Profiling Multiomic Biomarkers using Particle Detection Counters and Spectral-FLIM MicroscopyAbstract
Profiling multiomic biomarkers in bulk and in situ provides critical information which
enables basic research and clinical applications. Unfortunately, most existing profiling methods
are limited due to either low multiplexing, sensitivity, costs, or assay complexity. This thesis aims
to develop two core technologies that address 1) bulk profiling issues with sensitivity and low
throughput as well as 2) in situ profiling issues with low multiplexing capabilities, costs, and
limited throughput. To address the first issue, this work introduces a novel liquid biopsy approach
that utilizes a platform technology called Integrated Comprehensive Droplet Digital Detection
(IC3D). This integrated approach combines microfluidic droplet partitioning technology,
fluorescent multiplexed PCR chemistry, and our own unique and rapid particle counting
technology to deliver ultrasensitive and ultrafast detection of colorectal cancer-specific genomic
biomarkers from minimally processed blood samples. xv
To address the second issue, this work introduces a new spatial multi-omics technology
termed Multi Omic Single-scan Assay with Integrated Combinatorial Analysis (MOSAICA) that
integrates a) in situ labeling of molecular markers (e.g. mRNA, proteins) in cells or tissues with
combinatorial fluorescence spectral and lifetime encoded probes, and b) spectra and timeresolved fluorescence imaging and analysis to enable rapid, high-throughput, and cost-effective
spatial profiling of multi-omics biomarkers. By utilizing both time and intensity domains for
labeling and imaging, this technology seeks to discriminate a vast repertoire of lifetime and
spectral components simultaneously within the same pixel or image of a sample to enable highly
increased multiplexing capabilities with standard optical systems. Overall, these two
technologies represent simple, versatile, and scalable tools which enable more rapid, sensitive,
and/or multiplexed protein/transcriptomic analysis.
Design of Bacterial Strain-Specific qPCR Assays Using NGS Data and Publicly Available Resources and Its Application to Track Biocontrol StrainsAbstract
Biological control is emerging as a feasible alternative to chemical pesticides in agriculture. Measuring the microbial biocontrol agent (mBCA) populations in the environment is essential for an accurate environmental and health risk assessment and for optimizing the usage of an mBCA-based plant protection product. We hereby show a workflow to obtain a large number of qPCR markers suitable for robust strain-specific quantification. The workflow starts from whole genome sequencing data and consists of four stages: (i) identifying the strain-specific sequences, (ii) designing specific primer/probe sets for qPCR, and (iii) empirically verifying the performance of the assays. The first two stages involve exclusively computer work, but they are intended for researchers with little or no bioinformatic background: Only a knowledge of the BLAST suite tools and work with spreadsheets are required; a familiarity with the Galaxy environment and next-generation sequencing concepts are strongly advised. All bioinformatic work can be implemented using publicly available resources and a regular desktop computer (no matter the operating system) connected to the Internet. The workflow was tested with five bacterial strains from four different genera under development as mBCAs and yielded thousands of candidate markers and a triplex qPCR assay for each candidate mBCA. The qPCR assays were successfully tested in soils of different natures, water from different sources, and with samples from different plant tissues. The mBCA detection limits and population dynamics in the different matrices are similar to those in qPCR assays designed by other means. In summary, a new accessible, cost-effective, and robust workflow to obtain a large number of strain-specific qPCR markers is presented.
Detection of freshwater mussels (Unionidae) using environmental DNA in riverine systemsAbstract
Environmental DNA (eDNA) methods are being developed for use in conservation biology to improve upon conventional species survey techniques. Validation of eDNA methods in different environmental contexts is required if they are to be widely adopted. One potential application of eDNA methods is for the detection of freshwater mussels (Bivalvia: Unionidae), which are among the most imperiled species in North America. Conventional unionid survey methods are highly invasive and can be difficult to conduct due to issues with morphological identification and their cryptic use of habitat. eDNA methods can potentially provide a non‐invasive, extremely specific, and highly sensitive alternative. Here, we examine the effectiveness of eDNA methods at detecting an imperiled unionid, the wavy‐rayed lampmussel (Lampsilis fasciola), in lotic systems with moderate discharge. We developed a novel qPCR assay for the detection of L. fasciola eDNA, which included a custom internal positive control to check for PCR inhibition. We used different experimental densities of caged L. fasciola specimens as a point source of eDNA within two rivers of the Grand River watershed in Southern Ontario. Sampling occurred at set distances downstream of the cage using purpose‐built sampling equipment. Detection was obtained at the cage (i.e., point of eDNA shedding) but not downstream at distances ≥10 m during stream discharges of approximately 1,632–2,332 L/s. The results indicate that eDNA is diluted rapidly in rivers with moderate discharge and that high‐resolution spatial sampling efforts may be necessary to obtain meaningful eDNA‐based distribution data of unionids, and other sessile organisms, present at low density in lotic systems.
T-cell Receptor Excision Circles in Newborns with Heart DefectsAbstract
In the fetus, the cardiac neural crest gives rise to both the thymus and the conotruncus of the heart. In newborn screening for severe T-cell lymphopenia neonates with congenital heart defects may be detected. In this study, we investigated the occurrence of T-cell lymphopenia in neonates with or without 22q11.2 deletion syndrome (del) suffering from heart defects. This retrospective cohort study included 125 patients with heart defects. T-cell receptor excision circles (TRECs), a measure for T-cell lymphopenia, were quantified by RT-PCR using stored newborn screening blood spots. Three patient groups were compared: non-conotruncal defects (n = 57), conotruncal defects (n = 42), and 22q11.2 del with conotruncal defects (n = 26). Significantly lower TREC values were detected in patients with 22q11.2 del and conotruncal heart defects compared to those with non-syndromic conotruncal (p < 0.001) and non-conotruncal (p < 0.001) defects. In contrast, no significant difference was found between patients with non-syndromic conotruncal and non-conotruncal heart defects (p = 0.152). Low TREC levels were obtained in neonates treated with heart surgery/intervention within 2 weeks after birth and in those with a fatal outcome (p = 0.02) independent of patient group. A correlation was found between low TREC numbers and oxygen saturation, SpO2 below 95% (p = 0.017). The SpO2 was significantly lower in the non-syndromic conotruncal group compared to non-conotruncal (p < 0.001) and 22q11.2 del group (p = 0.015). No correlation was found between low neonatal TRECs and infections needing hospitalization later in life (p = 0.135). Patients with 22q11.2 del and conotruncal defects have significantly lower TREC levels compared to patients with heart defects without this syndrome.
MAPK-induced miR-29 restrains melanoma progression by targeting MAFGAbstract
The tumor suppressive miR-29 family of microRNAs is encoded by two clusters, miR-29b1∼a and miR-29b2∼c, which are regulated by oncogenic and tumor suppressive stimuli, including p53. Here we investigated whether MAPK hyperactivation-induced oncogenic stress regulates miR-29 abundance and how this signaling axis impacts melanoma development. Using mouse embryonic fibroblasts and human melanocytes, we found that oncogenic MAPK signaling stimulates p53-independent and p53-dependent transcription of pri-miR-29b1∼a and pri-miR-29b2∼c, respectively. Expression analyses revealed that while pri-miR-29a∼b1 remains elevated, pri-miR-29b2∼c levels decrease during melanoma progression. Using a rapid mouse modeling platform, we showed that inactivation of miR-29 in vivo accelerates the development of frank melanomas and decreases overall survival. We identified MAFG as a relevant miR-29 target that has oncogenic potential in melanocytes and is required for growth of melanoma cells. Our findings suggest that MAPK-driven miR-29 induction constitutes a tumor suppressive barrier by targeting MAFG, which is overcome by attenuation of miR-29b2∼c expression.
Rickettsia felis identified in two fatal cases of acute meningoencephalitisAbstract
Rickettsia felis has recently emerged worldwide as a cause of human illness. Typically causing mild, undifferentiated fever, it has been implicated in several cases of non-fatal neurological disease in Mexico and Sweden. Its distribution and pathogenicity in Southeast Asia is poorly understood.
We retroactively tested cerebrospinal fluid (CSF) or sera from 64 adult patients admitted to hospital in North Sulawesi, Indonesia with acute neurological disease. Rickettsia felis DNA was identified in the CSF of two fatal cases of meningoencephalitis using multi-locus sequence typing semi-nested PCR followed by Sanger sequencing. DNA from both cases had 100% sequence homologies to the R. felis reference strain URRWXCal2 for the 17-kDa and ompB genes, and 99.91% to gltA.
The identification of R. felis in the CSF of two fatal cases of meningoencephalitis in Indonesia suggests the distribution and pathogenicity of this emerging vector-borne bacteria might be greater than generally recognized. Typically Rickettsia are susceptible to the tetracyclines and greater knowledge of R. felis endemicity in Indonesia should lead to better management of some acute neurological cases.
The retinoic acid receptor (RAR) α-specific agonist Am80 (tamibarotene) and other RAR agonists potently inhibit hepatitis B virus transcription from cccDNAAbstract
Chronic infection with the human Hepatitis B virus (HBV) is a major global health problem. Hepatitis D virus (HDV) is a satellite of HBV that uses HBV envelope proteins for cell egress and entry. Using infection systems encoding the HBV/HDV receptor human sodium taurocholate co-transporting polypeptide (NTCP), we screened 1181 FDA-approved drugs applying markers for interference for HBV and HDV infection. As one primary hit we identified Acitretin, a retinoid, as an inhibitor of HBV replication and HDV release. Based on this, other retinoic acid receptor (RAR) agonists with different specificities were found to interfere with HBV replication, verifying that the retinoic acid receptor pathway regulates replication. Of the eight agonists investigated, RARα-specific agonist Am80 (tamibarotene) was most active. Am80 reduced secretion of HBeAg and HBsAg with IC50s < 10 nM in differentiated HepaRG-NTCP cells. Similar effects were observed in primary human hepatocytes. In HepG2-NTCP cells, profound Am80-mediated inhibition required prolonged treatment of up to 35 days. Am80 treatment of cells with an established HBV cccDNA pool resulted in a reduction of secreted HBsAg and HBeAg, which correlated with reduced intracellular viral RNA levels, but not cccDNA copy numbers. The effect lasted for >12 days after removal of the drug. HBV genotypes B, D, and E were equally inhibited. By contrast, Am80 did not affect HBV replication in transfected cells or HepG2.2.15 cells, which carry an integrated HBV genome. In conclusion, our results indicate a persistent inhibition of HBV transcription by Am80, which might be used for drug repositioning.
Differentiation between wild boar and domestic pig in food by targeting two gene loci by real-time PCRAbstract
Studies indicate that many meat products are not authentic, most frequently because the meat species differ from those given on the food labels. At present, DNA based methods play the most important role in meat species authentication. Discrimination of wild boar and domestic pig meat in food is challenging because it is differentiation on the subspecies level. We developed and validated two singleplex real-time PCR assays targeting SNP rs81416363 on chromosome 9 and a duplex real-time PCR assay targeting SNP g.299084751 C > T in the NR6A1 gene located on chromosome 1. The singleplex real-time PCR assays led to some ambiguous results for Mangalica and Krškopolje pig breeds and wild boar individuals from Germany, the duplex real-time PCR assay particularly for the Turopolje pig breed. We demonstrate that the probability of misclassification can be substantially reduced if the results of both the singleplex real-time PCR assays and the duplex real-time PCR assay are taken into consideration. 86 (91.5%) of a total of 94 individuals, comprising 64 domestic pigs (14 different breeds and 6 cross-breeds) and 30 wild boars (from Austria, Germany, Romania, USA and Estonia), were classified correctly.
Vector competence, vectorial capacity of Nyssorhynchus darlingi and the basic reproduction number of Plasmodium vivax in agricultural settlements in the Amazonian Region of BrazilAbstract
Brazilian malaria control programmes successfully reduced the incidence and mortality rates from 2005 to 2016. Since 2017, increased malaria has been reported across the Amazon. Few field studies focus on the primary malaria vector in high to moderate endemic areas, Nyssorhynchus darlingi, as the key entomological component of malaria risk, and on the metrics of Plasmodium vivax propagation in Amazonian rural communities.
Human landing catch collections were carried out in 36 houses of 26 communities in five municipalities in the Brazilian states of Acre, Amazonas and Rondônia states, with API (> 30). In addition, data on the number of locally acquired symptomatic infections were employed in mathematical modelling analyses carried out to determine Ny. darlingi vector competence and vectorial capacity to P. vivax; and to calculate the basic reproduction number for P. vivax.
Entomological indices and malaria metrics ranged among localities: prevalence of P. vivax infection in Ny. darlingi, from 0.243% in Mâncio Lima, Acre to 3.96% in Machadinho D’Oeste, Rondônia; daily human-biting rate per person from 23 ± 1.18 in Cruzeiro do Sul, Acre, to 66 ± 2.41 in Lábrea, Amazonas; vector competence from 0.00456 in São Gabriel da Cachoeira, Amazonas to 0.04764 in Mâncio Lima, Acre; vectorial capacity from 0.0836 in Mâncio Lima, to 1.5 in Machadinho D’Oeste. The estimated R0 for P. vivax (PvR0) was 3.3 in Mâncio Lima, 7.0 in Lábrea, 16.8 in Cruzeiro do Sul, 55.5 in São Gabriel da Cachoeira, and 58.7 in Machadinho D’Oeste. Correlation between P. vivax prevalence in Ny. darlingi and vector competence was non-linear whereas association between prevalence of P. vivax in mosquitoes, vectorial capacity and R0 was linear and positive.
In spite of low vector competence of Ny. darlingi to P. vivax, parasite propagation in the human population is enhanced by the high human-biting rate, and relatively high vectorial capacity. The high PvR0 values suggest hyperendemicity in Machadinho D’Oeste and São Gabriel da Cachoeira at levels similar to those found for P. falciparum in sub-Saharan Africa regions. Mass screening for parasite reservoirs, effective anti-malarial drugs and vector control interventions will be necessary to shrinking transmission in Amazonian rural communities, Brazil.
Real‐time PCR assays for rapid detection of Zeugodacus cucumis and Bactrocera jarvisi (Diptera: Tephritidae) for quarantine applicationAbstract
Zeugodacus cucumis and Bactrocera jarvisi are pests of fruit and vegetable crops and cause damage to horticulture industries. Immature stages of these two fruit fly species have been intercepted in New Zealand a number of times. Identification to species was not possible using morphological characters; thus, it is important to develop an assay for their species‐level identification. Here, the real‐time PCR assays for rapid identification of Z. cucumis and B. jarvisi were developed and validated. The PCR protocols demonstrated their specificity by amplifying the two target species successfully, with no cross‐reactions observed in the tested tephritid species. The in silico test of the primer and probe binding sites of the two assays also demonstrated the assays’ specificity by no mismatches present in the binding regions of the target species, but 1–4 mismatches in the binding regions of the non‐target fruit fly species. The thresholds of detection for the two assays are as low as 10 copies/µl of the target DNA, indicating that the assays have a very high sensitivity. The application of the real‐time PCR assays has greatly assisted in routine pest identifications at the New Zealand border and surveillance programme. Therefore, the assays have the potential to be used by diagnostic agencies and research organizations worldwide.
Digital Loop-Mediated Isothermal Amplification on a Commercial MembraneAbstract
In this work, we report digital loop-mediated isothermal
amplification (LAMP) or reverse-transcription LAMP (RT-LAMP) on a
commercial membrane, without the need for complex chip fabrication or use
of specialized equipment. Due to the pore size distribution, the theoretical
error for digital LAMP on these membranes was analyzed, using a
combination of Random Distribution Model and Multivolume Theory. A
facile peel-off process was developed for effective droplet formation on the
commercial track-etched polycarbonate (PCTE) membrane. Each pore
functions as an individual nanoreactor for single DNA amplification.
Absolute quantification of bacteria genomic DNA was realized with a
dynamic range from 11 to 1.1 × 105 copies/μL. One-step digital RT-LAMP
was also successfully performed on the membrane for the quantification of MS2 virus in wastewater. With the introduction of
new probes, the positive pores can be easily distinguished from negative ones with 100 times difference in fluorescence
intensities. Finally, the cost of a disposable membrane is less than $0.10/piece, which, to the best of our knowledge, is the most
inexpensive way to perform digital LAMP. The membrane system offers opportunities for point-of-care users or common
laboratories to perform digital quantification, single cell analysis, or other bioassays in an inexpensive, flexible, and simplified
A Brief Review of Non-Avian Reptile Environmental DNA (eDNA), with a Case Study of Painted Turtle (Chrysemys picta) eDNA Under Field ConditionsAbstract
Environmental DNA (eDNA) is an increasingly used non-invasive molecular tool for detecting species presence and monitoring populations. In this article, we review the current state of non-avian reptile eDNA work in aquatic systems, and present a field experiment on detecting the presence of painted turtle (Chrysemys picta) eDNA. Thus far, turtle and snake eDNA studies have shown mixed results in detecting the presence of these animals under field conditions. However, some instances of low detection rates and non-detection occur for these non-avian reptiles, especially for squamates. We explored non-avian reptile eDNA quantification by sampling four lentic ponds with different densities (0 kg/ha, 6 kg/ha, 9 kg/ha, and 13 kg/ha) of painted turtles over three months to detect differences in eDNA using a qPCR assay amplifying the COI gene of the mtDNA genome. Only one sample of the highest-density pond amplified eDNA for a positive detection. Yet, estimates of eDNA concentration from pond eDNA were rank-order correlated with turtle density. We present the “shedding hypothesis”—the possibility that animals with hard, keratinized integument do not shed as much DNA as mucus-covered organisms—as a potential challenge for eDNA studies. Despite challenges with eDNA inhibition and availability in water samples, we remain hopeful that eDNA can be used to detect freshwater turtles in the field. We provide key recommendations for biologists wishing to use eDNA methods for detecting non-avian reptiles.
Quantification of Hepatitis B Virus Covalently Closed Circular DNA in Infected Cell Culture Models by Quantitative PCRAbstract
Persistence of the human hepatitis B virus (HBV) requires the maintenance of covalently closed circular (ccc)DNA, the episomal genome reservoir in nuclei of infected hepatocytes. cccDNA elimination is a major aim in future curative therapies currently under development. In cell culture based in vitro studies, both hybridization- and amplification-based assays are currently used for cccDNA quantification. Southern blot, the current gold standard, is time-consuming and not practical for a large number of samples. PCR-based methods show limited specificity when excessive HBV replicative intermediates are present. We have recently developed a real-time quantitative PCR protocol, in which total cellular DNA plus all forms of viral DNA are extracted by silica column. Subsequent incubation with T5 exonuclease efficiently removes cellular DNA and all non-cccDNA forms of viral DNA while cccDNA remains intact and can reliably be quantified by PCR. This method has been used for measuring kinetics of cccDNA accumulation in several in vitro infection models and the effect of antivirals on cccDNA. It allowed detection of cccDNA in non-human cells (primary macaque and swine hepatocytes, etc.) reconstituted with the HBV receptor, human sodium taurocholate cotransporting polypeptide (NTCP). Here we present a detailed protocol of this method, including a work flowchart, schematic diagram and illustrations on how to calculate “cccDNA copies per (infected) cell”.
Linking the resistome and plasmidome to the microbiomeAbstract
The rapid spread of antibiotic resistance among bacterial pathogens is a serious human health threat. While a range of environments have been identified as reservoirs of antibiotic resistance genes (ARGs), we lack understanding of the origins of these ARGs and their spread from environment to clinic. This is partly due to our inability to identify the natural bacterial hosts of ARGs and the mobile genetic elements that mediate this spread, such as plasmids and integrons. Here we demonstrate that the in vivo proximity-ligation method Hi-C can reconstruct a known plasmid-host association from a wastewater community, and identify the in situ host range of ARGs, plasmids, and integrons by physically linking them to their host chromosomes. Hi-C detected both previously known and novel associations between ARGs, mobile genetic elements and host genomes, thus validating this method. We showed that IncQ plasmids and class 1 integrons had the broadest host range in this wastewater, and identified bacteria belonging to Moraxellaceae, Bacteroides, and Prevotella, and especially Aeromonadaceae as the most likely reservoirs of ARGs in this community. A better identification of the natural carriers of ARGs will aid the development of strategies to limit resistance spread to pathogens.
Development of event-specific qPCR detection methods for genetically modified alfalfa events J101, J163 and KK179Abstract
Genetically modified alfalfa is authorized for cultivation in several countries since 2005. On the other hand,
cultivation in or export to the European Union is not allowed and thus neither certified reference material nor
official event-specific detection methods are available. Therefore, based on patent sequence information, eventspecific real-time PCR detection methods targeting the junction sequence of the alfalfa genome and the transgenic insert of the respective events J101, J163 and KK179 were developed. Newly developed plasmids were
used as reference material for assay optimization and in-house validation. Plasmid standards were quantified
using digital droplet PCR and LOD95%, PCR efficiency, robustness and specificity of the assays were determined
using real-time PCR. A LOD95% of 10 copies per PCR reaction was observed and PCR efficiencies of 95–97 %
were achieved. Different real-time PCR instruments and PCR conditions were applied to test for robustness of the
assays using DNA at a concentration of 30 copies per μL for each gm alfalfa event. All replicates were positive
independent of the instrument or the PCR condition. DNA from certified reference material of different genetically modified crops as well as reference materials of the three events was used to experimentally test for
specificity. No unspecific amplification signal was observed for any of the assays. Validation results were in line
with the “Minimum Performance Requirements for Analytical Methods of GMO Testing” of the European
Network of GMO Laboratories. Furthermore, an inter-laboratory comparison study was conducted to show the
transferability and applicability of the methods and to verify the assay performance parameters.
A European interlaboratory trial to evaluate the performance of different PCR methods for Mycoplasma bovis diagnosisAbstract
Background: Several species-specific PCR assays, based on a variety of target genes are currently used in the
diagnosis of Mycoplasma bovis infections in cattle herds with respiratory diseases and/or mastitis. With this diversity
of methods, and the development of new methods and formats, regular performance comparisons are required to
ascertain diagnostic quality. The present study compares PCR methods that are currently used in six national
veterinary institutes across Europe. Three different sample panels were compiled and analysed to assess the
analytical specificity, analytical sensitivity and comparability of the different PCR methods. The results were also
compared, when appropriate, to those obtained through isolation by culture. The sensitivity and comparability
panels were composed of samples from bronchoalveolar fluids of veal calves, artificially contaminated or naturally
infected, and hence the comparison of the different methods included the whole workflow from DNA extraction
to PCR analysis.
Results: The participating laboratories used i) five different DNA extraction methods, ii) seven different real-time
and/or end-point PCRs targeting four different genes and iii) six different real-time PCR platforms. Only one
commercial kit was assessed; all other PCR assays were in-house tests adapted from published methods. The
analytical specificity of the different PCR methods was comparable except for one laboratory where Mycoplasma
agalactiae was tested positive. Frequently, weak-positive results with Ct values between 37 and 40 were obtained
for non-target Mycoplasma strains. The limit of detection (LOD) varied from 10 to 103 CFU/ml to 103 and 106 CFU/
ml for the real-time and end-point assays, respectively. Cultures were also shown to detect concentrations down
to 102 CFU/ml. Although Ct values showed considerable variation with naturally infected samples, both between
laboratories and tests, the final result interpretation of the samples (positive versus negative) was essentially the
same between the different laboratories.
Conclusion: With a few exceptions, all methods used routinely in the participating laboratories showed comparable
performance, which assures the quality of diagnosis, despite the multiplicity of the methods.
Effect of Thermotherapy on the Acquisition of Candidatus Liberibacter Asiaticus by the Asian Citrus Psyllid (Hemiptera: Liviidae)Abstract
The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is the most detrimental insect pest of citrus crops due to its role as a vector of Candidatus Liberibacter asiaticus (Las), the bacterial causal agent of huanglongbing, also known as citrus greening disease. Trees infected with Las decline rapidly and fruit production decreases until eventual tree death. Few treatment options for infected trees are available for disease management. A technique called “thermotherapy” is under development to reduce bacterial titers in infected trees; however, the effect of these treatments on the transmission cycle of Las is not known. Field and laboratory assays were conducted to determine whether thermotherapy treatment reduced Las acquisition by D. citri. Trees in the field were treated with a mobile heat treatment system. Potted trees in the laboratory were treated in a steam chamber. We monitored acquisition rates in D. citri following thermal treatment of Las-positive Citrus sinensis (L.) (Rutaceae). Psyllid acquisition and Las titer in thermotherapy-treated trees were compared with untreated Las-positive and untreated Las-negative trees. Our results confirmed the efficacy of whole-tree thermotherapy on Las in potted citrus trees. In contrast, thermotherapy did not significantly reduce plant Las titers or acquisition of Las by D. citri under field conditions. These results suggest that further development of field application methods is needed to determine the utility of thermotherapy as a tool for huanglongbing management.
Using discrete and online ATP measurements to evaluate regrowthpotential following ozonation and (non)biological drinking watertreatmentAbstract
Water utilities must control microbial regrowth in the distribution system to protect public health. In thisstudy, an adenosine triphosphate (ATP)-based biomass production potential test using indigenous bac-terial communities were used to evaluate regrowth potential following ozonation with either bio-filtration (BF) or sustained chlorination (SCl2). Two full-scale water treatment plants with differentupstream processes (i.e., WTP-BF: ozonation, coagulation/flocculation, biofiltration, UV irradiation,chlorination; and WTP-SCl2: ozonation, chlorination, coagulation/flocculation,filtration, chlorination)were compared. Characterization of indigenous bacteria using 16S rRNA gene sequencing, qPCR, andcellular ATP (cATP) showed microbial diversity changes across treatment, biomass sloughing from bio-filters (effluent cATP¼30±1 ng/L), and disinfection by chlorine (cATP<1 ng/L). For both WTPs, 14-daycumulative biomass production (CBPt¼Ptt¼0ATPðtÞDt) was highest for ozonated water samples(CBP14¼1.2103e3.0103d ngATP/L). CBP further increased with increasing ozone dose due to pro-duction of more biodegradable carbon. Growth promotion by carbon was confirmed from the con-sumption of ozonation byproducts (carboxylic acids, aldehydes) and the increase in CBP (9.5102e2.9103d ngATP/L) after addition of 50e300mgC/L acetate. Ozone followed by sustained chlorination(WTP-SCl2) effectively controlled biomass growth across the treatment process (CBP14<10 d ngATP/L). Incontrast, ozone followed by biofiltration (WTP-BF) reduced regrowth potential by 30% (biofilter influentCBP14¼1.3103d ngATP/L; biofilter effluent CBP14¼9.3102d ngATP/L). After adding chlorine to thebiofilter effluent, CBP14was reduced to<10 d ngATP/L. Lastly, online ATP measurements confirmed thediscrete measurements and improved identification of the cATP peak and growth phases of indigenousbacteria.
Using environmental DNA to extend the window of early detection for dreissenid musselsAbstract
Tools that bolster early detection of invasive dreissenid mussels are needed to
prevent their spread across western North America. In this study, we assessed if
environmental DNA (eDNA) can extend the seasonal window for dreissenid mussel
early detection beyond that of plankton tows, which are limited to warmer seasons
when mussel larvae are present. We focused eDNA sampling efforts at multiple sites
in Tiber Reservoir (Montana) where dreissenid mussel abundance is hypothesized
to be low. Samples were collected in June and October 2017, when water
temperatures were cooler than thermal optima for dreissenid reproduction, and in
July 2017 when water temperatures were warmer and conducive for reproduction.
We detected dreissenid mussel DNA in June, July and October even though no
dreissenid mussels were observed using non-molecular tools in 2017. A subset of
positive and negative eDNA samples was analyzed by an independent lab and
results were corroborated. We then estimated the effort needed for 95% probability
detection of dreissenid DNA at each site within Tiber Reservoir and found that as
many as 27, 14, and 34 samples needed to be collected in June, July and October,
respectively. To further validate the utility of eDNA, we also present ancillary
eDNA results from other waters in the Flathead Reservation (Montana) where
dreissenid mussels have never been detected and from waters with established
zebra mussel populations in the upper Mississippi River, which were sampled in
the spring when water temperatures were cooler than thermal optima for dreissenid
reproduction. All Flathead Reservation samples were negative for dreissenid mussel
DNA, while all upper Mississippi River samples were positive. This study adds to a
growing body of research that demonstrates eDNA is a highly sensitive tool for
dreissenid mussel surveillance in newly invaded waters, including colder seasons when
non-molecular tools are likely to be less effective or more challenging to employ
Immunoserology of European seabass (Dicentrarchus labrax) and white grouper (Epinephelus aeneus) as a non-lethal diagnostic tool for viral nervous necrosisAbstract
Viral nervous necrosis (VNN) is a lethal fish disease that has spread worldwide over the last two decades, causing severe losses in aquaculture. Diagnosis of the infection is generally made by sampling brain tissue, which involves sacrificing often valuable fish. Aiming at developing a non-lethal diagnostic method, the immune responses to an experimental nervous necrosis virus (NNV) infection in sea bass Dicentrarchus labrax and white grouper Epinephelus aeneus, two species most susceptible to the disease, were studied. RT-qPCR revealed presence of NNV in the fish brain within 24 h post-infection, the virus titer remaining high up to 30–35 days post-infection. In D. labrax blood, the virus was detectable within the first 5 days, after which its presence declined rapidly. Mx gene expression correlated to the virus presence in the blood and brain. An indirect ELISA was developed that quantified anti-NNV IgM in the fish blood. In D. labrax, anti-NNV IgM titer increased significantly within 5 days post-infection, and presence of specific IgM was detectable for 180 days. A sandwich ELISA was developed for E. aeneus. In this latter species, anti-NNV IgM titer increased significantly within the first 12 days and was detectable for 208 further days. The sandwich ELISA can be used as a diagnostic tool for detecting NNV exposure in all fish species for which specific antibodies against their IgMs are not yet commercially available. Our immunoserological method can reliably be used for diagnosis of VNN infection and does not require sacrificing the fish.
Preweaned heifer management on US dairy operations: Part IV. Factors associated with the presence of Escherichia coli O157 in preweaned dairy heifersAbstract
Dairy calves shed pathogenic Escherichia coli O157 (O157) in feces and are a potential route of exposure for human infections. As part of the National Animal Health Monitoring System's (NAHMS) Dairy 2014 study, we evaluated farm, animal, and environmental factors associated with O157 presence in dairy heifer calves. For this O157 study, calves were enrolled from 100 dairy operations in 13 states. Each operation collected data from calves from birth to weaning over an 18-mo period. A single fecal sample was collected from 487 calves in western states and from 871 calves in eastern states (n = 1,358 total), and O157 was detected in 2.5% (n = 34) of fecal samples. Descriptive statistics and univariable screening were used to determine which farm practices, environmental factors, and calf health measures were associated with O157 detection. Multilevel logistic models, controlling for dairy operation, were created using backward elimination of screened variables. The final O157 main effects model included variables for source of colostrum, temperature-humidity index (THI), and serum IgG concentration. Higher serum IgG was associated with lower odds of O157 shedding, whereas calves fed colostrum from their own dam had higher odds of O157 shedding than calves fed colostrum from pooled sources. Interaction models showed that THI level modified the effect of colostrum source on O157 shedding; calves with a THI indicative of heat stress had a significantly increased presence of O157 when fed colostrum from a first-lactation dam. The THI level also modified the effects of serum IgG. Calves with thermoneutral or heat stress THI values had increased presence of O157 with poor (<10 g/L) or adequate (10–15 g/L) serum IgG levels compared with those having excellent (≥15 g/L) serum IgG levels. These results highlight factors that influence the presence of O157 in preweaned dairy heifer calves and may be used to guide practices that mitigate shedding through improved animal husbandry.
M3-subtype muscarinic receptor activation stimulates intracellular calcium oscillations and aldosterone production in human adrenocortical HAC15 cellsAbstract
A previous body of work in bovine and rodent models shows that cholinergic agonists modulate the secretion of steroid hormones from the adrenal cortex. In this study we used live-cell Ca2+ imaging to investigate cholinergic activity in the HAC15 human adrenocortical carcinoma cell line. The cholinergic agonists carbachol and acetylcholine triggered heterogeneous Ca2+ oscillations that were strongly inhibited by antagonists with high affinity for the M3 muscarinic receptor subtype, while preferential block of M1 or M2 receptors was less effective. Acute exposure to carbachol and acetylcholine modestly elevated aldosterone secretion in HAC15 cells, and this effect was also diminished by M3 inhibition. HAC15 cells expressed relatively high levels of mRNA for M3 and M2 receptors, while M1 and M5 mRNA were much lower. In conclusion, our data extend previous findings in non-human systems to implicate the M3 receptor as the dominant muscarinic receptor in the human adrenal cortex.
Suitability of group-level oral fluid sampling in ruminant populations for lumpy skin disease virus detectionAbstract
The geographic expansion of Lumpy skin disease (LSD) from the near East into the European Union highlighted again the need for appropriate disease detection tools applicable to animal host populations where access to individual animals is difficult. This is of particular importance considering that the clinical manifestation of LSD is often mild making early disease detection challenging under the above-mentioned conditions. Building on positive experiences of group-level oral fluid sampling for pathogen detection as it is known to work for swine herds and wild boar, the concept was transferred to ruminants. Two groups of six cattle were infected experimentally with Lumpy skin disease virus (LSDV) under controlled conditions. Blood as well as oropharyngeal and nasal swab samples were collected at regular intervals. Group samples were obtained by placing cotton gauze around a salt lick block provided commonly as dietary supplement. Pieces of the gauze with visible signs of manipulation were tested in parallel to samples obtained from individual animals. Genome load analysis by qPCR technology revealed LSDV detection window starting from day 2 post infection until day 28 post infection, the end of the animal trial. At the individual level, detection periods varied between animals and type of sample and included intermitted detection. The accumulative character of the alternative sampling method makes it suitable to detect LSDV DNA at group-level even at times of the infection where a selective sampling of individuals from a group – as normally done in LSD surveillance – would have most likely failed in the detection.
Salmonella-mediated inflammation eliminates competitors for fructose-asparagine in the gut.Abstract
Salmonella enterica elicits intestinal inflammation to gain access to nutrients. One of these nutrients is fructose-asparagine (F-Asn). The availability of F-Asn to Salmonella during infection is dependent upon the pathogenicity islands, SPI1 and SPI2, which in turn are required to provoke inflammation. Here, we determined that F-Asn is present in mouse chow at approximately 400 pmol/mg dry weight. F-Asn is also present in the intestinal tract of germ-free mice at 2700 pmol/mg dry weight, and in the intestinal tract of conventional mice at 15 pmol/mg. These findings suggest that the mouse intestinal microbiota consumes F-Asn. We utilized heavy-labeled precursors of F-Asn to monitor its formation in the intestine, in the presence or absence of inflammation, and none was observed. Finally, we determined that some members of the class Clostridia encode F-Asn utilization pathways and are eliminated from highly inflamed Salmonella-infected mice. Collectively, our studies identify the source of F-Asn as the diet, and that Salmonella-mediated inflammation is required to eliminate competitors and allow the pathogen near-exclusive access to this nutrient.
Development of a new qPCR method for specific detection and quantification of genetically modified maize MON863Abstract
Analysis of non-transgenic seed samples for the presence of genetically modified maize MON863 revealed unexpected amplification signals using an official qPCR method. These amplification signals only occured when using other master mix products as in the original validation process. DNA sequence data from an unspecific amplicon could be mapped to mitochondrial maize DNA reference sequence. Oligo sequence analysis revealed that forward primer and probe both can hybridize to the mitochondrial maize DNA leading to unspecific amplification signals in qPCR. Therefore, we designed and validated a new qPCR method for event MON863 with a LOD of 5 copies per reaction. The method shows high specificity as no unspecific amplification signal was detected after analysis of reference material for different genetically modified crops and conventional maize samples. Robustness tests were performed in two different laboratories and no effects on method performance could be observed when using different master mixes and qPCR devices, as well as with variation in oligonucleotide concentrations.
Development of a quantitative loop-mediated isothermal amplification assay for the field detection of Erysiphe necatorAbstract
Plant pathogen detection systems have been useful tools to monitor inoculum presence and initiate management schedules. More recently, a loop-mediated isothermal amplification (LAMP) assay was successfully designed for field use in the grape powdery mildew pathosystem; however, false negatives or false positives were prevalent in grower-conducted assays due to the difficulty in perceiving the magnesium pyrophosphate precipitate at low DNA concentrations. A quantitative LAMP (qLAMP) assay using a fluorescence resonance energy transfer-based probe was assessed by grape growers in the Willamette Valley of Oregon. Custom impaction spore samplers were placed at a research vineyard and six commercial vineyard locations, and were tested bi-weekly by the lab and by growers. Grower-conducted qLAMP assays used a beta-version of the Smart-DART handheld LAMP reaction devices (Diagenetix, Inc., Honolulu, HI, USA), connected to Android 4.4 enabled, Bluetooth-capable Nexus 7 tablets for output. Quantification by a quantitative PCR assay was assumed correct to compare the lab and grower qLAMP assay quantification. Growers were able to conduct and interpret qLAMP results; however, the Erysiphe necator inoculum quantification was unreliable using the beta-Smart-DART devices. The qLAMP assay developed was sensitive to one spore in early testing of the assay, but decreased to >20 spores by the end of the trial. The qLAMP assay is not likely a suitable management tool for grape powdery mildew due to losses in sensitivity and decreasing costs and portability for other, more reliable molecular tools.
Climate change favours specific fungal communities in boreal peatlandsAbstract
Fungi play a pivotal role in the carbon sequestration potential of boreal peatlands through the process of decomposition. As such, climate-driven changes in the diversity and community composition of peatland fungal communities could have substantial impacts on carbon release from these ecosystems, especially in subsurface peat that represents an important global carbon stock. We used Illumina MiSeq sequencing of rDNA to examine fungal communities after 18 months in intact peatland mesocosms subjected to conditions associated with Canada's future climate, including: warming, elevated atmospheric CO2 and lowered water table. Warming was the main driver of changes in fungal communities across three depths of the peat profile with both Ascomycota- and Basidiomycota-dominated groups becoming more homogenous under warming conditions. Specific changes in fungal functional groups, however, were temperature dependent with potential cellulose decomposers and mycorrhizal root-associated fungi from Basidiomycota dominant under warming of +4 °C, whereas there was a prevalence of potential lignocellulose decomposers and mycorrhizal root-associated fungi from Ascomycota under +8 °C warming. These climate change-induced shifts in the structure of fungal communities in favour of recalcitrant compound decomposers observed across a depth gradient, may reduce long-term carbon storage of boreal peatlands under future climate change scenarios.
Efficacy of hyperbaric oxygen therapy in bacterial biofilm eradicationAbstract
Objective: Chronic wounds typically require several concurrent therapies, such as debridement, pressure offloading, and systemic and/or topical antibiotics. The aim of this study was to examine the efficacy of hyperbaric oxygen therapy (HBOT) towards reducing or eliminating bacterial biofilms in vitro and in vivo. Method: Efficacy was determined using in vitro grown biofilms subjected directly to HBOT for 30, 60 and 90 minutes, followed by cell viability determination using propidium monoazide-polymerase chain reaction (PMA-PCR). The efficacy of HBOT in vivo was studied by searching our chronic patient wound database and comparing time-to-healing between patients who did and did not receive HBOT as part of their treatment. Results: In vitro data showed small but significant decreases in cell viability at the 30- and 90-minute time points in the HBOT group. The in vivo data showed reductions in bacterial load for patients who underwent HBOT, and ~1 week shorter treatment durations. Additionally, in patients' chronic wounds there was a considerable emergence of anaerobic bacteria and fungi between intermittent HBOT treatments. Conclusion: The data demonstrate that HBOT does possess a certain degree of biofilm killing capability. Moreover, as an adjuvant to standard treatment, more favourable patient outcomes are achieved through a quicker time-to-healing which reduces the chance of complications. Furthermore, the data provided insights into biofilm adaptations to challenges presented by this treatment strategy which should be kept in mind when treating chronic wounds. Further studies will be necessary to evaluate the benefits and mechanisms of HBOT, not only for patients with chronic wounds but other chronic infections caused by bacterial biofilms.
Red deer (Cervus elaphus)-specific real-time PCR assay for the detection of food adulterationAbstract
We present a red deer-specific real-time PCR assay which, combined with a reference real-time PCR assay published previously, allows the quantification of the red deer content in food products. Thus, it can be applied to detect food adulteration. The primer/probe system of the red deer-specific real-time PCR assay amplifies a 87 bp long fragment of the protein kinase C iota gene. To eliminate cross-reactivity with closely related species, the forward primer was designed to contain one deliberate base mismatch adjacent to one red deer-specific base. The red deer-specific real-time PCR assay did not show cross-reactivity with 23 animal and 50 plant species tested. LOD and LOQ, determined by analyzing a serially diluted DNA extract containing 1% (w/w) red deer DNA in pig DNA, were 0.05% and 0.4%, respectively. The accuracy was validated by analyzing DNA mixtures, meat extract mixtures, meat mixtures and model game sausages with known red deer content. The highest accuracy was obtained when the calibration mixture was similar to the analyzed sample in both the composition and concentration of the animal species of interest. High recoveries were not only obtained for raw samples but also after subjection to thermal treatment, including brewing (15 min at 75–78 °C), boiling (90 min at 100 °C) and microwave treatment (15 s, 40 s or 2 min at 650 W). The deer-specific real-time PCR assay was found to be robust with respect to small deviations in the reaction volume or the annealing temperature and the use of another real-time PCR instrument.
Assessment of fecal pollution in Lake Pontchartrain, LouisianaAbstract
Water quality in Lake Pontchartrain was deteriorating and recreational activities along the beach were restricted by the end of the 20th Century. A microbial source tracking (MST) study was conducted to determine the fecal contamination sources at public beach of the lake, so that effective pollution control strategies can be developed. Water samples were collected over an eight-month period at ten locations along the lake in 2016 and 2017. E. coli and Enterococcus were detected in 90.6% (culture) and 97.5% (qPCR), 95.8% (culture) and 91.8% (qPCR) of water samples from all sampling sites, respectively. Significant positive relationship between E. coli and Enterococcus results was observed for both qPCR and culture methods. HF183 marker was detected in 94.3% water samples (149 of 158), with concentrations ranging from 29.0 to 6073.5GC/100ml and from 129.8 to 38,465.6GC/100ml in summer and winter, respectively. The results also indicate that significant rainfall events have the potential to supply considerable loads of fecal bacteria to lake waters. Further research is needed to determine the contribution of other animals to fecal contamination in the region.
Development and validation of a fallow deer (Dama dama)-specific TaqMan real-time PCR assay for the detection of food adulteration - ScienceDirectAbstract
The aim of the present study was to develop a real-time PCR assay for the identification and quantification of fallow deer (Dama dama) in food to detect food adulteration. Despite high sequence homology among different deer species, a fallow deer-specific primer/probe system targeting a fragment of the nuclear MC1-R gene was designed. This primer/probe system did not amplify DNA from 19 other animals and 50 edible plant species. Moderate cross-reactivity was observed for sika deer, red deer, roe deer, reindeer and wild boar. The LOD and LOQ of the real-time PCR assay were 0.1% and 0.4%, respectively. To validate the assay, DNA mixtures, meat extract mixtures, meat mixtures and model game sausages were analyzed. Satisfactory quantitative results were obtained when the calibration mixture was similar to the analyzed sample in both the composition and concentration of the animal species of interest.
Three-color crystal digital PCRAbstract
Digital PCR is an exciting new field for molecular analysis, allowing unprecedented precision in the quantification of nucleic acids, as well as the fine discrimination of rare molecular events in complex samples. We here present a novel technology for digital PCR, Crystal Digital PCR™, which relies on the use of a single chip to partition samples into 2D droplet arrays, which are then subjected to thermal cycling and finally read using a three-color fluorescence scanning device. This novel technology thus allows three-color multiplexing, which entails a different approach to data analysis. In the present publication, we present this innovative workflow, which is both fast and user-friendly, and discuss associated data analysis issue, such as fluorescence spillover compensation and data representation. Lastly, we also present proof-of-concept of this three-color detection system, using a quadriplex assay for the detection of EGFR mutations L858R, L861Q and T790M.
Mitochondrial DNA deletion percentage in sun exposed and non sun exposed skinAbstract
The percentages of mitochondrial genomes carrying the mtDNA3895 and the mtDNA4977 (common) deletion were quantified in sun exposed and non sun exposed skin biopsies, for five cohorts of patients varying either in sun exposure profile, age or skin cancer status.
Non-melanoma skin cancer diagnoses are rising in Ireland and worldwide  but most risk prediction is based on subjective visual estimations of sun exposure history. A quantitative objective test for pre-neoplastic markers may result in better adherence to sun protective behaviours. Mitochondrial DNA (mtDNA) is known to be subject to the loss of a significant proportion of specific sections of genetic code due to exposure to ultraviolet light in sunlight. Although one such deletion has been deemed more sensitive, another, called the mtDNA4977 or common deletion, has proved to be a more useful indicator of possible risk in this study.
Quantitative molecular analysis was carried out to determine the percentage of genomes carrying the deletion using non sun exposed and sun exposed skin biopsies in cohorts of patients with high or low sun exposure profiles and two high exposure groups undergoing treatment for NMSC.
Results indicate that mtDNA deletions correlate to sun exposure; in groups with high sun exposure habits a significant increase in deletion number in exposed over non sun exposed skin occurred. An increase in deletion percentage was also seen in older cohorts compared to the younger group.
The mtDNA3895 deletion was detected in small amounts in exposed skin of many patients, the mtDNA4977 common deletion, although present to some extent in non sun exposed skin, is suggested to be the more reliable and easily detected marker. In all cohorts except the younger group with relatively lower sun exposure, the mtDNA4977 deletion was more frequent in sun exposed skin samples compared to non-sun exposed skin.
Temperature modulates liver lipid accumulation in Atlantic salmon (Salmo salar L.) fed low dietary levels of long-chain n-3 fatty acidsAbstract
Atlantic salmon (Salmo salar) were fed five graded levels of eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), from 1.4 to 5.2% of total fatty acids (FA, 5–17 mg kg−1 feed), and grew from ~160 g to ~3000 g, with the period from 1450 g onwards conducted both at 6 °C and at 12 °C. All fish appeared healthy, and there were no diet-related differences in haematological or plasma parameters, as well as intestinal histological or gut microbiota analysis. Fish reared at 6 °C had higher accumulation of storage lipids in the liver compared to fish reared at 12 °C. Liver lipids also increased with decreasing dietary EPA + DHA at 6 °C, while there was no such relationship at 12 °C. Gene expression of SREBP1 and 2, LXR, FAS and CPT1 could not explain the differences in liver lipid accumulation. In liver polar lipids, DHA was found to be reduced when dietary EPA + DHA was <2.7% of FAs, while the level of EPA in the membranes was not affected. In conclusion, reducing dietary EPA + DHA from 5.2 to 1.4% of total FAs had a minor impact on fish health. Temperature was the factor that most affected the liver lipid accumulation, but there was also an interaction with dietary components.