Detection of Anatoxins in Human Urine by Liquid Chromatography Triple Quadrupole Mass Spectrometry and ELISA.

Harmful cyanobacterial blooms are becoming more common and persistent around the world. When in bloom, various cyanobacterial strains can produce anatoxins in high concentrations, which, unlike other cyanobacterial toxins, may be present in clear water. Potential human and animal exposures to anatoxins occur mainly through unintentional ingestion of contaminated algal mats and water. To address this public health threat, we developed and validated an LC-MS/MS method to detect anatoxins in human urine to confirm exposures. Pooled urine was fortified with anatoxin-a and dihydroanatoxin at concentrations from 10.0 to 500 ng/mL to create calibrators and quality control samples. Samples were diluted with isotopically labeled anatoxin and solvent prior to LC-MS/MS analysis. This method can accurately quantitate anatoxin-a with inter- and intraday accuracies ranging from 98.5 to 103% and relative standard deviations < 15%, which is within analytical guidelines for mass spectrometry methods. Additionally, this method qualitatively detects a common degradation product of anatoxin, dihydroanatoxin, above 10 ng/mL. We also evaluated a commercial anatoxin-a ELISA kit for potential diagnostic use; however, numerous false positives were detected from unexposed individual human urine samples. In conclusion, we have developed a method to detect anatoxins precisely and accurately in urine samples, addressing a public health area of concern, which can be applied to future exposure events.

Maternal and Neonatal Effects of Maternal Oral Exposure to Perfluoro-2-methoxyacetic Acid (PFMOAA) during Pregnancy and Early Lactation in the Sprague-Dawley Rat.

Perfluoro-2-methoxyacetic acid (PFMOAA) is a short-chain perfluoroalkyl ether carboxylic acid that has been detected at high concentrations (∼10 µg/L) in drinking water in eastern North Carolina, USA, and in human serum and breastmilk in China. Despite documented human exposure there are almost no toxicity data available to inform risk assessment of PFMOAA. Here we exposed pregnant Sprague-Dawley rats to a range of PFMOAA doses (10-450 mg/kg/d) via oral gavage from gestation day (GD) 8 to postnatal day (PND) 2 and compared results to those we previously reported for perfluorooctanoic acid (PFOA) and hexafluoropropylene oxide-dimer acid (HFPO-DA or GenX). Newborn pups displayed reduced birthweight (≥30 mg/kg), depleted liver glycogen concentrations (all doses), hypoglycemia (≥125 mg/kg), and numerous significantly altered genes in the liver associated with fatty acid and glucose metabolism similar to gene changes produced by HFPO-DA. Pup survival was significantly reduced at ≥125 mg/kg, and at necropsy on PND2 both maternal and neonatal animals displayed increased liver weights, increased serum aspartate aminotransferase (AST), and reduced serum thyroid hormones at all doses (≥10 mg/kg). Pups also displayed highly elevated serum cholesterol at all doses. PFMOAA concentrations in serum and liver increased with maternal oral dose in both maternal and F1 animals and were similar to those we reported for PFOA but considerably higher than HFPO-DA. We calculated 10% effect levels (ED10 or EC10) and relative potency factors (RPF; PFOA = index chemical) among the three compounds based on maternal oral dose and maternal serum concentration (µM). Reduced pup liver glycogen, increased liver weights and reduced thyroid hormone levels (maternal and pup) were the most sensitive end points modeled. PFMOAA was ∼3-7-fold less potent than PFOA for most end points based on maternal serum RPFs, but slightly more potent for increased maternal and pup liver weights. PFMOAA is a maternal and developmental toxicant in the rat producing a constellation of adverse effects similar to PFOA and HFPO-DA.

Dose additive maternal and offspring effects of oral maternal exposure to a mixture of three PFAS (HFPO-DA, NBP2, PFOS) during pregnancy in the Sprague-Dawley rat.

Simultaneous exposure to multiple per- and polyfluoroalkyl substances (PFAS) is common in humans across the globe. Individual PFAS are associated with adverse health effects, yet the nature of mixture effects after exposure to two or more PFAS remains unclear. Previously we reported that oral administration of hexafluoropropylene oxide-dimer acid (HFPO-DA, or GenX), Nafion byproduct 2 (NBP2), or perfluorooctane sulfonate (PFOS) individually during pregnancy produced maternal and F1 effects. Here, we hypothesized that responses to the combined exposure to these three PFAS would be dose additive. Pregnant Sprague-Dawley rats were exposed to a fixed-ratio equipotent mixture where the top dose contained each PFAS at their ED50 for neonatal mortality (100 % dose = PFOS 3 mg/kg; NBP2 10 mg/kg; HFPO-DA 110 mg/kg), followed by a dilution series (33.3, 10, 3.3, and 1 %) and vehicle controls (0 % dose). Consistent with the single chemical studies, dams were exposed from gestation day (GD)14-18 or from GD8-postnatal day (PND2). Fetal and maternal livers on GD18 displayed multiple significantly upregulated genes associated with lipid and carbohydrate metabolism at all dose levels, while dams displayed significantly increased liver weight (≥3.3 % dose) and reduced serum thyroid hormones (≥33.3 % dose). Maternal exposure from GD8-PND2 significantly reduced pup bodyweights at birth (≥33.3 % dose) and PND2 (all doses), increased neonatal liver weights (≥3.3 % dose), increased pup mortality (≥3.3 % dose), and reduced maternal bodyweights and weight gain at the top dose. Echocardiography of adult F1 males and females identified significantly increased left ventricular anterior wall thickness (~10 % increase), whereas other cardiac morphological, functional, and transcriptomic measures were unaffected. Mixture effects in maternal and neonatal animals conformed to dose addition using a relative potency factor (RPF) analysis. Results support dose addition-based cumulative assessment approaches for estimating combined effects of PFAS co-exposure.

Cumulative maternal and neonatal effects of combined exposure to a mixture of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) during pregnancy in the Sprague-Dawley rat.

Globally, biomonitoring data demonstrate virtually all humans carry residues of multiple per- and polyfluoroalkyl substances (PFAS). Despite pervasive co-exposure, limited mixtures-based in vivo PFAS toxicity research has been conducted. Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) are commonly detected PFAS in human and environmental samples and both produce adverse effects in laboratory animal studies, including maternal and offspring effects when orally administered during pregnancy and lactation. To evaluate the effects of combined exposure to PFOA and PFOS, we orally exposed pregnant Sprague-Dawley rats from gestation day 8 (GD8) to postnatal day 2 (PND2) to PFOA (10-250 mg/kg/d) or PFOS (0.1-5 mg/kg/d) individually to characterize effects and dose response curve parameters, followed by a variable-ratio mixture experiment with a constant dose of PFOS (2 mg/kg/d) mixed with increasing doses of PFOA (3-80 mg/kg/d). The mixture study design was intended to: 1) shift the PFOA dose response curves for endpoints shared with PFOS, 2) allow comparison of dose addition (DA) and response addition (RA) model predictions, 3) conduct relative potency factor (RPF) analysis for multiple endpoints, and 4) avoid overt maternal toxicity. Maternal serum and liver concentrations of PFOA and PFOS were consistent between the individual chemical and mixture experiments. Combined exposure with PFOS significantly shifted the PFOA dose response curves towards effects at lower doses compared to PFOA-only exposure for multiple endpoints and these effects were well predicted by dose addition. For endpoints amenable to mixture model analyses, DA produced equivalent or better estimates of observed data than RA. All endpoints evaluated were accurately predicted by RPF and DA approaches except for maternal gestational weight gain, which produced less-than-additive results in the mixture. Data support the hypothesis of cumulative effects on shared endpoints from PFOA and PFOS co-exposure and dose additive approaches for predictive estimates of mixture effects.

Prenatal ozone exposure programs a sexually dimorphic susceptibility to high-fat diet in adolescent Long Evans rats.

Altered fetal growth, which can occur due to environmental stressors during pregnancy, may program a susceptibility to metabolic disease. Gestational exposure to the air pollutant ozone is associated with fetal growth restriction in humans and rodents. However, the impact of this early life ozone exposure on offspring metabolic risk has not yet been investigated. In this study, fetal growth restriction was induced by maternal inhalation of 0.8 ppm ozone on gestation days 5 and 6 (4 hr/day) in Long Evans rats. To uncover any metabolic inflexibility, or an impaired ability to respond to a high-fat diet (HFD), a subset of peri-adolescent male and female offspring from filtered air or ozone exposed dams were fed HFD (45% kcal from fat) for 3 days. By 6 weeks of age, male and female offspring from ozone-exposed dams were heavier than offspring from air controls. Furthermore, offspring from ozone-exposed dams had greater daily caloric consumption and reduced metabolic rate when fed HFD. In addition to energy imbalance, HFD-fed male offspring from ozone-exposed dams had dyslipidemia and increased adiposity, which was not evident in females. HFD consumption in males resulted in the activation of the protective 5'AMP-activated protein kinase (AMPKα) and sirtuin 1 (SIRT1) pathways in the liver, regardless of maternal exposure. Unlike males, ozone-exposed female offspring failed to activate these pathways, retaining hepatic triglycerides following HFD consumption that resulted in increased inflammatory gene expression and reduced insulin signaling genes. Taken together, maternal ozone exposure in early pregnancy programs impaired metabolic flexibility in offspring, which may increase susceptibility to obesity in males and hepatic dysfunction in females.

Variability of Microcystin-LR Standards Available from Seven Commercial Vendors.

Microcystins (MCs) are a large group of heptapeptide cyanobacterial toxins commonly produced in harmful algal blooms (HABs) and associated with adverse health effects in wildlife, livestock, pets, and humans. MC chemical standards are extracted from cyanobacteria biomass rather than produced synthetically and are used in water assessment methods and toxicological studies. MC standards are generally supplied in less than 1 mg quantities, and verification of the mass can only be accomplished by analytical chemistry methods using a certified reference of the specific MC for comparison. Analytical quantification of MCs in environmental samples and toxicology studies using accurate doses of test chemicals administered to experimental animals rely on the availability and accuracy of chemical standards. To check the accuracy and purity of available standards, seven individual microcystin-LR (MCLR) standards were purchased from separate commercial vendors and analyzed to determine the actual mass supplied and identify the presence of potential contaminants. To determine the effect of varying toxin mass in toxicological studies, each MCLR standard was administered to CD-1 mice in doses based on mass purchased, by a single 40 µg/kg intraperitoneal injection. The measured mass purchased varied from the vendor label mass by more than 35% for two of the seven MCLR standards. Contaminants, including trifluoroacetic acid (TFA), were identified in four of the seven samples. Comparative in vivo hepatotoxicity between vendor samples closely reflected the actual amount of MCLR present in each standard and demonstrated the toxicological impact of varying cyanotoxin mass.

A rapid assessment bioaccumulation screening (RABS) study design for emerging per-and polyfluoroalkyl substances in mice exposed to industrially impacted surface water.

The shift away from PFOS and PFOA production in the past 20 years towards shorter chain and replacement PFAS has led to the environmental release of complex mixtures of emerging PFAS for which bioaccumulation potential and toxicology are largely unknown. The rate at which emerging PFAS can be prioritized for research in these complex mixtures is often limited by the lack of available chemical standards. We developed a study design that rapidly assesses which emerging PFAS in an environmentally derived mixture have the potential for mammalian bioaccumulation and thus prioritize these emerging chemicals for standard synthesis and toxicity testing. Surface water was collected at an impacted site downstream of an industrial fluorochemical manufacturing outfall and concentrated 100-fold via weak anion exchange, solid-phase extraction. The concentrated extract contained 13 previously identified emerging PFAS, including hexafluoropropylene oxide-dimer acid (HFPO-DA). BALB/c mice were orally dosed with surface water concentrate once a day for seven days. Twenty-four hours after the last dose, liver, serum, urine, and feces were collected and the emerging PFAS were semi-quantified based on peak area counts. Of the 13 emerging PFAS, Nafion byproduct-2 (Nafion BP2), Hydro-EVE, PFO4DA, and PFO5DoA had the largest increases in percent composition when comparing serum and liver to the dosing solution, suggesting that these PFAS may have the highest bioaccumulation potential. This finding supports other studies that detected bioaccumulation of the same four PFAS in human serum collected from communities with contaminated drinking water. In the future, the Rapid Assessment Bioaccumulation Screening (RABS) study design can be extended to other complex industrial chemical mixtures impacting surface water in order to better inform chemical prioritization for acquisition and in vitro/in vivo toxicity testing of the potential pollutants.

Developmental toxicity of Nafion byproduct 2 (NBP2) in the Sprague-Dawley rat with comparisons to hexafluoropropylene oxide-dimer acid (HFPO-DA or GenX) and perfluorooctane sulfonate (PFOS).

Nafion byproduct 2 (NBP2) is a polyfluoroalkyl ether sulfonic acid that was recently detected in surface water, drinking water, and human serum samples from monitoring studies in North Carolina, USA. We orally exposed pregnant Sprague-Dawley rats to NBP2 from gestation day (GD) 14-18 (0.1-30 mg/kg/d), GD17-21, and GD8 to postnatal day (PND) 2 (0.3-30 mg/kg/d) to characterize maternal, fetal, and postnatal effects. GD14-18 exposures were also conducted with perfluorooctane sulfonate (PFOS) for comparison to NBP2, as well as data previously published for hexafluoropropylene oxide-dimer acid (HFPO-DA or GenX). NBP2 produced stillbirth (30 mg/kg), reduced pup survival shortly after birth (10 mg/kg), and reduced pup body weight (10 mg/kg). Histopathological evaluation identified reduced glycogen stores in newborn pup livers and hepatocyte hypertrophy in maternal livers at ≥ 10 mg/kg. Exposure to NBP2 from GD14-18 reduced maternal serum total T3 and cholesterol concentrations (30 mg/kg). Maternal, fetal, and neonatal liver gene expression was investigated using RT-qPCR pathway arrays, while maternal and fetal livers were also analyzed using TempO-Seq transcriptomic profiling. Overall, there was limited alteration of genes in maternal or F1 livers from NBP2 exposure with significant changes mostly occurring in the top dose group (30 mg/kg) associated with lipid and carbohydrate metabolism. Metabolomic profiling indicated elevated maternal bile acids for NBP2, but not HFPO-DA or PFOS, while all three reduced 3-indolepropionic acid. Maternal and fetal serum and liver NBP2 concentrations were similar to PFOS, but ∼10-30-fold greater than HFPO-DA concentrations at a given maternal oral dose. NBP2 is a developmental toxicant in the rat, producing neonatal mortality, reduced pup body weight, reduced pup liver glycogen, reduced maternal thyroid hormones, and altered maternal and offspring lipid and carbohydrate metabolism similar to other studied PFAS, with oral toxicity for pup loss that is slightly less potent than PFOS but more potent than HFPO-DA.

Application of whole-genome sequencing for norovirus outbreak tracking and surveillance efforts in Orange County, CA.

Noroviruses are the leading cause of acute gastroenteritis and foodborne illness in the United States. Traditional Sanger sequencing of short genomic regions (~300-600 bp) is the primary method for differentiation of this pathogen; however, whole-genome sequencing (WGS) offers a valuable approach to further characterize strains of this virus. The objective of this study was to investigate the ability of WGS compared to Sanger sequencing to differentiate norovirus strains and enhance outbreak investigation and surveillance efforts. WGS results for 41 norovirus-positive stool samples from 15 different outbreaks occurring from 2012 to 2019 in Orange County, CA, were analyzed for this study. All samples were genotyped with both WGS and Sanger sequencing based on the B-C region. WGS generated nearly full-length viral genome sequences (7029-7768 bp) with 4x to 35,378x coverage. Phylogenetic analysis of WGS data enabled differentiation of genotypically similar strains from separate outbreaks. Single nucleotide variation (SNV) analysis on a subset of strains revealed nucleotide variations (15-79 nt) among isolates from multiple outbreaks of GII.4 Sydney_2015[P31] and GII.17[P17]. Overall, the results demonstrated that coupling norovirus genotype identification with WGS enables enhanced genetic differentiation of strains and provides valuable information for outbreak investigation and surveillance efforts.

Dose-Response Study of Microcystin Congeners MCLA, MCLR, MCLY, MCRR, and MCYR Administered Orally to Mice.

Microcystins are common freshwater cyanobacterial toxins that affect liver function. The toxicities of five microcystin congeners (microcystin-LA (MCLA), MCLR, MCLY, MCRR, and MCYR) commonly observed in harmful algal blooms (HABs) were evaluated in BALB/c mice after a single oral administration of doses ranging from those that were no observed adverse effect levels (NOAELs) to lowest observed adverse effect levels (LOAELs). Animals were monitored for changes in behavior and appearance, and euthanized 24 h after dosing. Test endpoints included clinical changes, necropsy observations, and serum indicators of hepatic toxicity and general homeostasis. Doses were 0.5-7 mg/kg MCLA, 0.5-11 mg/kg MCLR, 1-7 mg/kg MCLY, 7-22 mg/kg MCRR, and 3-11 mg/kg MCYR. MCLA at 3 mg/kg elevated liver/body weight ratio and liver score, ALT, AST, and GLDH, indicating hepatic toxicity, reduced serum glucose and highly elevated total serum bilirubin. MCLR and MCLY induced similar effects with LOAELs of 5 mg/kg, although a greater extent and severity of effects were observed in MCLR animals. MCRR exposure at 22 mg/kg was associated with reduced serum glucose. MCYR induced scattered liver effects at 7 mg/kg and reduced serum glucose levels at 5 mg/kg. The results indicate significant differences in congener-induced toxicity after microcystin exposure.

Hexafluoropropylene oxide-dimer acid (HFPO-DA or GenX) alters maternal and fetal glucose and lipid metabolism and produces neonatal mortality, low birthweight, and hepatomegaly in the Sprague-Dawley rat.

Hexafluoropropylene oxide dimer acid (HFPO-DA or GenX) is an industrial replacement for the straight-chain perfluoroalkyl substance (PFAS), perfluorooctanoic acid (PFOA). Previously we reported maternal, fetal, and postnatal effects from gestation day (GD) 14-18 oral dosing in Sprague-Dawley rats. Here, we further evaluated the perinatal toxicity of HFPO-DA by orally dosing rat dams with 1-125 mg/kg/d (n = 4 litters per dose) from GD16-20 and with 10-250 mg/kg/d (n = 5) from GD8 - postnatal day (PND) 2. Effects of GD16-20 dosing were similar to those previously reported for GD14-18 dosing and included increased maternal liver weight, altered maternal serum lipid and thyroid hormone concentrations, and altered expression of peroxisome proliferator-activated receptor (PPAR) pathway genes in maternal and fetal livers. Dosing from GD8-PND2 produced similar effects as well as dose-responsive decreased pup birth weight (≥30 mg/kg), increased neonatal mortality (≥62.5 mg/kg), and increased pup liver weight (≥10 mg/kg). Histopathological evaluation of newborn pup livers indicated a marked reduction in glycogen stores and pups were hypoglycemic at birth. Quantitative gene expression analyses of F1 livers revealed significant alterations in genes related to glucose metabolism at birth and on GD20. Maternal serum and liver HFPO-DA concentrations were similar between dosing intervals, indicating rapid clearance, however dams dosed GD8 - PND2 had greater liver weight and gestational weight gain effects at lower doses than GD16-20 dosing, indicating the importance of exposure duration. Comparison of neonatal mortality dose-response curves between HFPO-DA and previously published perfluorooctane sulfonate (PFOS) data indicated that, based on serum concentration, the potency of these two PFAS are similar in the rat. Overall, HFPO-DA is a developmental toxicant in the rat and the spectrum of adverse effects is consistent with prior PFAS toxicity evaluations, such as PFOS and PFOA.

The Comparative Toxicity of 10 Microcystin Congeners Administered Orally to Mice: Clinical Effects and Organ Toxicity.

Microcystins (MCs) are common cyanobacterial toxins that occur in freshwaters worldwide. Only two of the >200 MC variants have been tested for potential toxicity after oral exposure. This paper reports on the toxicity of 10 different MC congeners identified in algal blooms, microcystin-LR (MCLR), MCLA, MCLF, MCLW, MCLY, MCRR, [Asp3]MCRR, [Asp3,Dhb7]MCRR, MCWR, and MCYR after single administrations to BALB/c mice. In a preliminary MCLR dose-response study of 3 to 9 mg/kg doses, ≥5 mg/kg induced clinical changes, increased serum levels of ALT, AST, and GLDH, liver congestion, increased liver/body weight ratios, and reduced serum glucose and total protein. Based on the extent of these effects, the 10 congeners were administered as single 7 mg/kg oral doses and toxicity evaluated. The greatest toxicity was observed with MCLA and MCLR including a high percentage of moribundity. In addition to eliciting effects similar to those listed above for MCLR, MCLA also induced serum alterations indicative of jaundice. MCLY, and MCYR induced changes like those noted with MCLR, but to lesser extents. MCLW and MCLF exhibited some serum and morphological changes associated with hepatic toxicity, while there were few indications of toxicity after exposures to MCRR, [Asp3]MCRR, [Asp3,Dhb7]MCRR, or MCWR. These data illustrate a wide spectrum of hepatic effects and different potencies of these MC congeners.

Toxicity of Balb-c mice exposed to recently identified 1,1,2,2-tetrafluoro-2-[1,1,1,2,3,3-hexafluoro-3-(1,1,2,2-tetrafluoroethoxy)propan-2-yl]oxyethane-1-sulfonic acid (PFESA-BP2).

1,1,2,2-tetrafluoro-2-[1,1,1,2,3,3-hexafluoro-3-(1,1,2,2-tetrafluoroethoxy)propan-2-yl]oxyethane-1-sulfonic acid (PFESA-BP2) was first detected in 2012 in the Cape Fear River downstream of an industrial manufacturing facility. It was later detected in the finished drinking water of municipalities using the Cape Fear River for their water supply. No toxicology data exist for this contaminant despite known human exposure. To address this data gap, mice were dosed with PFESA-BP2 at 0, 0.04, 0.4, 3, and 6 mg/kg-day for 7 days by oral gavage. As an investigative study, the final dose groups evolved from an original dose of 3 mg/kg which produced liver enlargement and elevated liver enzymes. The dose range was extended to explore a no effect level. PFESA-BP2 was detected in the sera and liver of all treated mice. Treatment with PFESA-BP2 significantly increased the size of the liver for all mice at 3 and 6 mg/kg-day. At the 6 mg/kg-day dose, the liver more than doubled in size compared to the control group. Male mice treated with 3 and 6 mg/kg-day and females treated with 6 mg/kg-day demonstrated significantly elevated serum markers of liver injury including alanine aminotransferase (ALT), glutamate dehydrogenase (GLDH), and liver/body weight percent. The percent of PFESA-BP2 in serum relative to the amount administered was similar in male and female mice, ranged from 9 to 13 %, and was not related to dose. The percent accumulation in the liver of the mice varied by sex (higher in males), ranged from 30 to 65 %, and correlated positively with increasing dose level.

Outbreak of Tattoo-associated Nontuberculous Mycobacterial Skin Infections.

BACKGROUND: On 29 April 2015, the Florida Department of Health in Miami-Dade County (DOH Miami-Dade) was notified by a local dermatologist of 3 patients with suspected nontuberculous mycobacterial (NTM) infection after receiving tattoos at a local tattoo studio. METHODS: DOH Miami-Dade conducted interviews and offered testing, described below, to tattoo studio clients reporting rashes. Culture of clinical isolates and identification were performed at the Florida Bureau of Public Health Laboratories. Characterization of NTM was performed by the Centers for Disease Control and Prevention and the US Food and Drug Administration (FDA), respectively. Whole-genome sequencing (WGS) and single-nucleotide polymorphism (SNP) analyses were used to construct a phylogeny among 21 Mycobacterium isolates at the FDA. RESULTS: Thirty-eight of 226 interviewed clients were identified as outbreak-associated cases. Multivariate logistic regression revealed that individuals who reported gray tattoo ink in their tattoos were 8.2 times as likely to report a rash (95% confidence interval, 3.1-22.1). Multiple NTM species were identified in clinical and environmental specimens. Phylogenetic results from environmental samples and skin biopsies indicated that 2 Mycobacterium fortuitum isolates (graywash ink and a skin biopsy) and 11 Mycobacterium abscessus isolates (5 from the implicated bottle of graywash tattoo ink, 2 from tap water, and 4 from skin biopsies) were indistinguishable. In addition, Mycobacterium chelonae was isolated from 5 unopened bottles of graywash ink provided by 2 other tattoo studios in Miami-Dade County. CONCLUSIONS: WGS and SNP analyses identified the tap water and the bottle of graywash tattoo ink as the sources of the NTM infections.

Quantification of Microcystin-LR in Human Urine by Immunocapture Liquid Chromatography Tandem Mass Spectrometry.

Microcystins are toxins produced by many cyanobacteria species, which are often released into waterways during blue-green algal blooms in freshwater and marine habitats. The consumption of microcystin-contaminated water is a public health concern as these toxins are recognized tumor promoters and are hepatotoxic to humans and animals. A method to confirm human exposures to microcystins is needed; therefore, our laboratory has developed an immunocapture liquid chromatography tandem mass spectrometry (LC-MS/MS) method targeting the conserved adda portion of microcystins for the quantitation of a prevalent and highly toxic congener of microcystin, microcystin-LR (MC-LR). An acute exposure method was initially evaluated for accuracy and precision by analyzing calibrators and quality control (QC) samples ranging from 0.500 to 75.0 ng/mL in urine. All calibrators and QC samples characterized were within 15% of theoretical concentrations. An analysis of acutely exposed mouse urine samples using this method identified MC-LR levels from 10.7 to 33.9 ng/mL. Since human exposures are anticipated to result from low-dose or chronic exposures, a high-sensitivity method was validated with 20 calibration curves and QC samples ranging from 0.0100 to 7.50 ng/mL. Relative standard deviations (RSDs) and inaccuracies of these samples were within 15%, meeting United States Food and Drug Administration (FDA) guidelines for analytical methods, and the limit of detection was 0.00455 ng/mL. In conclusion, we have developed a method which can be used to address public health concerns by precisely and accurately measuring MC-LR in urine samples.

pH dependent octanol-water partitioning coefficients of microcystin congeners.

Hazardous algal blooms can generate toxic compounds with significant health impacts for exposed communities. The ubiquitous class of algal toxins known as microcystins exhibits significant heterogeneity in its peptide structure, which has been minimally studied, given the significant impact this has on hydrophobicity, acid/base character and related environmental fate and health effects. Octanol-water partition coefficients for the microcystin congeners MCLR, MCRR, MCLY, MCLF, and MCLA were calculated over an environmentally and physiologically relevant pH range. Microcystin-LR log(Kow) partition coefficient values were found to be consistent with previously established literature values, 1.67 to -1.41 between pH 1 and 8. Microcystin RR was found to be pH insensitive with a log(Kow) of -0.7. The remaining congeners exhibit similar pH dependence as MCLR, with systematic increases in hydrophobicity driven by the introduction of more hydrophobic residues to their variable amino acid region. The variation in pH dependent hydrophobicity suggests increased propensity for bioaccumulation and alternate environmental fates for differing microcystin forms, requiring further investigation.

Improved TaqMan real-time assays for detecting hepatitis A virus.

Rapid advancement in genomics and bioinformatics in recent years holds great promise for research and development in many disciplines including public health. For the detection of pathogens, methods based on nucleic acid amplification need to be re-evaluated periodically to ensure the validity of signature primers and probes as more and more outbreak strains are sequenced and collected into databases in public domains. In this study, a previous assay designed computationally for detecting hepatitis A virus (HAV) was re-examined. Alignment of 57 complete or near complete HAV genomes allowed identification of conserved sequences for developing new primers and TaqMan probes. Two sets of real-time reverse transcription PCR reagents were developed by targeting highly conserved regions with primers and probes having optimal melting temperatures and minimum secondary structures. These two assays had 10 to 1000 fold lower detection limits than the previous assay when tested using representative human HAV genotypes IA, IB, and IIIA. The better of the two improved assays had a detection limit of 3.7 × 10-2 to 6.6 × 10-2 TCID50 or less. The improved detection sensitivity was likely due to improvement in the following four areas: 1) The Gardner1 probe has a single nucleotide mismatch at the 5' end in all 19 strains of genotypes IIIA and IIIB. 2) For the Gardner1 forward primer, there is a mismatch corresponding to the 3' end of the oligonucleotides in two strains belonging to genotype IA. 3) The Gardner1 probe had a melting temperature of 66.2 °C, which is less than the optimum of 68-70 °C (Dorak, 2006). 4) The Gardner1 forward and reverse primers had high potential of forming primer dimers. The improved HAV detection assays developed in this study would support better food safety surveillance initiatives and response to disease outbreaks of viral food-borne illness.

Interlaboratory Validation for a Real-Time PCR Salmonella Detection Method Using the ABI 7500 FAST Real-Time PCR System.

Sixteen FERN (Food Emergency Response Network) member laboratories collaborated in this study to verify extension of the real-time PCR Salmonella detection method originally designed for the single-tube Cepheid SmartCycler II and validated against the Salmonella method of the U. S. Food and Drug Administration Bacteriological Analytical Manual to the Applied Biosystems (ABI) 7500 FAST Real-Time PCR system multiwell plate platform. Four foods were selected for this study: chili powder, soft cheese, fish, and tomatoes; these foods represent products that are commonly analyzed for the presence of Salmonella for regulatory purposes. Each food consisted of six uninoculated control samples, six samples inoculated with low Salmonella levels (target 1 to 5 CFU/25 g), and six samples inoculated with high levels (target 10 to 50 CFU/25 g). All samples were tested for Salmonella using the 24-h quantitative PCR (qPCR) method for detecting Salmonella, which utilizes modified buffered peptone water as the sole enrichment medium and an internal control for the qPCR. Each of these 18 samples was individually analyzed for Salmonella by the collaborating laboratories using both the ABI 7500 FAST system (alternative method) and the SmartCycler II system (reference method). Statistical analysis of the data revealed no significant difference (P ≥ 0.05) between these two qPCR platforms except for the chili powder samples. The differences noted with chili powder (P = 0.0455) were attributed to the enhanced sensitivity of the ABI 7500 FAST system compared with the SmartCycler II system. The detection limit of both qPCR methods was 0.02 to 0.15 CFU/g. These results provide a solid basis for extending the 24-h qPCR Salmonella method to the ABI 7500 FAST system for high-throughput detection of Salmonella in foods.

Rapid detection and differentiation of human noroviruses using RT-PCR coupled to electrospray ionization mass spectrometry.

The goal of this study was to develop an assay for the detection and differentiation of noroviruses using RT-PCR followed by electrospray ionization mass spectrometry (ESI-MS). Detection of hepatitis A virus was also considered. Thirteen primer pairs were designed for use in this assay and a reference database was created using GenBank sequences and reference norovirus samples. The assay was tested for inclusivity and exclusivity using 160 clinical norovirus samples, 3 samples of hepatitis A virus and 3 other closely related viral strains. Results showed that the assay was able to detect norovirus with a sensitivity of 92% and a specificity of 100%. Norovirus identification at the genogroup level was correct for 98% of samples detected by the assay and for 75% of a subset of samples (n = 32) compared at the genotype level. Identification of norovirus genotypes is expected to improve as more reference samples are added to the database. The assay was also capable of detecting and genotyping hepatitis A virus in all 3 samples tested. Overall, the assay developed here allows for detection and differentiation of noroviruses within one working day and may be used as a tool in surveillance efforts or outbreak investigations.

Use of porcine vaginal tissue ex-vivo to model environmental effects on vaginal mucosa to toxic shock syndrome toxin-1.

Menstrual toxic shock syndrome (mTSS) is a rare, recognizable, and treatable disease that has been associated with tampon use epidemiologically. It involves a confluence of microbial risk factors (Staphylococcus aureus strains that produce the superantigen-TSST-1), as well as environmental characteristics of the vaginal ecosystem during menstruation and host susceptibility factors. This paper describes a series of experiments using the well-characterized model of porcine vaginal mucosa ex-vivo to assess the effect of these factors associated with tampon use on the permeability of the mucosa. The flux of radiolabeled TSST-1 and tritiated water ((3)H2O) through porcine vaginal mucosa was determined at various temperatures, after mechanical disruption of the epithelial surface by tape stripping, after treatment with surfactants or other compounds, and in the presence of microbial virulence factors. Elevated temperatures (42, 47 and 52°C) did not significantly increase flux of (3)H2O. Stripping of the epithelial layers significantly increased the flux of labeled toxin in a dose-dependent manner. Addition of benzalkonium chloride (0.1 and 0.5%) and glycerol (4%) significantly increased the flux of (3)H2O but sodium lauryl sulfate at any concentration tested did not. The flux of the labeled toxin was significantly increased in the presence of benzalkonium chloride but not Pluronic® L92 and Tween 20 and significantly increased with addition of α-hemolysin but not endotoxin. These results show that the permeability of porcine vagina ex-vivo to labeled toxin or water can be used to evaluate changes to the vaginal environment and modifications in tampon materials, and thus aid in risk assessment.