Mixed infections with Chlamydia and porcine epidemic diarrhea virus - a new in vitro model of chlamydial persistence

BACKGROUND: Chlamydiae induce persistent infections, which have been associated with a wide range of chronic diseases in humans and animals. Mixed infections with Chlamydia and porcine epidemic diarrhea virus (PEDV) may result in generation of persistent chlamydial infections. To test this hypothesis, an in vitro model of dual infection with cell culture-adapted PEDV and Chlamydia abortus or Chlamydia pecorum in Vero cells was established. RESULTS: Infected cultures were investigated by immunofluorescence (IF), transmission electron microscopy (TEM) and re-infection experiments. By IF, Chlamydia-infected cells showed normal inclusions after 39 hpi. Dual infections with Chlamydia abortus revealed a heterogenous mix of inclusion types including small inclusions consisting of aberrant bodies (ABs), medium-sized inclusions consisting of ABs and reticulate bodies and normal inclusions. Only aberrant inclusions were observable in dual infection experiments with Chlamydia pecorum and PEDV. TEM examinations of mixed infections with Chlamydia abortus and Chlamydia pecorum revealed aberrant chlamydial inclusions containing reticulate-like, pleomorphic ABs, which were up to 2 microm in diameter. No re-differentiation into elementary bodies (EBs) was detected. In re-infection experiments, co-infected cells produced fewer EBs than monoinfected cells. CONCLUSIONS: In the present study we confirm that PEDV co-infection alters the developmental cycle of member species of the family Chlamydiaceae, in a similar manner to other well-described persistence induction methods. Interestingly, this effect appears to be partially species-specific as Chlamydia pecorum appears more sensitive to PEDV co-infection than Chlamydia abortus, as evidenced by TEM and IF observations of a homogenous population of aberrant inclusions in PEDV - Chlamydia pecorum co-infections

Mixtures of Chemical Pollutants at European Legislation Safety Concentrations: How Safe Are They?

The risk posed by complex chemical mixtures in the environment to wildlife and humans is increasingly debated, but has been rarely tested under environmentally relevant scenarios. To address this issue, two mixtures of 14 or 19 substances of concern (pesticides, pharmaceuticals, heavy metals, polyaromatic hydrocarbons, a surfactant, and a plasticizer), each present at its safety limit concentration imposed by the European legislation, were prepared and tested for their toxic effects. The effects of the mixtures were assessed in 35 bioassays, based on 11 organisms representing different trophic levels. A consortium of 16 laboratories was involved in performing the bioassays. The mixtures elicited quantifiable toxic effects on some of the test systems employed, including i) changes in marine microbial composition, ii) microalgae toxicity, iii) immobilization in the crustacean Daphnia magna, iv) fish embryo toxicity, v) impaired frog embryo development, and vi) increased expression on oxidative stress-linked reporter genes. Estrogenic activity close to regulatory safety limit concentrations was uncovered by receptor-binding assays. The results highlight the need of precautionary actions on the assessment of chemical mixtures even in cases where individual toxicants are present at seemingly harmless concentration

Mixtures of chemical pollutants at European legislation safety concentrations: how safe are they?

The risk posed by complex chemical mixtures in the environment to wildlife and humans is increasingly debated, but has been rarely tested under environmentally relevant scenarios. To address this issue, two mixtures of 14 or 19 substances of concern (pesticides, pharmaceuticals, heavy metals, polyaromatic hydrocarbons, a surfactant, and a plasticizer), each present at its safety limit concentration imposed by the European legislation, were prepared and tested for their toxic effects. The effects of the mixtures were assessed in 35 bioassays, based on 11 organisms representing different trophic levels. A consortium of 16 laboratories was involved in performing the bioassays. The mixtures elicited quantifiable toxic effects on some of the test systems employed, including i) changes in marine microbial composition, ii) microalgae toxicity, iii) immobilization in the crustacean Daphnia magna, iv) fish embryo toxicity, v) impaired frog embryo development, and vi) increased expression on oxidative stress-linked reporter genes. Estrogenic activity close to regulatory safety limit concentrations was uncovered by receptor-binding assays. The results highlight the need of precautionary actions on the assessment of chemical mixtures even in cases where individual toxicants are present at seemingly harmless concentrations

Comparison of yeast estrogen screening on HPTLC and in microtiter plates

In contrast to testing whole mixtures in microtiter plates, separation on HPTLC plates with subsequent bioassay detection has the potential to reveal multiple toxic chemicals and help identify responsible entities. The planar-YES gas been used to screen various samples on HPTLC plates, but there has been little to no comparison to teh official method. Herein, the p-YES and the microtiter YES were compared in their sensitivity to screen for endocrine active compounds related to food packaging materials

Performance of measuring estrogenicity with planar-YES compared to 96-well plate YES

The yeast estrogen screen (YES) performed directly on high performance thin-layer chromatography plates (planar-YES) is a promising tool for screening and effect-directed analysis (EDA) of (xeno)estrogens, and it has recently experienced several improvements. The planar-YES eliminates the need for carrier solvents, and can be performed immediately after chromatographic separation. However, there has been no direct comparison of the planar-YES to a 96-well plate version of the YES To fill this gap, we tested several chemicals in the planar-YES and a 96 well plate YES (lyticase-YES, L-YES). We demonstrate that the detection limit for the reference substance (17β-estradiol) in the planar-YES is an order of magnitude lower than in the L-YES. For both assays, we present the bioactivity of 20 chemicals chosen for their relationship to food packaging materials. Fourteen of the 20 chemicals gave positive responses. The bioassays may be susceptible to losing test substances when the extract is transferred into the assay, e.g. through evaporation in the planar-YES or sorption to the test chambers in the L-YES. Therefore, we explore the bioactivity of the 20 chemicals with respect to their physico-chemical properties. This work helps us understand the advantages and limitations of both assays for estrogen screening and EDA

Estrogenic activity of food contact materials : evaluation of 20 chemicals using a yeast estrogen screen on HPTLC or 96-well plates

Food contact materials (FCM) may contain complex mixtures of estrogenic chemicals. A yeast estrogen screen performed on high performance thin-layer chromatography plates (planar-YES, P-YES) is promising for analysis of such mixtures, as it could allow for better elucidation of effects compared with established methods in microtiter plates. However, the P-YES has not been directly compared with established methods. We compared the performance of a microtiter plate YES (lyticase-YES, L-YES) to P-YES on silica gel HPTLC plates using 17β-estradiol (E2), 20 chemicals representative of migrants from plastic FCM, and three migrates of coated metal food cans. Effective doses (ED10, ED50) and estradiol equivalencies were calculated for each chemical. Thirteen chemicals had calculable EDs in the L-YES or P-YES, with average EDs 13-fold (range 0.63–36) more potent in P-YES than in the L-YES. Normalized to E2, the median estrogenicity was within 1.5-fold (0.43–8.8) between the assays. Therefore, P-YES was as or more sensitive than L-YES but potencies relative to E2 were comparable between assays. With chromatography, the P-YES detected estrogenicity in coated metal cans, effects that were unmeasurable in L-YES. With the sample preparation methods used in this study, both YES assays are sufficiently sensitive to detect bisphenol A below the specific migration limit for plastic packaging (0.05 mg/kg food). This study demonstrates that P-YES outperforms L-YES because it is more sensitive, provides comparable estradiol equivalents, and circumvents confounding mixture effects. The P-YES will be useful for routine monitoring of FCM and toxicant identification in problematic materials

Effects of treated wastewater on the ecotoxicity of small streams - Unravelling the contribution of chemicals causing effects.

Wastewater treatment plant effluents are important point sources of micropollutants. To assess how the discharge of treated wastewater affects the ecotoxicity of small to medium-sized streams we collected water samples up- and downstream of 24 wastewater treatment plants across the Swiss Plateau and the Jura regions of Switzerland. We investigated estrogenicity, inhibition of algal photosynthetic activity (photosystem II, PSII) and growth, and acetylcholinesterase (AChE) inhibition. At four sites, we measured feeding activity of amphipods (Gammarus fossarum) in situ as well as water flea (Ceriodaphnia dubia) reproduction in water samples. Ecotoxicological endpoints were compared with results from analyses of general water quality parameters as well as a target screening of a wide range of organic micropollutants with a focus on pesticides and pharmaceuticals using liquid chromatography high-resolution tandem mass spectrometry. Measured ecotoxicological effects in stream water varied substantially among sites: 17β-estradiol equivalent concentrations (EEQbio, indicating the degree of estrogenicity) were relatively low and ranged from 0.04 to 0.85 ng/L, never exceeding a proposed effect-based trigger (EBT) value of 0.88 ng/L. Diuron equivalent (DEQbio) concentrations (indicating the degree of photosystem II inhibition in algae) ranged from 2.4 to 1576 ng/L and exceeded the EBT value (70 ng/L) in one third of the rivers studied, sometimes even upstream of the WWTP. Parathion equivalent (PtEQbio) concentrations (indicating the degree of AChE inhibition) reached relatively high values (37 to 1278 ng/L) mostly exceeding the corresponding EBT (196 ng/L PtEQbio). Decreased feeding activity by amphipods or decreased water flea reproduction downstream compared to the upstream site was observed at one of four investigated sites only. Results of the combined algae assay (PSII inhibition) correlated best with results of chemical analysis for PSII inhibiting herbicides. Estrogenicity was partly and AChE inhibition strongly underestimated based on measured steroidal estrogens respectively organophosphate and carbamate insecticides. An impact of dissolved organic carbon on results of the AChE inhibition assay was obvious. For this assay more work is required to further explore the missing correlation of bioassay data with chemical analytical data. Overall, the discharge of WWTP effluent led to increased estrogenicity, PSII and AChE inhibition downstream, irrespective of upstream land use

Evaluation of Three ISO Estrogen Receptor Transactivation Assays Applied to 52 Domestic Effluent Samples

Estrogens are released to the aquatic environment by wastewater treatment plant (WWTP) effluents and can affect wildlife. In the last three decades, many in vitro assay platforms have been developed to detect and quantify estrogenicity in water. In 2018, the International Organization for Standardization (ISO) standardized protocols became available for three types of in vitro estrogen receptor transactivation assays (ERTAs) detecting estrogenicity in 96-well plates (ISO19040 1-3). Two ERTAs-lyticase Yeast Estrogen Screen (L-YES) and Arxula YES (A-YES)-use genetically modified yeast strains, whereas the third utilizes stably transfected human cells. One human cell based assay is ER alpha-CALUX, which is based on a genetically modified human bone osteosarcoma cell line. In the present study, we characterized the performance, comparability, and effectiveness of these three ERTAs, including an evaluation involving proposed water quality thresholds (effect-based trigger values [EBTs]). For a robust evaluation, we collected 52 effluent samples over three sampling campaigns at 15 different WWTPs in Switzerland. Estrogen receptor transactivation assay results were correlated and compared with results from chemical analysis targeting known estrogens. The three ERTAs showed comparable data over all campaigns. However, the selection of EBTs plays a significant role in the interpretation and comparison of bioassay results to distinguish between acceptable and unacceptable water quality. Applying a fixed cross-assay EBT for effluent of 4 ng L-1 resulted in varying numbers of threshold exceedances ranging between zero and four samples depending on the ERTA used. Using assay-specific EBTs showed exceedances in eight samples (ER alpha-CALUX) and in one sample (A-YES), respectively. Thus, proposed EBTs do not produce similar risk profiles across samples and further refinement of assay-specific EBTs is needed to account for assay-specific differences and to enable the application of ERTAs as effect-based methods in environmental monitoring. Environ Toxicol Chem 2022;00:1-15. (c) 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.ISSN:0277-2248ISSN:1029-0486ISSN:0092-986

Probiotic gut-borne Saccharomyces cerevisiae reduces liver toxicity caused by aflatoxins in weanling piglets

The present study was conducted to investigate the aflatoxin B1 (AFB1) liver toxicity and gut histomorphometry after gut borne-Saccharomyces cerevisiae supplementation to AFB1-contaminated piglet diets. Thirty-two male mixed-breed piglets (weaned at 21 days old) were housed in individual pens and allowed to acclimate for 7 days. Animals were randomly assigned to four treatments of 22 days: T1 - low AFB1 levels diet (L, 31.6 μg/kg); T2 - L + S. cerevisiae 1 g/kg; T3 - high AFs levels diet (H, 495 μg/kg); T4 - H + S. cerevisiae 1 g/kg. The addition of probiotic yeast was able to reduce 72% of residual AFB1 present in the liver. The liver histopathology of piglets fed AFB1 showed a typical macroscopic and microscopic pattern of subclinical aflatoxicosis that was prevented by the yeast. Also, the addition of the yeast was able to decrease the alanine-aminotransferase (25.5±0.71 U) and aspartate-aminotransferase (26.5±6.10 U) even showing values lower than the control ones. The apparent absorption area showed the greater surface when the probiotic was present alone compared to the control (T1), whereas when present together with the toxin demonstrated a modulatory effect. The addition of probiotic gut-borne S. cerevisiae in the pig diets was effective in counteracting the toxic effects of harmful AFB1 in livers besides a tendency to improve the histomorphometric parameters and modulating the toxic effect of AFB1 on intestine. These results are promising for the production of feed additives that will be used in animal feed, since the probiotic action and the decontamination of mycotoxins in the same product are complemented.Fil: Poloni, Valeria Lorena. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Microbiología e Inmunología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Magnoli, Alejandra Paola. Universidad Nacional de Río Cuarto. Facultad de Agronomía y Veterinaria. Departamento de Producción Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Fochesato, Analía Silvia. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Microbiología e Inmunología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Poloni, Luis Alberto. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Cristofolini, Andrea Lorena. Universidad Nacional de Río Cuarto. Facultad de Agronomía y Veterinaria. Departamento de Patología Animal. Área de Microscopia Electrónica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Merkis, Cecilia Inés. Universidad Nacional de Río Cuarto. Facultad de Agronomía y Veterinaria. Departamento de Patología Animal. Área de Microscopia Electrónica; ArgentinaFil: Schifferli Riquelme, Carlos Antonio. Universidad San Sebastián; ChileFil: Schifferli Maldonado, Fabian Andres. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Microbiología e Inmunología; ArgentinaFil: Montenegro, Mariana Angélica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones y Transferencia de Villa María. Universidad Nacional de Villa María. Centro de Investigaciones y Transferencia de Villa María; ArgentinaFil: Cavaglieri, Lilia Reneé. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Microbiología e Inmunología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentin

Effect-based tools for monitoring estrogenic mixtures: Evaluation of five in vitro bioassays

In vitro estrogen receptor transactivation assays (ERTAs) are increasingly used to measure the overall estrogenic activity of environmental water samples, which may serve as an indicator of exposure of fish or other aquatic organisms to (xeno)estrogens. Another potential area of application of ERTAs is to assist the monitoring of the potent steroids 17 beta-estradiol (E2) and 17 alpha-ethinylestradiol (EE2) under the Water Framework Directive (WFD) watch-list mechanism. Chemical analysis of E2 and EE2 is currently hampered by limits of quantification being mostly above the proposed annual average Environmental Quality Standards (AA-EQS) of 0.4 and 0.035 ng/L, respectively. Sensitive ERTAs could circumvent current detection challenges by measuring total estrogenic activity expressed as E2-equivalent (EEQ) concentrations. However, the use of different ERTAs results in different EEQ concentrations for the same sample. Reasons for these differences are known, but it remains unclear how to use and interpret bioassay results in a harmonised way. The aim of this study was to compare the intra- and inter-day variability of EEQ measurements using five different ERTAs (YES, ERa-CALUX, MELN, T47D-KBluc and GeneBLAzer-ER alpha) with regard to their applicability as effect-based tools in environmental monitoring. Environmentally relevant artificial mixtures of (xeno)estrogens were prepared to represent samples with higher (i.e. multiple times the AA-EQS for E2) or lower pollution levels (i.e. around the AA-EQS for E2). Mixtures were tested either directly or following solid phase extraction (SPE). The SPE step was included, as environmental samples typically require enrichment before analysis. Samples were analysed repeatedly to test intra-day and inter-day variability. Estrogenicity was quantified using the 10% effect level (PC10) of the positive control (E2) and expressed as EEQ concentrations. The average coefficient of variation (CV) of EEQ concentrations for the five ERTAs and all samples was 32%. CV was lower for intra-day experiments (30%) compared to inter-day experiments (37%). Sample extraction using SPE did not lead to additional variability; the intra-day CV for SPE extracted samples was 28%. Of the five ERTAs, ERa-CALUX had the best precision and repeatability (overall CV of 13%). (C) 2016 Elsevier Ltd. All rights reserved