Remote sensing monitoring of mining tailings in the fluvial-estuarine-coastal ocean continuum of the Lower Doce River Valley (Brazil).

Water clarity is a key parameter of aquatic ecosystems impacted by mining tailings. Tracking down tailings dispersion along the river basin requires a regional monitoring approach. The longitudinal fluvial connectivity, river-estuary-coastal ocean, and the lateral connectivity, river-floodplain-alluvial lakes are interconnected by hydrological flows, particularly during high fluvial discharge. The present study aims to track the dispersal of iron ore tailing spill, from the collapse of the Fundão dam (Mariana, MG, Brazil), on November 5, 2015, in the Lower Doce River Valley. A semi-empirical model of turbidity data, as a water clarity proxy, and multispectral remote sensing data (MSI Sentinel-2), based on different hydrological conditions and well-differentiated water types, yielded an accuracy of 92%. Five floods (> 3187m3 s-1) and five droughts (< 231m3 s-1) events occurred from 2013 to 2020. The flood of January 2016 occurred one month after the mining slurries reached the coast, intruding tailings on some alluvial and coastal plain lakes with highly turbid waters (> 400 NTU). A fluvial plume is formed in the inner shelf adjoining the river mouth on high flow. The dispersion of river plume was categorized as plume core (turbidity > 200 NTU), plume core and inner shelf waters (100-199 NTU), other shelf water (50-99 NTU), and offshore waters (< 50 NTU). Fluvial discharge and local winds are the main drivers for river plume dispersion and transport of terrigenous material along the coast. This work provides elements for evaluating the impact of mining tailings and an approach for remote sensing regional monitoring of surface water quality.

Welfare Assessment of Adult Laboratory Zebrafish: A Practical Guide.

Teleost fish such as Danio rerio (zebrafish) have been successfully used in biomedical research since decades. Genetically altered fish lines obtained by state-of-the-art genetic technologies are serving as well-known model organisms. In Europe, following Directive 2010/63/EU, generation, breeding, and husbandry of new genetically altered lines of laboratory animals require governmental state approval in case pain, suffering, distress, or long-lasting harm to the offspring derived by breeding of these lines cannot be excluded. The identification and assessment of pain, distress, or harm, according to a severity classification of mild, moderate, severe, or humane endpoint, became a new challenging task for all scientists, animal technicians, and veterinarians for daily work with laboratory zebrafish. In this study, we describe the performance of the assessment of welfare parameters of selected pathologic phenotypes and abnormalities frequently found in laboratory fish facilities based on veterinary, biological, and physiological aspects by using a dedicated score sheet. In a colony of zebrafish, we evaluated the frequency of genotype-independent abnormalities observed within 3 years. We give examples for severity classification and measures once an abnormality has been identified according to the 3Rs (Replacement, Reduction and Refinement).

Determination of 3-iodothyronamine (3-T1AM) in mouse liver using liquid chromatography-tandem mass spectrometry.

3-iodothyronamine (3-T1AM) has been suggested as a novel chemical messenger and potent trace amine-associated receptor 1 ligand in the CNS that occurs naturally as endogenous metabolite of the thyroid hormones. Discrepancies and variations in 3-T1AM plasma and tissue concentrations have nonetheless caused controversy regarding the existence and biological role of 3-T1AM. These discussions are at least partially based on potential analytical artefacts caused by differential decay kinetics of 3-T1AM and the widely used deuterated quantification standard D4-T1AM. Here, we report a novel LC-MS/MS method for the quantification of 3-T1AM in biological specimens using stable isotope dilution with 13C6-T1AM, a new internal standard that showed pharmacodynamic properties comparable to endogenous 3-T1AM. The method detection limit (MDL) and method quantification limit (MQL) of 3-T1AM were 0.04 and 0.09 ng/g, respectively. The spike-recoveries of 3-T1AM were between 85.4% and 94.3%, with a coefficient of variation of 3.7-5.8%. The intra-day and inter-day variations of 3-T1AM were 8.45-11.2% and 3.58-5.73%, respectively. Endogenous 3-T1AM liver values in C57BL/6J mice were 2.20 ± 0.49 pmol/g with a detection frequency of 50%. Higher liver 3-T1AM values were found when C57BL/6J mice were treated with N-acetyl-3-iodothyronamine or O-acetyl-3-iodothyronamine. Overall, our new stable isotope dilution LC-MS/MS method improves both the sensitivity and selectivity compared with existing methods. The concomitant possibility to quantify additional thyroid hormones such as thyroxine, 3,5,3'-triiodo-L-thyronine, 3,3',5'-triiodo-L-thyronine, 3,3'-diiodo-L-thyronine, and 3,5-diiodo-L-thyronine further adds to the value of our novel method in exploring the natural occurrence and fate of 3-T1AM in biological tissues and fluids.

Detection of Murine Astrovirus and Myocoptes musculinus in individually ventilated caging systems: Investigations to expose suitable detection methods for routine hygienic monitoring.

Murine Astrovirus is one of the most prevalent viral agents in laboratory rodent facilities worldwide, but its influence on biomedical research results is poorly examined. Due to possible influence on research results and high seroprevalence rates in mice, it appears useful to include this virus into routine health monitoring programs. In order to establish exhaust air particle PCR as a reliable detection method for Murine Astrovirus infections in mice kept in individually ventilated cages (IVC) and compare the method to sentinel mice monitoring regarding reproducibility and detection limit, we conducted a study with defined Murine Astrovirus cage prevalence. In parallel, the efficacy of both detection strategies (soiled-bedding sentinel (SBS) and exhaust air dust (EAD) analysis) was tested for Myocoptes musculinus. The fur mite was used as a reference organism during the whole study period to ensure the validity of this method. Because some publications already demonstrated successful detection of several pathogens, including murine fur mite species, via EAP-PCR. Detection of Murine Astrovirus infections at low prevalence is possible with both methods tested. Detection by exhaust air particles (EAP) is faster, more sensitive and more reliable compared to soiled bedding sentinels (SBS). Exhaust air particle PCR also detected the reference organism Myocoptes musculinus, which was not detected at all by sentinel mice, not even by high sensitivity fur swab qPCR. In conclusion, Murine Astrovirus can be detected by both exhaust air particle PCR and soiled bedding sentinels. We recommend exhaust air particle PCR as the better detection technique for Murine Astrovirus, because it is more reliable. Environmental samples are the method of choice for detection of Myocoptes musculinus because relying on soiled bedding sentinels harbors a big risk of missing existing infestations.

Combining fish and environmental PCR for diagnostics of diseased laboratory zebrafish in recirculating systems.

Precise knowledge of the health status of experimental fish is crucial to obtain high scientific and ethical standards in biomedical research. In addition to the use of sentinel fish, the examination of diseased fish is a fundamental part of all health monitoring concepts. PCR assays offer excellent sensitivity and the ability to test a broad variety of pathogenic agents in different sample types. Recently, it was shown that analysis of environmental samples such as water, sludge or detritus from static tanks can complement PCR analysis of fish and is actually more reliable for certain pathogens. In our study, we investigated whether the analysis of filtered water mixed with detritus of tanks including fish showing clinical signs of illness is suitable to complement health monitoring programs in recirculating systems. The obtained data indicate that pathogens such as Pseudoloma neurophilia or Myxidium streisingeri were exclusively or mainly found in fish, while mycobacteria were predominantly present in environmental samples. A combination of both sample types seems to be required for the detection of a broad range of infectious agents in zebrafish colonies using real-time PCR technology.

Environmental samples make soiled bedding sentinels dispensable for hygienic monitoring of IVC-reared mouse colonies.

Accurate knowledge of the health status of experimental animals is pivotal to high scientific and ethical standards in biomedical research. Individually ventilated cages (IVCs) are becoming the predominant system for housing laboratory mice, as they prevent cage-to-cage infections. However, this feature constitutes a major drawback for hygienic monitoring of mouse colonies, as traditional screening programs build on reliable transmission of infectious agents from experimental animals to sentinel mice commonly tested as representatives for the mouse colonies. In recent years, the laboratory animal community has realized that sentinels are ineffectual for screening mouse colonies in IVC systems because infections are often not transmitted to sentinels and therefore remain undetected. Furthermore, sentinel monitoring results in high numbers of used animals. In contrast, environmental monitoring provides a more reliable approach to identify and exclude pathogens in rodent colonies. In recent studies we provided evidence that polymerase chain reaction analysis of exhaust air particles is superior to soiled bedding sentinels for different agents. In this study, we show that testing pooled environmental samples generates more meaningful information compared to soiled bedding sentinels during routine hygienic monitoring in different barriers.

Understanding gene functions and disease mechanisms: Phenotyping pipelines in the German Mouse Clinic.

Since decades, model organisms have provided an important approach for understanding the mechanistic basis of human diseases. The German Mouse Clinic (GMC) was the first phenotyping facility that established a collaboration-based platform for phenotype characterization of mouse lines. In order to address individual projects by a tailor-made phenotyping strategy, the GMC advanced in developing a series of pipelines with tests for the analysis of specific disease areas. For a general broad analysis, there is a screening pipeline that covers the key parameters for the most relevant disease areas. For hypothesis-driven phenotypic analyses, there are thirteen additional pipelines with focus on neurological and behavioral disorders, metabolic dysfunction, respiratory system malfunctions, immune-system disorders and imaging techniques. In this article, we give an overview of the pipelines and describe the scientific rationale behind the different test combinations.

Murine norovirus detection in the exhaust air of IVCs is more sensitive than serological analysis of soiled bedding sentinels.

One limitation to housing rodents in individually ventilated cages (IVCs) is the ineffectiveness of traditional health monitoring programs that test soiled bedding sentinels every quarter. Aerogen transmission does not occur with this method. Moreover, the transmission of numerous pathogens in bedding is uncertain, and sentinel susceptibility to various pathogens varies. A novel method using particle collection from samples of exhaust air was developed in this study which was also systematically compared with routine health monitoring using soiled bedding sentinels. We used our method to screen these samples for the presence of murine norovirus (MNV), a mouse pathogen highly prevalent in laboratory animal facilities. Exhaust air particles from prefilters of IVC racks with known MNV prevalence were tested by quantitative reverse transcription polymerase chain reaction (RT-qPCR). MNV was detected in exhaust air as early as one week with one MNV-positive cage per rack, while sentinels discharged MNV RNA without seroconverting. MNV was reliably and repeatedly detected in particles collected from samples of exhaust air in all seven of the three-month sampling rounds, with increasing MNV prevalence, while sentinels only seroconverted in one round. Under field conditions, routine soiled bedding sentinel health monitoring in our animal facility failed to identify 67% ( n = 85) of positive samples by RT-qPCR of exhaust air particles. Thus, this method proved to be highly sensitive and superior to soiled bedding sentinels in the reliable detection of MNV. These results represent a major breakthrough in hygiene monitoring of rodent IVC systems and contribute to the 3R principles by reducing the number of animals used and by improving experimental conditions.

Exhaust Air Dust Monitoring is Superior to Soiled Bedding Sentinels for the Detection of Pasteurella pneumotropica in Individually Ventilated Cage Systems.

Reliable detection of unwanted organisms is essential for meaningful health monitoring in experimental animal facilities. Currently, most rodents are housed in IVC systems, which prevent the aerogenic transmission of pathogens between cages. Typically soiled-bedding sentinels (SBS) exposed to soiled bedding collected from a population of animals within an IVC rack are tested as representatives, but infectious agents often go undetected due to inefficient transmission. Pasteurellaceae are among the most prevalent bacterial pathogens isolated from experimental mice, and the failure of SBS to detect these bacteria is well established. In this study, we investigated whether analysis of exhaust air dust (EAD) samples by using a sensitive and specific real-time PCR assay is superior to conventional SBS monitoring for the detection of Pasteurella pneumotropica (Pp) infections. In a rack with a known prevalence of Pp-positive mice, weekly EAD sampling was compared with the classic SBS method over 3 mo. In 6 rounds of testing, with a prevalence of 5 infected mice in each of 7 cages in a rack of 63 cages, EAD PCR detected Pp at every weekly time point; SBS failed to detect Pp in all cases. The minimal prevalence of Pp-infected mice required to obtain a reliable positive result by EAD PCR testing was determined to be 1 in 63 cages. Reliable detection of Pp was achieved after only 1 wk of exposure. Analysis of EAD samples by real-time PCR assay provides a sensitive, simple, and reliable approach for Pp identification in laboratory mice.

High-Resolution Melting Curve Analysis for Identification of Pasteurellaceae Species in Experimental Animal Facilities.

Pasteurellaceae are among the most prevalent bacterial pathogens isolated from mice housed in experimental animal facilities. Reliable detection and differentiation of Pasteurellaceae are essential for high-quality health monitoring. In this study, we combined a real-time PCR assay amplifying a variable region in the 16S rRNA sequence with high-resolution melting curve analysis (HRM) to identify and differentiate among the commonly isolated species Pasteurella pneumotropica biotypes "Jawetz" and "Heyl", Actinobacillus muris, and Haemophilus influenzaemurium. We used a set of six reference strains for assay development, with the melting profiles of these strains clearly distinguishable due to DNA sequence variations in the amplicon. For evaluation, we used real-time PCR/HRM to test 25 unknown Pasteurellaceae isolates obtained from an external diagnostic laboratory and found the results to be consistent with those of partial 16S rRNA sequencing. The real-time PCR/HRM method provides a sensitive, rapid, and closed-tube approach for Pasteurellaceae species identification for health monitoring of laboratory mice.