Comparing DNA isolation and sequencing strategies for 16S rRNA gene amplicon analysis in biofilm containing environments.

Bacteria are primarily responsible for biological water treatment processes in constructed wetland systems. Gravel in constructed wetlands serves as an essential substrate onto which complex bacterial biofilms may successfully grow and evolve. To fully understand the bacterial community in these systems it is crucial to properly isolate biofilms and process DNA from such substrates. This study looked at how best to isolate bacterial biofilms from gravel substrates in terms of bacterial richness. It considered factors including the duration of agitation during extraction, extraction temperature, and enzyme usage. Further, the 16S taxonomy data subsequently produced from Illumina MiSeq reads (using the SILVA 132 ribosomal RNA (rRNA) database on the DADA2 pipeline) were compared with the 16S data produced from Oxford Nanopore Technologies (ONT) MinION reads (using the NCBI 16S database on the EPI2ME pipeline). Finally, performance was tested by comparing the taxonomy data generated from the Illumina MiSeq and ONT MinION reads using the same (SILVA 132) database. We found no significant differences in the effective number of species observed when using different bacterial biofilm detachment techniques. However, enzyme treatment enhanced the total concentration of DNA. In terms of wetland community profiles, relative abundance differences within each sample type were clearer at the genus level. For genus-level taxonomic classification, MinION sequencing with the EPI2ME pipeline (NCBI database) produced bacterial abundance information that was poorly correlated with that from the Illumina MiSeq and DADA2 pipelines (SILVA132 database). When using the same database for each sequencing technology (SILVA132), the correlation between relative abundances at genus-level improved from negligible to moderate. This study provides detailed information of value to researchers working on constructed wetlands regarding efficient biofilm detachment techniques for DNA isolation and 16 s metabarcoding platforms for sequencing and data analysis.

Maximising signal-to-noise ratios in environmental DNA-based monitoring.

Man's impacts on global ecosystems are increasing and there is a growing demand that these activities be appropriately monitored. Monitoring requires measurement of a response metric ('signal') that changes maximally and consistently in response to the monitored activity irrespective of other factors ('noise'), thus maximising the signal-to-noise ratio. Indices derived from time-consuming morphology-based taxonomic identification of organisms are a core part of many monitoring programmes. Metabarcoding is an alternative to morphology-based identification and involves the sequencing of short fragments of DNA ('markers') from multiple taxa simultaneously. DNA suitable for metabarcoding includes that extracted from environmental samples (eDNA). Metabarcoding outputs DNA sequences that can be identified (annotated) by matching them against archived annotated sequences. However, sequences from most organisms are not archived - preventing annotation and potentially limiting metabarcoding in monitoring applications. Consequently, there is growing interest in using unannotated sequences as response metrics in monitoring programmes. We compared the sequences from three commonly used markers (16S (V3/V4 regions), 18S (V1/V2 regions) and COI) and, sampling along steep impact gradients, showed that the 16S and COI sequences were associated with the largest and smallest signal-to-noise ratio respectively. We trialled four separate, intuitive, noise-reduction approaches and demonstrated that removing less frequent sequences improved the signal-to-noise ratio, partitioning an additional 25 % from noise to explanatory factors in non-parametric ANOVA (NPA) and reducing dispersion in the data. For the 16S marker, retaining only the most frequently observed sequence, per sample, resulting in nine sequences across 150 samples, generated a near-maximal signal-to-noise ratio (95 % of the variance explained in NPA). We recommend that NPA, combined with rigorous elimination of less frequent sequences, be used to pre-filter sequences/taxa being used in monitoring applications. Our approach will simplify downstream analysis, for example the identification of key taxa and functional associations.

Quantitation of γ-aminobutyric acid in equine plasma by hydrophilic interaction liquid chromatography with tandem mass spectrometry.

γ-Aminobutyric acid is the principal inhibitory neurotransmitter in the central nervous system and regulates the neuronal excitability. There has been anecdotal evidence that γ-aminobutyric acid has been used within a few hours prior to competition in equine sports to calm down nervous horses. However, regulating the use of γ-aminobutyric acid is challenging because it is an endogenous substance in the horse. γ-Aminobutyric acid is usually present at low ng/mL levels in equine plasma; therefore, a sensitive method has to be developed to quantify these low background levels. Measuring low concentrations of endogenous γ-aminobutyric acid is essential to establish a threshold that can be used to differentiate levels attributable to exogenous administrations of γ-aminobutyric acid. A hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry method was developed and validated for the quantitation of γ-aminobutyric acid in equine plasma. Calibrators were prepared in artificial surrogate matrix consisting of 35 mg/mL equine serum albumin in phosphate buffered saline. Samples were prepared by protein precipitation with acetonitrile. Utilizing this methodology, a total of 403 equine plasma samples collected post-competition from horses participating in equestrian events in Canada were analyzed.

Home Use of a Pyrethroid-Containing Pesticide and Facial Paresthesia in a Toddler: A Case Report.

Paresthesias have previously been reported among adults in occupational and non-occupational settings after dermal contact with pyrethroid insecticides. In this report, we describe a preverbal 13-month-old who presented to his primary care pediatrician with approximately 1 week of odd facial movements consistent with facial paresthesias. The symptoms coincided with a period of repeat indoor spraying at his home with a commercially available insecticide containing two active ingredients in the pyrethroid class. Consultation by the Northwest Pediatric Environmental Health Specialty Unit and follow-up by the Washington State Department of Health included urinary pyrethroid metabolite measurements during and after the symptomatic period, counseling on home clean up and use of safer pest control methods. The child's symptoms resolved soon after home cleanup. A diagnosis of pesticide-related illness due to pyrethroid exposure was made based on the opportunity for significant exposure (multiple applications in areas where the child spent time), supportive biomonitoring data, and the consistency and temporality of symptom findings (paresthesias). This case underscores the vulnerability of children to uptake pesticides, the role of the primary care provider in ascertaining an exposure history to recognize symptomatic illness, and the need for collaborative medical and public health efforts to reduce significant exposures in children.

Biogeography of Paenibacillus larvae, the causative agent of American foulbrood, using a new multilocus sequence typing scheme.

American foulbrood is the most destructive brood disease of honeybees (Apis mellifera) globally. The absence of a repeatable, universal typing scheme for the causative bacterium Paenibacillus larvae has restricted our understanding of disease epidemiology. We have created the first multilocus sequence typing scheme (MLST) for P. larvae, which largely confirms the previous enterobacterial repetitive intergenic consensus (ERIC)-polymerase chain reaction (PCR)-based typing scheme's divisions while providing added resolution and improved repeatability. We have used the new scheme to determine the distribution and biogeography of 294 samples of P. larvae from across six continents. We found that of the two most epidemiologically important ERIC types, ERIC I was more diverse than ERIC II. Analysis of the fixation index (FST ) by distance suggested a significant relationship between genetic and geographic distance, suggesting that population structure exists in populations of P. larvae. Interestingly, this effect was only observed within the native range of the host and was absent in areas where international trade has moved honeybees and their disease. Correspondence analysis demonstrated similar sequence type (ST) distributions between native and non-native countries and that ERIC I and II STs mainly have differing distributions. The new typing scheme facilitates epidemiological study of this costly disease of a key pollinator.

Development and validation of a hydrophilic interaction liquid chromatography with tandem mass spectrometry method for the simultaneous detection and quantification of etilefrine and oxilofrine in equine blood plasma and urine.

A sensitive hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry method was developed and validated for the simultaneous detection and quantification of etilefrine and oxilofrine in equine blood plasma and urine. The method is highly sensitive and specific with good precision and accuracy. In plasma the limit of detection and limit of quantification are 0.03 and 0.1 ng/mL, respectively, for both analytes. In urine the limit of detection and limit of quantification are 0.3 and 1 ng/mL, respectively, for both analytes. The suitability of the method for doping control analysis in equine species is demonstrated by analyzing postadministration samples collected after a single intravenous administration of 50 mg etilefrine to a standardbred mare. Etilefrine was detected up to 120 h in urine and up to 48 h in plasma. Etilefrine is highly conjugated in equine urine whereas it exists in the free form in equine plasma. Therefore, enzyme hydrolysis prior to sample preparation is recommended for the detection and quantification of etilefrine and oxilofrine in equine urine.

Detection of efaproxiral (RSR13) and its metabolites in equine by liquid chromatography tandem mass spectrometry.

Efaproxiral (RSR 13) is an experimental synthetic allosteric modifier of haemoglobin (Hb) that acts by increasing the release of oxygen from Hb to the surrounding tissues. It has been shown to increase maximum oxygen uptake (VO(2max)) in a canine skeletal muscle model. The ability to increase maximal muscle oxygen uptake makes efaproxiral a potential performance-enhancing agent and is therefore prohibited by the World Anti-Doping Agency. In this study, a method for the detection and elimination of efaproxiral in equine plasma and urine after a 2.5 g intravenous administration of efaproxiral is described. Post administration plasma and urine samples were collected up to 120 h. Efaproxiral was detected up to 120 h in urine and up to 78 h in plasma. In plasma, the peak concentration was 42 µg/ml and detected at 5 min post administration. In urine, the peak concentration was 2.8 mg/ml and detected at 0-1 h post administration. A validated liquid chromatography tandem mass spectrometry method was used for the quantitation of efaproxiral in equine plasma and urine. The limit of detection of the method is 0.05 ng/ml in plasma and 0.1 ng/ml in urine. The method is highly sensitive and specific with good precision, accuracy and recovery. The manuscript also describes the systematic identification of efaproxiral metabolites detected in post administration equine urine samples. The metabolites were identified by use of enhanced mass spectra and enhanced product ion scans. Both positive and negative mode ionizations were utilized for metabolite identification and plausible fragmentation pathways were proposed for the phase 1 metabolite identified. In addition to free efaproxiral, one phase 1 metabolite and two phase 2 metabolites were identified in post administration urine.

Detection of myo-inositol tris pyrophosphate (ITPP) in equine following an administration of ITPP.

Myo-Inositol tris pyrophosphate (ITPP) is a powerful allosteric modulator of haemoglobin that increases oxygen-releasing capacity of red blood cells. It is capable of crossing the red blood cell membrane unlike its open polyphosphate analog myo-inositol hexakisphosphate (IHP). Systemic administration of ITPP enhanced the exercise capacity in mice. There have been rumours of its abuse in the horse racing industry to enhance the performance of racing horses. In this paper, the detection of ITPP in equine plasma and urine after an administration of ITPP is reported. A Standardbred mare was administered 200 mg of ITPP intravenously. Urine and plasma samples were collected up to 120 h post administration and analyzed for ITPP by liquid chromatography-tandem mass spectrometry. ITPP was detected in post administration plasma samples up to 6 hours. The peak concentration was detected at 5 min post administration. In urine, ITPP was detected up to 24 h post administration. The peak concentration was detected at 1.5 h post administration.

Severe acute illness in a toddler exposed to multiple agricultural pesticides and an insect repellent.

Acute severe pesticide-related illness among farm worker children is rarely reported. The authors report a toddler with acute onset of apnea, cyanosis, somnolence, hypotonia, tachycardia, and miosis who required hospitalization. Health care providers suspected pesticide poisoning, but were unable to determine the causal agent. Investigation by a public health program documented four pesticide exposures that occurred within one-half hour of acute illness. This case illustrates the importance of a thorough environmental/occupational exposure history and obtaining biological samples. It also documents the need to strengthen the Worker Protection Standard for agricultural workers and the importance of reporting and investigating pesticide-related illness.

Detection and elimination profile of cathinone in equine after norephedrine (Propalin®) administration using a validated liquid chromatography-tandem mass spectrometry method.

Cathinone is the principal psychostimulant present in the leaves of khat shrub, which are widely used in East Africa and the Arab peninsula as an amphetamine-like stimulant. Cathinone readily undergoes metabolism in vivo to form less potent cathine and norephedrine as the metabolites. However, the presence of cathine and norephedrine in biological fluids cannot be used as an indicator of cathinone administration. The metabolism of pseudoephedrine and ephedrine, commonly used in cold and allergy medications, also produces cathine and norephedrine, respectively, as the metabolites. Besides, cathine and norephedrine may also originate from the ingestion of nutritional supplemental products containing extracts of Ephedra species. In Canada, ephedrine and norephedrine are available for veterinary use, whereas cathinone is not approved for human or veterinary use. In this article, the detection of cathinone in equine after administration of norephedrine is reported. To the best of our knowledge, this is the first such report in any species where administration of norephedrine or ephedrine generates cathinone as the metabolite. This observation is quite significant, because in equine detection of cathinone in biological fluids could be due to administration of the potent stimulant cathinone or the nonpotent stimulant norephedrine. A single oral dose of 450 mg norephedrine was administered to four Standardbred mares. Plasma and urine samples were collected up to 120 h after administration. The amount of cathinone and norephedrine detected in post administration samples was quantified using a highly sensitive, specific, and validated liquid chromatography-tandem mass spectrometry method. Using these results, we constructed elimination profiles for cathinone and norephedrine in equine plasma and urine. A mechanism that generates a geminal diol as an intermediate is postulated for this in vivo conversion of norephedrine to cathinone. Cathinone was also detected in samples collected after a single intramuscular administration of 200 mg ephedrine and oral administration of 300 mg ephedrine in equine.

Pyrethrin and pyrethroid illnesses in the Pacific northwest: a five-year review.

OBJECTIVE: Pyrethrin and pyrethroid insecticides are commonly applied in homes and businesses and on some agricultural crops. This research used a two-state regional approach to analyze reports of acute pesticide poisonings due to pyrethrin and pyrethroid insecticides. METHODS: The Washington State Department of Health and the Oregon Public Health Division collected pesticide poisoning surveillance data from 2001 through 2005. Cases were included if they involved exposure to at least one pyrethrin or pyrethroid insecticide. Descriptive statistics were calculated; differences between categories were assessed using Chi-square analysis. RESULTS: A total of 407 cases fit our definition. Overall, the rate of poisoning in Oregon was significantly higher than in Washington (incidence rate ratio 1.70, 95% confidence interval 1.40, 2.07), and rates for both states generally increased during the time period. For both states, most exposures resulted in low severity illnesses (92%), and most were classified as possible cases (73%). Only about one-fourth of cases were related to a person's work. The most common category of clinical signs and symptoms of illness was respiratory (52% of cases), followed by neurological (40% of cases). Exposure route was predominantly inhalation; there was no association between route and case severity. There was a significant association between illness severity and losing time from work or regular activities (p<0.0001). CONCLUSIONS: Although the majority of pyrethrin and pyrethroid poisoning cases were low in severity, adverse reactions have occurred, as transpired in Oregon in 2005. Regional analysis has the potential to improve the surveillance system and provide unique opportunities for targeting preventive interventions.

Acute pesticide poisoning among agricultural workers in the United States, 1998-2005.

BACKGROUND: Approximately 75% of pesticide usage in the United States occurs in agriculture. As such, agricultural workers are at greater risk of pesticide exposure than non-agricultural workers. However, the magnitude, characteristics and trend of acute pesticide poisoning among agricultural workers are unknown. METHODS: We identified acute pesticide poisoning cases in agricultural workers between the ages of 15 and 64 years that occurred from 1998 to 2005. The California Department of Pesticide Regulation and the SENSOR-Pesticides program provided the cases. Acute occupational pesticide poisoning incidence rates (IR) for those employed in agriculture were calculated, as were incidence rate ratios (IRR) among agricultural workers relative to non-agricultural workers. RESULTS: Of the 3,271 cases included in the analysis, 2,334 (71%) were employed as farmworkers. The remaining cases were employed as processing/packing plant workers (12%), farmers (3%), and other miscellaneous agricultural workers (19%). The majority of cases had low severity illness (N = 2,848, 87%), while 402 (12%) were of medium severity and 20 (0.6%) were of high severity. One case was fatal. Rates of illness among various agricultural worker categories were highly variable but all, except farmers, showed risk for agricultural workers greater than risk for non-agricultural workers by an order of magnitude or more. Also, the rate among female agricultural workers was almost twofold higher compared to males. CONCLUSION: The findings from this study suggest that acute pesticide poisoning in the agricultural industry continues to be an important problem. These findings reinforce the need for heightened efforts to better protect farmworkers from pesticide exposure.

Acute pesticide-related illness among emergency responders, 1993-2002.

BACKGROUND: Emergency responders are among the first to arrive at a pesticide-related release event. Magnitude, severity, and risk factor information on acute pesticide poisoning among those workers is needed. METHODS: Survey data collected from the SENSOR-Pesticides, CDPR and HSEES programs between 1993 and 2002 from 21 states were reviewed. Acute occupational pesticide-related illness incidence rates for each category of emergency responder were calculated, as were incidence rate ratios (IRR) among emergency responders compared to all other workers employed in non-agricultural industries. RESULTS: A total of 291 cases were identified. Firefighters accounted for 111 cases (38%), law enforcement officers for 104 cases (36%), emergency medical technicians for 34 cases (12%), and 42 cases (14%) were unspecified emergency responders. Among the 200 cases with information on activity responsible for exposure, most were exposed while performing activities related to a pesticide release event (84%) and not involving patient care, while the remainder involved exposure to pesticide-contaminated patients. A majority of cases were exposed to insecticides (51%). Most had low severity illnesses (90%). The incidence rate was highest for firefighters (39.1/million) and law enforcement officers (26.6/million). The IRRs were also elevated for these professions (firefighters, IRR = 2.67; law enforcement officers, IRR = 1.69). CONCLUSIONS: The findings suggest the need for greater efforts to prevent acute occupational pesticide-related illness among emergency responders.