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Isothermal recombinant polymerase amplification and CRIPSR (CAS12A) assay detection of Renibacterium salmoninarum as an example for wildlife pathogen detection in environmental DNA samples

Improving rapid detection methods for pathogens is important for research as we collectively aim to improve the health of ecosystems globally. In the northern hemisphere, the success of salmon ( Oncorhynchus spp.) populations is vitally important to the larger marine, aquatic, and terrestrial ecosystems they inhabit. This has led to managers cultivating salmon in hatcheries and aquaculture to bolster their populations, but young salmon face many challenges, including diseases such as bacterial kidney disease (BKD). Early detection of the BKD causative agent, Renibacterium salmoninarum , is useful for managers to avoid outbreaks in hatcheries and aquaculture stocks to enable rapid treatment with targeted antibiotics. Isothermal amplification and CRIPSR-Cas12a systems may enable sensitive, relatively rapid, detection of target DNA molecules from environmental samples compared to quantitative PCR (qPCR) and culture methods. We used these technologies to develop a sensitive and specific rapid assay to detect R. salmoninarum from water samples using isothermal recombinase polymerase amplification (RPA) and an AsCas12a RNA-guided nuclease detection. The assay was specific to R. salmoninarum (0/10 co-occurring or closely related bacteria detected) and sensitive to 0.0128 pg/µL of DNA (approximately 20–40 copies/µL) within 10 min of Cas activity. This assay successfully detected R. salmoninarum environmental DNA in 14/20 water samples from hatcheries with known quantification for the pathogen via previous qPCR (70% of qPCR-positive samples). The RPA-CRISPR/AsCas12a assay had a limit of detection (LOD) of >10 copies/µL in the hatchery water samples and stochastic detection below 10 copies/µL, similar to but slightly higher than the qPCR assay. This LOD enables 37 C isothermal detection, potentially in the field, of biologically relevant levels of R. salmoninarum in water. Further research is needed to develop easy-to-use, cost-effective, sensitive RPA/CRISPR-AsCas12a assays for rapidly detecting low concentrations of wildlife pathogens in environmental samples.

Journal of Wildlife Diseases

Regional occurrence of aqueous tungsten and relations with antimony, arsenic and molybdenum concentrations (Sardinia, Italy)

T ungsten (W) is rarely found in natural waters, yet it can be introduced into the food chain and cause potentially toxic effects. Uptake of W by plants and vegetables, or trace presence of W in drinking water are possible vectors for ingestion of W by humans. The latter is recognized as a possible cause of lymphatic leukemia. Increased uses of W might result in a degradation of water resources, with attendant adverse effects on biota and human health. Therefore, this study was aimed at investigating regional occurrence and speciation of W in aquatic systems in Sardinia, Italy, factors affecting W mobility and possible relations with other oxyanion-forming trace elements such as Sb, As and Mo. Although our results are specifically from Sardinia, the implications are broader and should prompt future studies in other areas with known high W concentrations. A total of 350 sample sites are reported here, including surface waters, groundwaters, mine drainages, thermal waters and local seawater. The waters were analyzed for major and trace components, including W, Sb, As and Mo. The waters showed a variety of major chemical compositions and W concentrations. High concentrations of W were found in some mine waters and drainages from slag heaps, with W, Sb and As up to 140, 5000 and 800 μg L −1 , respectively. The highest concentrations of W occurred under slightly alkaline pH and oxygenated conditions, and were likely due to the dissolution of scheelite [CaWO 4 ] hosted in materials with which the water came into contact. High W concentrations also were observed in thermal waters, under alkaline pH and reducing conditions, and sometimes coincided with relatively high concentrations either of As or Mo. Previous studies of W geochemistry have focused on WO 4 2− as the major dissolved form of W. For this study, we have augmented the thermodynamic database in PHREEQC to include possible formation of many other W-bearing complexes gleaned from the literature. The results of the speciation calculations with the newly added complexation reactions shows that the neutral species CaWO 4 ° and MgWO 4 ° are particularly dominant in most W-bearing waters and lead to undersaturation with respect to scheelite and other W-bearing minerals. Assessing W contamination in water systems and establishing W limits in drinking water may prevent potential adverse effects of W on human and ecosystem health.

Sardinia

Sentinel-2 for chlorophyll-a water quality monitoring: A review of validation evidence and application potential

Water quality monitoring is integral to preserving the health of freshwater ecosystems, and satellite remote sensing has emerged as one monitoring method. Sentinel-2, in particular, has been valuable for water quality monitoring due to its 5-day global temporal revisit time and spatial resolution that ranges from 10 to 60 metres. Sentinel-2 can be used to measure and monitor chlorophyll-a, which historically has been used as an indicator of water quality, eutrophication and harmful algal blooms. Our goal was to review aquatic chlorophyll-a Sentinel-2 research to assess the types of validation evidence reported. Validation evidence is defined here as the set of information key to assessing algorithm performance, and include the spatial and temporal scales of satellite validation, reported in situ sampling method context information, demonstration of validation results through plots, and appropriate algorithm performance metrics. We highlight how the body of literature collectively contributes to advancing a national scale chlorophyll-a product that could support future resource management applications. Our review of 122 published studies indicated that much of the validation evidence corresponded to early stages, as defined by the NASA data maturity framework, due to a limited focus on individual lakes and limited detail on methodology for reproducibility. Prioritizing data accessibility for both in situ data and satellite workflows used in published studies; reporting methods with transparency and consistency; and using standard algorithm performance metrics could provide a consistent framework to support and enhance the utility of satellite inland water quality research. These three quality assurance mechanisms can promote effective evaluation of approaches for remote sensing of chlorophyll-a. Adopting these quality criteria could enable the integration of validation evidence from multiple studies, supporting more spatially and temporally representative products that would advance these approaches towards maturation for broader application.

International Journal of Remote Sensing

Reduced Myxobolus cerebralis actinospore production in a Colorado reservior may be linked to changes in Tubifex tubifex population structure

Elucidating the dynamics of a parasitic infection requiring two hosts in a natural ecosystem can be a daunting task. Myxobolus cerebralis (Mc), the myxozoan parasite that causes whirling disease in some salmonids, was detected in the Colorado River upstream of Windy Gap Reservoir (WGR) in 1988. Subsequently, whirling disease was implicated in the decline of wild Rainbow Trout Oncorhynchus mykiss in the river when WGR was identified as a point source of Mc triactinomyxons (TAMs). Between 1997 and 2004, numerous investigations began to elucidate the etiology of Mc in WGR. During this period, Mc TAM production in WGR declined more than 90%. Explanations for the decline have included differences in stream discharge between years, changes in the thermal regime of the lake, severe drought, changes in the fish population structure in WGR, and reductions in the prevalence and severity of Mc infection in salmonids in the Colorado and Fraser rivers upstream of WGR. All of these have been discredited as explanations for the reduced TAM production. In 2005, a new study was conducted to replicate the studies completed in 1998. In this paper, the results of a new real-time polymerase chain reaction assay utilized to quantify the mitochondrial 16S rDNA specific to each of four lineages of Tubifex tubifex in pooled samples of 50 oligochaetes are presented. These results suggest that compared with 1998, the densities of aquatic oligochaetes and T. tubifex have increased, TAM production has been greatly reduced, and the decline is congruent with the dominance of lineages I, V, and VI of T. tubifex—three lineages that are refractory or highly resistant to Mc infection—in the oligochaete population. While it is possible that the resistant lineages function as biofilters that deactivate Mc myxospores, the reason for the decline in TAM production in WGR remains an enigma.

Colorado

Myxobolus cerebralis in native cutthroat trout of the Yellowstone Lake ecosystem

The exotic parasite Myxobolus cerebralis was first detected in native adult Yellowstone cutthroat trout Oncorhynchus clarkii bouvierii from Yellowstone Lake in 1998, seriously threatening the ecological integrity of this pristine, naturally functioning ecosystem. We immediately began to assess the prevalence and spatial extent of M. cerebralis infection in Yellowstone cutthroat trout within Yellowstone Lake and to determine the infection risk of age-0 Yellowstone cutthroat trout, the relative abundance and actinospore production of lubificid worms, and the basic environmental characteristics of tributaries. During 1999-2001, juvenile and adult Yellowstone cutthroat trout were infected throughout Yellowstone Lake; the highest prevalence (15.3-16.4%) occurred in the northern and central regions. Exposure studies in 13 streams indicated that Pelican and Clear creeks and the Yellowstone River were positive for M. cerebralis; the highest prevalence (100%) and severity was found in Pelican Creek during mid-July. Sexually mature individuals of the oligochaete Tubifex tubifex were most abundant in early summer, were genetically homogenous, and were members of a lineage known to produce moderate to high levels of M. cerebralis triactinomyxons. Only 20 of the 3,037 sampled tubificids produced actinospores after 7 d in culture, and none of the actinospores were M. cerebralis. However, one non-actinospore-producing T. tubifex from Pelican Creek tested positive for M. cerebralis by polymerase chain reaction. Stream temperatures at Pelican Creek, a fourth-order, low-gradient stream, were over 20??C during the first exposure period, suggesting that T. tubifex were capable of producing triactinomyxons at elevated temperatures in the wild. Although the infection of otherwise healthy adult Yellowstone cutthroat trout within Yellowstone Lake suggests some resistance, our sentinel cage exposures indicated that this subspecies may be more susceptible to whirling disease than previous laboratory challenges have indicated, and M. cerebralis may be contributing to a significant recent decline in this population. ?? Copyright by the American Fisheries Society 2006.

Wyoming

Identical sequence types of Yersinia ruckeri associated with lethal disease in wild-caught invasive Blue Catfish and cultured hybrid catfish (Channel Catfish ♀ × Blue Catfish ♂) from disparate aquatic ecosystems

Objective The Blue Catfish Ictalurus furcatus is commonly raised in warmwater aquaculture in the United States to produce Channel Catfish I. punctatus × Blue Catfish hybrids. It is also a prominent aquatic invasive species of concern in the mid-Atlantic region of the United States. Here, Yersina ruckeri was isolated from moribund Blue Catfish and hybrid catfish from disparate regions of the USA. The goal of the research here was to compare these Y. ruckeri strains to each other and other known strains for which adequate sequence data was available. In addition, we sought to determine if the strain from Blue Catfish was pathogenic to Rainbow Trout Oncorhynchus mykiss . Methods Moribund hybrid catfish from culture ponds in Mississippi were processed for diagnostic evaluation in March 2016. In April 2022, a moribund Blue Catfish specimen was collected from a tributary of the Nanticoke River in Maryland. Bacterial isolates were identified and characterized using biochemical tests, antimicrobial sensitivity screening, serotyping, and complete or partial genome sequencing. Disease pathology was described via histology. The isolate from Blue Catfish was used in challenge experiments to determine if it was pathogenic to Rainbow Trout. Multilocus sequencing typing was conducted using the PubMLST database. Results Biochemical testing was consistent with Y. ruckeri . A draft genome of the Y. ruckeri isolate was assembled based on Oxford Nanopore Technology sequencing and identified a single genomic replicon (3,791,418 bp) consistent in size to other Y. ruckeri genomes and a pLT plasmid (60, 933 bp). The challenge study demonstrated no significant virulence of this isolate for Rainbow Trout ( Y. ruckeri ). This isolate was most similar to other strains isolated from ictalurids. Notably, the gyrase B gene from this isolate was identical to that of archived strains isolated from moribund Mississippi hybrid catfish aquaculture during 2016 and these isolates share identical PubMLST sequence type profiles. Similarly, they shared a pLT plasmid that differed by only 6 bp. This plasmid has never been reported from trout isolates and appears to be unique to ictalurids. Conclusions Analyses here provide preliminary genetic evidence that geographically distant (Maryland and Mississippi, USA) isolates of Y. ruckeri from ictalurids are genetically similar to each other and Y. ruckeri (strain SC09) that infects ictalurids in China. This strain is not a biothreat to Rainbow Trout at typical culture temperatures.

Journal of Aquatic Animal Health