristics, with potential uses for increased surveillance capabilities and as a tool in understanding the dynamics of disease circulation in the communities.

Comparative analysis of Adsorption-Extraction (AE) and Nanotrap® Magnetic Virus Particles (NMVP) workflows for the recovery of endogenous enveloped and non-enveloped viruses in wastewater.

In this study, two virus concentration methods, namely Adsorption-Extraction (AE) and Nanotrap® Magnetic Virus Particles (NMVP) along with commercially available extraction kits were used to quantify endogenous pepper mild mottle virus (PMMoV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in nucleic acid extracted from 48 wastewater samples collected over six events from eight wastewater treatment plants (WWTPs). The main aim was to determine which workflow (i.e., concentration and extraction methods) produces greater concentrations of endogenous PMMoV and SARS-CoV-2 gene copies (GC) in comparison with each other. Turbidity and total suspended solids (TSS) of wastewater samples within and among the eight WWTPs were highly variable (41-385 NTU and 77-668 mg/L TSS). In 58 % of individual wastewater samples, the log10 GC concentrations of PMMoV were greater by NMVP workflow compared to AE workflow. Paired measurements of PMMoV GC/10 mL from AE and NMVP across all 48 wastewater samples were weakly correlated (r = 0.455, p = 0.001) and demonstrated a poor linear relationship (r2 = 0.207). The log10 GC concentrations of SARS-CoV-2 in 69 % of individual samples were greater by AE workflow compared to NMVP workflow. In contrast to PMMoV, the AE and NMVP derived SARS-CoV-2 GC counts were strongly correlated (r = 0.859, p < 0.001) and demonstrated a strong linear relationship (r2 = 0.738). In general, the PMMoV GC achieved by the NMVP workflow decreased with increasing turbidity, but the PMMoV GC by the AE workflow did not appear to be as sensitive to either turbidity or TSS levels. These findings suggest that wastewater sample turbidity or suspended solids concentration, and the intended target for analysis should be considered when validating an optimal workflow for wastewater surveillance of viruses.

Comparison of RT-qPCR and RT-dPCR Platforms for the Trace Detection of SARS-CoV-2 RNA in Wastewater.

We compared reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and RT digital PCR (RT-dPCR) platforms for the trace detection of SARS-CoV-2 RNA in low-prevalence COVID-19 locations in Queensland, Australia, using CDC N1 and CDC N2 assays. The assay limit of detection (ALOD), PCR inhibition rates, and performance characteristics of each assay, along with the positivity rates with the RT-qPCR and RT-dPCR platforms, were evaluated by seeding known concentrations of exogenous SARS-CoV-2 in wastewater. The ALODs using RT-dPCR were approximately 2-5 times lower than those using RT-qPCR. During sample processing, the endogenous (n = 96) and exogenous (n = 24) SARS-CoV-2 wastewater samples were separated, and RNA was extracted from both wastewater eluates and pellets (solids). The RT-dPCR platform demonstrated a detection rate significantly greater than that of RT-qPCR for the CDC N1 and CDC N2 assays in the eluate (N1, p = 0.0029; N2, p = 0.0003) and pellet (N1, p = 0.0015; N2, p = 0.0067) samples. The positivity results also indicated that for the analysis of SARS-CoV-2 RNA in wastewater, including the eluate and pellet samples may further increase the detection sensitivity using RT-dPCR.

RT-qPCR and ATOPlex sequencing for the sensitive detection of SARS-CoV-2 RNA for wastewater surveillance.

During the coronavirus disease 2019 (COVID-19) pandemic, wastewater surveillance has become an important tool for monitoring the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within communities. In particular, reverse transcription-quantitative PCR (RT-qPCR) has been used to detect and quantify SARS-CoV-2 RNA in wastewater, while monitoring viral genome mutations requires separate approaches such as deep sequencing. A high throughput sequencing platform (ATOPlex) that uses a multiplex tiled PCR-based enrichment technique has shown promise in detecting variants of concern (VOC) while also providing virus quantitation data. However, detection sensitivities of both RT-qPCR and sequencing can be impacted through losses occurring during sample handling, virus concentration, nucleic acid extraction, and RT-qPCR. Therefore, process limit of detection (PLOD) assessments are required to estimate the gene copies of target molecule to attain specific probability of detection. In this study, we compare the PLOD of four RT-qPCR assays (US CDC N1 and N2, China CDC N and ORF1ab) for detection of SARS-CoV-2 to that of ATOPlex sequencing by seeding known concentrations of gamma-irradiated SARS-CoV-2 into wastewater. Results suggest that among the RT-qPCR assays, US CDC N1 was the most sensitive, especially at lower SARS-CoV-2 seed levels. However, when results from all RT-qPCR assays were combined, it resulted in greater detection rates than individual assays, suggesting that application of multiple assays is better suited for the trace detection of SARS-CoV-2 from wastewater samples. Furthermore, while ATOPlex offers a promising approach to SARS-CoV-2 wastewater surveillance, this approach appears to be less sensitive compared to RT-qPCR under the experimental conditions of this study, and may require further refinements. Nonetheless, the combination of RT-qPCR and ATOPlex may be a powerful tool to simultaneously detect/quantify SARS-CoV-2 RNA and monitor emerging VOC in wastewater samples.

Monitoring of SARS-CoV-2 in sewersheds with low COVID-19 cases using a passive sampling technique.

Monitoring SARS-CoV-2 RNA in sewer systems, upstream of a wastewater treatment plant, is an effective approach for understanding potential COVID-19 transmission in communities with higher spatial resolutions. Passive sampling devices provide a practical solution for frequent sampling within sewer networks where the use of autosamplers is not feasible. Currently, the design of upstream sampling is impeded by limited understanding of the fate of SARS-CoV-2 RNA in sewers and the sensitivity of passive samplers for the number of infected individuals in a catchment. In this study, passive samplers containing electronegative membranes were applied for at least 24-h continuous sampling in sewer systems. When monitoring SARS-CoV-2 along a trunk sewer pipe, we found RNA signals decreased proportionally to increasing dilutions, with non-detects occurring at the end of pipe. The passive sampling membranes were able to detect SARS-CoV-2 shed by >2 COVID-19 infection cases in 10,000 people. Moreover, upstream monitoring in multiple sewersheds using passive samplers identified the emergence of SARS-CoV-2 in wastewater one week ahead of clinical reporting and reflected the spatiotemporal spread of a COVID-19 cluster within a city. This study provides important information to guide the development of wastewater surveillance strategies at catchment and subcatchment levels using different sampling techniques.

Evaluation of process limit of detection and quantification variation of SARS-CoV-2 RT-qPCR and RT-dPCR assays for wastewater surveillance.

Effective wastewater surveillance of SARS-CoV-2 RNA requires the rigorous characterization of the limit of detection resulting from the entire sampling process - the process limit of detection (PLOD). Yet to date, no studies have gone beyond quantifying the assay limit of detection (ALOD) for RT-qPCR or RT-dPCR assays. While the ALOD is the lowest number of gene copies (GC) associated with a 95% probability of detection in a single PCR reaction, the PLOD represents the sensitivity of the method after considering the efficiency of all processing steps (e.g., sample handling, concentration, nucleic acid extraction, and PCR assays) to determine the number of GC in the wastewater sample matrix with a specific probability of detection. The primary objective of this study was to estimate the PLOD resulting from the combination of primary concentration and extraction with six SARS-CoV-2 assays: five RT-qPCR assays (US CDC N1 and N2, China CDC N and ORF1ab (CCDC N and CCDC ORF1ab), and E_Sarbeco RT-qPCR, and one RT-dPCR assay (US CDC N1 RT-dPCR) using two models (exponential survival and cumulative Gaussian). An adsorption extraction (AE) concentration method (i.e., virus adsorption on membrane and the RNA extraction from the membrane) was used to concentrate gamma-irradiated SARS-CoV-2 seeded into 36 wastewater samples. Overall, the US CDC N1 RT-dPCR and RT-qPCR assays had the lowest ALODs (< 10 GC/reaction) and PLODs (<3,954 GC/50 mL; 95% probability of detection) regardless of the seeding level and model used. Nevertheless, consistent amplification and detection rates decreased when seeding levels were < 2.32 × 103 GC/50 mL even for US CDC N1 RT-qPCR and RT-dPCR assays. Consequently, when SARS-CoV-2 RNA concentrations are expected to be low, it may be necessary to improve the positive detection rates of wastewater surveillance by analyzing additional field and RT-PCR replicates. To the best of our knowledge, this is the first study to assess the SARS-CoV-2 PLOD for wastewater and provides important insights on the analytical limitations for trace detection of SARS-CoV-2 RNA in wastewater.

Detection of the Omicron (B.1.1.529) variant of SARS-CoV-2 in aircraft wastewater.

On the 26th of November 2021, the World Health Organization (WHO) designated the newly detected B.1.1.529 lineage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) the Omicron Variant of Concern (VOC). The genome of the Omicron VOC contains more than 50 mutations, many of which have been associated with increased transmissibility, differing disease severity, and potential to evade immune responses developed for previous VOCs such as Alpha and Delta. In the days since the designation of B.1.1.529 as a VOC, infections with the lineage have been reported in countries around the globe and many countries have implemented travel restrictions and increased border controls in response. We putatively detected the Omicron variant in an aircraft wastewater sample from a flight arriving to Darwin, Australia from Johannesburg, South Africa on the 25th of November 2021 via positive results on the CDC N1, CDC N2, and del(69-70) RT-qPCR assays per guidance from the WHO. The Australian Northern Territory Health Department detected one passenger onboard the flight who was infected with SARS-CoV-2, which was determined to be the Omicron VOC by sequencing of a nasopharyngeal swab sample. Subsequent sequencing of the aircraft wastewater sample using the ARTIC V3 protocol with Nanopore and ATOPlex confirmed the presence of the Omicron variant with a consensus genome that clustered with the B.1.1.529 BA.1 sub-lineage. Our detection and confirmation of a single onboard Omicron infection via aircraft wastewater further bolsters the important role that aircraft wastewater can play as an independent and unintrusive surveillance point for infectious diseases, particularly coronavirus disease 2019.

Wastewater surveillance demonstrates high predictive value for COVID-19 infection on board repatriation flights to Australia.

Controlling importation and transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from overseas travelers is essential for countries, such as Australia, New Zealand, and other island nations, that have adopted a suppression strategy to manage very low community transmission. Wastewater surveillance of SARS-CoV-2 RNA has emerged as a promising tool employed in public health response in many countries globally. This study aimed to establish whether the surveillance of aircraft wastewater can be used to provide an additional layer of information to augment individual clinical testing. Wastewater from 37 long-haul flights chartered to repatriate Australians was tested for the presence of SARS-CoV-2 RNA. Children 5 years or older on these flights tested negative for coronavirus disease 19 (COVID-19) (deep nasal and oropharyngeal reverse-transcription (RT)-PCR swab) 48 h before departure. All passengers underwent mandatory quarantine for 14-day post arrival in Howard Springs, NT, Australia. Wastewater from 24 (64.9 %) of the 37 flights tested positive for SARS-CoV-2 RNA. During the 14 day mandatory quarantine, clinical testing identified 112 cases of COVID-19. Surveillance for SARS-CoV-2 RNA in repatriation flight wastewater using pooled results from three RT-qPCR assays demonstrated a positive predictive value (PPV) of 87.5 %, a negative predictive value (NPV) of 76.9 % and 83.7% accuracy for COVID-19 cases during the post-arrival 14-day quarantine period. The study successfully demonstrates that the surveillance of wastewater from aircraft for SARS-CoV-2 can provide an additional and effective tool for informing the management of returning overseas travelers and for monitoring the importation of SARS CoV-2 and other clinically significant pathogens.

Genomic insights into a population of introduced European rabbits Oryctolagus cuniculus in Australia and the development of genetic resistance to rabbit hemorrhagic disease virus.

The European rabbit (Oryctolagus cuniculus) is one of the most devastating invasive species in Australia. Since the 1950s, myxoma virus (MYXV) and rabbit haemorrhagic disease virus (RHDV) have been used to manage overabundant rabbit populations. Resistance to MYXV was observed within a few years of the release. More recently, resistance to lethal RHDV infection has also been reported, undermining the efficiency of landscape-scale rabbit control. Previous studies suggest that genetic resistance to lethal RHDV infection may differ locally between populations, yet the mechanisms of genetic resistance remain poorly understood. Here, we used genotyping by sequencing (GBS) data representing a reduced representation of the genome, to investigate Australian rabbit populations. Our aims were to understand the relationship between populations and identify possible genomic signatures of selection for RHDV resistance. One population we investigated had previously been reported to show levels of resistance to lethal RHDV infection. This population was compared to three other populations with lower or no previously reported RHDV resistance. We identified a set of novel candidate genes that could be involved in host-pathogen interactions such as virus binding and infection processes. These genes did not overlap with previous studies on RHDV resistance carried out in different rabbit populations, suggesting that multiple mechanisms are feasible. These findings provide useful insights into the different potential mechanisms of genetic resistance to RHDV virus which will inform future functional studies in this area.

Minimizing errors in RT-PCR detection and quantification of SARS-CoV-2 RNA for wastewater surveillance.

Wastewater surveillance for pathogens using reverse transcription-polymerase chain reaction (RT-PCR) is an effective and resource-efficient tool for gathering community-level public health information, including the incidence of coronavirus disease-19 (COVID-19). Surveillance of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in wastewater can potentially provide an early warning signal of COVID-19 infections in a community. The capacity of the world's environmental microbiology and virology laboratories for SARS-CoV-2 RNA characterization in wastewater is increasing rapidly. However, there are no standardized protocols or harmonized quality assurance and quality control (QA/QC) procedures for SARS-CoV-2 wastewater surveillance. This paper is a technical review of factors that can cause false-positive and false-negative errors in the surveillance of SARS-CoV-2 RNA in wastewater, culminating in recommended strategies that can be implemented to identify and mitigate some of these errors. Recommendations include stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, PCR inhibition assessment, inclusion of sample processing controls, and considerations for RT-PCR assay selection and data interpretation. Clear data interpretation guidelines (e.g., determination of positive and negative samples) are critical, particularly when the incidence of SARS-CoV-2 in wastewater is low. Corrective and confirmatory actions must be in place for inconclusive results or results diverging from current trends (e.g., initial onset or reemergence of COVID-19 in a community). It is also prudent to perform interlaboratory comparisons to ensure results' reliability and interpretability for prospective and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization and detection for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance continues to be demonstrated during this global crisis. In the future, wastewater should also play an important role in the surveillance of a range of other communicable diseases.

Efficacy of Intra-Operative Topical Wound Anaesthesia to Mitigate Piglet Castration Pain-A Large, Multi-Centred Field Trial.

Piglet castration results in acute pain and stress to the animal. There is a critical need for effective on-farm methods of pain mitigation. Local anaesthesia using Tri-Solfen® (Animal Ethics Pty Ltd., Melbourne, Australia), a topical local anaesthetic and antiseptic formulation instilled to the wound during surgery, is a newly evolving on-farm method to mitigate castration pain. To investigate the efficacy of Tri-Solfen®, instilled to the wound during the procedure, to alleviate subsequent castration-related pain in neonatal piglets, we performed a large, negatively controlled, randomised field trial in two commercial pig farms in Europe. Piglets (173) were enrolled and randomised to undergo castration with or without Tri-Solfen®, instilled to the wound immediately following skin incision. A 30 s wait period was then observed prior to completing castration. Efficacy was investigated by measuring pain-induced motor and vocal responses during the subsequent procedure and post-operative pain-related behaviour in treated versus untreated piglets. There was a significant reduction in nociceptive motor and vocal response during castration and in the post-operative pain-related behaviour response in Tri-Solfen®-treated compared to untreated piglets, in the first 30 min following castration. Although not addressing pain of skin incision, Tri-Solfen® is effective to mitigate subsequent acute castration-related pain in piglets under commercial production conditions.

Comparative analysis of rapid concentration methods for the recovery of SARS-CoV-2 and quantification of human enteric viruses and a sewage-associated marker gene in untreated wastewater.

To support public-health-related disease surveillance and monitoring, it is crucial to concentrate both enveloped and non-enveloped viruses from domestic wastewater. To date, most concentration methods were developed for non-enveloped viruses, and limited studies have directly compared the recovery efficiency of both types of viruses. In this study, the effectiveness of two different concentration methods (Concentrating pipette (CP) method and an adsorption-extraction (AE) method amended with MgCl2) were evaluated for untreated wastewater matrices using three different viruses (SARS-CoV-2 (seeded), human adenovirus 40/41 (HAdV 40/41), and enterovirus (EV)) and a wastewater-associated bacterial marker gene targeting Lachnospiraceae (Lachno3). For SARS-CoV-2, the estimated mean recovery efficiencies were significantly greater by as much as 5.46 times, using the CP method than the AE method amended with MgCl2. SARS-CoV-2 RNA recovery was greater for samples with higher titer seeds regardless of the method, and the estimated mean recovery efficiencies using the CP method were 25.1 ± 11% across ten WWTPs when wastewater samples were seeded with 5 × 104 gene copies (GC) of SARS-CoV-2. Meanwhile, the AE method yielded significantly greater concentrations of indigenous HAdV 40/41 and Lachno3 from wastewater compared to the CP method. Finally, no significant differences in indigenous EV concentrations were identified in comparing the AE and CP methods. These data indicate that the most effective concentration method varies by microbial analyte and that the priorities of the surveillance or monitoring program should be considered when choosing the concentration method.

Cryptosporidiosis Modulates the Gut Microbiome and Metabolism in a Murine Infection Model.

Cryptosporidiosis is a major human health concern globally. Despite well-established methods, misdiagnosis remains common. Our understanding of the cryptosporidiosis biochemical mechanism remains limited, compounding the difficulty of clinical diagnosis. Here, we used a systems biology approach to investigate the underlying biochemical interactions in C57BL/6J mice infected with Cryptosporidium parvum. Faecal samples were collected daily following infection. Blood, liver tissues and luminal contents were collected 10 days post infection. High-resolution liquid chromatography and low-resolution gas chromatography coupled with mass spectrometry were used to analyse the proteomes and metabolomes of these samples. Faeces and luminal contents were additionally subjected to 16S rRNA gene sequencing. Univariate and multivariate statistical analysis of the acquired data illustrated altered host and microbial energy pathways during infection. Glycolysis/citrate cycle metabolites were depleted, while short-chain fatty acids and D-amino acids accumulated. An increased abundance of bacteria associated with a stressed gut environment was seen. Host proteins involved in energy pathways and Lactobacillus glyceraldehyde-3-phosphate dehydrogenase were upregulated during cryptosporidiosis. Liver oxalate also increased during infection. Microbiome-parasite relationships were observed to be more influential than the host-parasite association in mediating major biochemical changes in the mouse gut during cryptosporidiosis. Defining this parasite-microbiome interaction is the first step towards building a comprehensive cryptosporidiosis model towards biomarker discovery, and rapid and accurate diagnostics.

Influence of Human Activities on Broad-Scale Estuarine-Marine Habitats Using Omics-Based Approaches Applied to Marine Sediments.

Rapid urban expansion and increased human activities have led to the progressive deterioration of many marine ecosystems. The diverse microbial communities that inhabit these ecosystems are believed to influence large-scale geochemical processes and, as such, analyzing their composition and functional metabolism can be a means to assessing an ecosystem's resilience to physical and chemical perturbations, or at the very least provide baseline information and insight into future research needs. Here we show the utilization of organic and inorganic contaminant screening coupled with metabolomics and bacterial 16S rRNA gene sequencing to assess the microbial community structure of marine sediments and their functional metabolic output. The sediments collected from Moreton Bay (Queensland, Australia) contained low levels of organic and inorganic contaminants, typically below guideline levels. The sequencing dataset suggest that sulfur and nitrite reduction, dehalogenation, ammonia oxidation, and xylan degradation were the major metabolic functions. The community metabolites suggest a level of functional homogeneity down the 40-cm core depth sampled, with sediment habitat identified as a significant driver for metabolic differences. The communities present in river and sandy channel samples were found to be the most active, with the river habitats likely to be dominated by photoheterotrophs that utilized carbohydrates, fatty acids and alcohols as well as reduce nitrates to release atmospheric nitrogen and oxidize sulfur. Bioturbated mud habitats showed overlapping faunal activity between riverine and sandy ecosystems. Nitrogen-fixing bacteria and lignin-degrading bacteria were most abundant in the sandy channel and bioturbated mud, respectively. The use of omics-based approaches provide greater insight into the functional metabolism of these impacted habitats, extending beyond discrete monitoring to encompassing whole community profiling that represents true phenotypical outputs. Ongoing omics-based monitoring that focuses on more targeted pathway analyses is recommended in order to quantify the flux changes within these systems and establish variations from these baseline measurements.

A genomic approach to identify and monitor a novel pyrethroid resistance mutation in the redlegged earth mite, Halotydeus destructor.

Resistance mechanisms are typically uncovered by identifying sequence variation in known candidate genes, however this strategy can be problematic for species with no reference data in known relatives. Here we take a genomic approach to identify resistance to pyrethroids in the redlegged earth mite, Halotydeus destructor, a member of the Penthalidae family of mites that are virtually uncharacterized genetically. Based on shallow genome sequencing followed by a genome assembly, we first identified contigs of the H. destructor parasodium channel gene. By linking variation in this gene to known resistant phenotypes, we located a single nucleotide polymorphism in resistant mites. This polymorphism results in a leucine (L) to phenylalanine (F) amino acid substitution in the II6 region (predicted) of the gene (L1024F). This novel mutation has not previously been linked to pyrethroid resistance, although other polymorphisms have been identified in the two-spotted spider mite, Tetranychus urticae at the same locus (L1024V). The sequencing approach was successful in generating a candidate polymorphism that was then validated using laboratory bioassays and field surveys. A high throughput Illumina-based sequencing diagnostic was developed to rapidly assess resistance allele frequencies in pools of mites sourced from hundreds of populations across Australia. Resistance was confirmed to be widespread in the southern wheatbelt region of Western Australia. Two different resistance mutations were identified in field populations, both resulting in the same amino acid substitution. The frequency and distribution of resistance amplicon haplotypes suggests at least two, and probably more independent origins of resistance.

Global transcriptomic profiling in barramundi (Lates calcarifer) from rivers impacted by differing agricultural land uses.

Most catchments discharging into the Great Barrier Reef lagoon have elevated loads of suspended sediment, nutrients, and pesticides, including photosystem II inhibiting herbicides, associated with upstream agricultural land use. To investigate potential impacts of declining water quality on fish physiology, RNA sequencing (RNASeq) was used to characterize and compare the hepatic transcriptomes of barramundi (Lates calcarifer) captured from 2 of these tropical river catchments in Queensland, Australia. The Daintree and Tully Rivers differ in upstream land uses, as well as sediment, nutrient, and pesticide loads, with the area of agricultural land use and contaminant loads lower in the Daintree. In fish collected from the Tully River, transcripts involved in fatty acid metabolism, amino acid metabolism, and citrate cycling were also more abundant, suggesting elevated circulating cortisol concentrations, whereas transcripts involved in immune responses were less abundant. Fish from the Tully also had an increased abundance of transcripts associated with xenobiotic metabolism. Previous laboratory-based studies observed similar patterns in fish and amphibians exposed to the agricultural herbicide atrazine. If these transcriptomic patterns are manifested at the whole organism level, the differences in water quality between the 2 rivers may alter fish growth and fitness. Environ Toxicol Chem 2017;36:103-112. © 2016 SETAC.

Altered levels of endocrine biomarkers in juvenile barramundi (Lates calcarifer; Bloch) following exposure to commercial herbicide and surfactant formulations.

Agricultural pesticides that are known endocrine disrupting chemicals have been detected in waters in the Great Barrier Reef catchment and lagoon. Altered transcription levels of liver vitellogenin (vtg) have been documented in wild populations of 2 Great Barrier Reef fisheries species and were strongly associated with pesticide-containing runoff from sugarcane plantations. The present study examined endocrine and physiological biomarkers in juvenile barramundi (Lates calcarifer) exposed to environmentally relevant concentrations of commercial herbicide (ATRADEX(®) WG Herbicide, DIUREX(®) WG Herbicide) and surfactant (ACTIVATOR(®) 90) formulations commonly used on sugarcane in the Great Barrier Reef catchment. Estrogenic biomarkers (namely, liver vtg messenger RNA and plasma 17ß-estradiol) increased following exposure to commercial mixtures but not to the analytical grade chemical, suggesting an estrogenic response to the additives. In contrast, brain aromatase (cyp19a1b) transcription levels, plasma testosterone and 11-ketotestosterone concentrations, and gill ventilation rates were not affected by any of the experimental exposures. These findings support the assertion that exposure to pesticide-containing runoff from sugarcane plantations is a potential causative agent of altered liver vtg transcription levels in wild barramundi. Whether exposure patterns in the Great Barrier Reef catchment and lagoon are sufficient to impair fish sexual and reproductive development and ultimately influence fish population dynamics remains to be determined. These findings highlight the need to consider both active and so-called inert ingredients in commercial pesticide formulations for environmental risk assessments.