A refinement to eRNA and eDNA-based detection methods for reliable and cost-efficient screening of pathogens in Atlantic salmon aquaculture.

Finfish aquaculture is one of the fastest-growing food production sectors in the world, and numerous infectious diseases are a constant challenge to the fish farming industry, causing decreased fish health and, consequently, economic losses. Specific and sensitive tools for pathogen detection are crucial for the surveillance of environmental samples to prevent the spread of fish pathogens in farms. Monitoring of waterborne pathogens through filtration of water and subsequent molecular detection of target-specific DNA or RNA sequence motifs is an animal-friendly method. This approach could reduce or even replace the sacrifice of fish for monitoring purposes in aquaculture and allow earlier implementation of disease control measures. Sampling methods might be a bottleneck, and there is a need for simple sampling methods that still ensure the best detection probability. In this study, we tested different filtration methods with spiked freshwater and seawater for a panel of fish pathogens to discern a suitable procedure that can be easily applied on-site by farm personnel without compromising detection probability. Specifically, we tested combinations of different filtration flow rates, lysis buffers, and filters for the detection of some of the pathogens relevant to the aquaculture industry. The results showed that a "sandwich" filtration method using two different filters and a flow rate of up to 4.0 L/min ensured good pathogen detection. The filters, consisting of a hydrophilic glass fibre filter with binder resin on the top and a hydrophilic mixed cellulose esters membrane at the bottom, achieved the best concentration and qPCR detection of both viral and bacterial fish pathogens. This up-and-coming tool allows the detection of very different fish pathogens during a single filtration step, and it can be combined with one single automated total nucleic acid extraction step for all the investigated pathogens, reducing both analysis costs and time.

Microcystin profiles in European noble crayfish Astacus astacus and water in Lake Steinsfjorden, Norway.

Lake Steinsfjorden, an important noble crayfish (Astacus astacus) habitat, is often affected by blooms of Planktothrix spp. that produce microcystins (MCs). A poor correlation between MCs by ELISA in the water and in crayfish tissue in a study in 2015 prompted further investigation by LC-HRMS. LC-HRMS analyses of filters from water samples and on selected crayfish tissue extracts from the 2015 study revealed the presence of known and previously unreported MCs. Crayfish samples from May and June 2015 were dominated by MCs from the Planktothrix bloom, whereas in September novel MCs that appeared to be metabolites of MC-LR were dominant, even though neither these nor MC-LR were detected in the water in 2015. A water sample from October 2016 also showed MCs typical of Planktothrix (i.e., [d-Asp3]- and [d-Asp3,Dhb7]MC-RR and -LR), but low levels of MC-RR and MC-LR were detected in the lake water for the first time. In late summer and autumn, the MC profiles of crayfish were dominated by the homonorvaline (Hnv) variant MC-LHnv, a putative metabolite of MC-LR. Taken together, ELISA, LC-HRMS and previous PCR analyses showed that although Planktothrix was part of the crayfish diet, it was not the sole source of MCs in the crayfish. Possibly, crayfish in Lake Steinsfjorden may be ingesting MCs from benthic cyanobacteria or from contaminated prey. Therefore, information on the cyanobacterial or MC content in the water column cannot safely be used to make predictions about MC concentrations in the crayfish in Lake Steinsfjorden. Interestingly, the results also show that targeted LC-MS analysis of the crayfish would at times have underestimated their MC content by nearly an order of magnitude, even if all previously reported MC variants had been included in the analysis.

Molecular detection of Aphanomyces astaci - An improved species specific qPCR assay.

The parasitic oomycete Aphanomyces astaci is the causative agent of crayfish plague, a devastating disease for European freshwater crayfish. Species specific quantitative real-time PCR (qPCR) can offer rapid detection of the pathogen. However, the well established A. astaci qPCR assay recommended by the World Organization for Animal Health (WOAH) amplifies the recently described Aphanomyces fennicus. Consequently, false-positive results may occur. This calls for the improvement of the established species specific A. astaci qPCR assay in order to avoid amplifying A. fennicus while screening for A. astaci. We developed an improved species specific A. astaci qPCR assay and validated the assay across three laboratories, using established procedures including different qPCR master mixes for each respective laboratory. Genomic DNA from A. astaci, A. fennicus and closely related Aphanomyces spp. was analysed and compared with both the improved and established assay. Additionally, DNA from crayfish tissue and environmental samples were analysed with both assays. The improved assay showed similar sensitivity with the established assay for all sample types, while proving highly specific for A. astaci avoiding amplification of A. fennicus and the other tested Aphanomyces spp. Environmental DNA (eDNA) samples collected at River Lierelva in Norway amplified with the established assay, but not with the improved assay indicating false positive. We were able to sequence a 530 bp fragment of the ITS region from these eDNA samples and the consensus sequence showed 99.9-100 % pairwise identity with A. fennicus and 97.2-98 % pairwise identity with A. astaci, suggesting that the occurrence of A. fennicus is not limited to Finland, where it was first discovered.

Mucosal immune and stress responses of Neoparamoeba perurans-infected Atlantic salmon (Salmo salar) treated with peracetic acid shed light on the host-parasite-oxidant interactions.

Treatment development for parasitic infestation is often limited to disease resolution as an endpoint response, and physiological and immunological consequences are not thoroughly considered. Here, we report the impact of exposing Atlantic salmon affected with amoebic gill disease (AGD) to peracetic acid (PAA), an oxidative chemotherapeutic. AGD-affected fish were treated with PAA either by exposing them to 5 ppm for 30 min or 10 ppm for 15 min. Unexposed fish from both infected and uninfected groups were also included. Samples for molecular, biochemical, and histological evaluations were collected at 24 h, 2 weeks, and 4 weeks post-treatment. Behavioral changes were observed during PAA exposure, and post-treatment mortality was higher in the infected and PAA treated groups, especially in 10 ppm for 15 min. Plasma indicators showed that liver health was affected by AGD, though PAA treatment did not exacerbate the infection-related changes. Transcriptome profiling in the gills showed significant changes, triggered by AGD and PAA treatments, and the effects of PAA were more notable 24 h after treatment. Genes related to immune pathways of B- and T- cells and protein synthesis and metabolism were downregulated, where the magnitude was more remarkable in 10 ppm for 15 min group. Even though treatment did not fully resolve the pathologies associated with AGD, 5 ppm for 30 min group showed lower parasite load at 4 weeks post-treatment. Mucous cell parameters (i.e., size and density) increased within 24 h post-treatment and were significantly higher at termination, especially in AGD-affected fish, with some treatment effects influenced by the dose of PAA. Infection and treatments resulted in oxidative stress-in the early phase in the gill mucosa, while systemic reactive oxygen species (ROS) dysregulation was evident at the later stage. Infected fish responded to elevated circulating ROS by increasing antioxidant production. Exposing the fish to a crowding stress revealed the interference in the post-stress responses. Lower cortisol response was displayed by AGD-affected groups. Collectively, the study established that PAA, within the evaluated treatment protocols, could not provide a convincing treatment resolution and, thus, requires further optimization. Nonetheless, PAA treatment altered the mucosal immune and stress responses of AGD-affected Atlantic salmon, shedding light on the host-parasite-treatment interactions. .

Cushing's Syndrome in a Pregnant Patient Secondary to Adrenal Adenoma: Treatment With Unilateral Adrenalectomy.

BACKGROUND: A pregnant woman presented with Cushing's syndrome (CS) secondary to adrenal adenoma and was treated with laparoscopic-assisted right adrenalectomy during the second trimester of pregnancy. SUMMARY: Our patient is a 33-year-old woman who presented with hypertension and cushingoid appearance at 21 weeks gestational age. Laboratory evidence indicated CS was likely, and imaging found a 2.3 x 3.0 cm right adrenal nodule as a suggested cause. Laparoscopic-assisted right adrenalectomy was performed at 23 weeks 4 days gestation without complication. Pathology of the removed specimen revealed an adrenal gland containing a 3.0 x 3.0 x 2.0 cm well-circumscribed proliferation of adrenal cortical cells without significant atypia. This report describes the rare occurrence of CS during pregnancy and subsequent successful surgical management. We review the diagnosis of CS during pregnancy and surgical considerations. CONCLUSION: CS, while a rare entity in the general population, is even more unique in the setting of pregnancy due to the negative effects of hypercortisolism on fertility. CS during pregnancy presents a diagnostically complicated scenario, along with specific considerations necessary for successful surgical management.

Detection of an invasive aquatic plant in natural water bodies using environmental DNA.

The ability to detect founding populations of invasive species or rare species with low number of individuals is important for aquatic ecosystem management. Traditional approaches use historical data, knowledge of the species' ecology and time-consuming surveys. Within the past decade, environmental DNA (eDNA) has emerged as a powerful additional tracking tool. While much work has been done with animals, comparatively very little has been done with aquatic plants. Here we investigated the transportation and seasonal changes in eDNA concentrations for an invasive aquatic species, Elodea canadensis, in Norway. A specific probe assay was developed using chloroplast DNA to study the fate of the targeted eDNA through space and time. The spatial study used a known source of Elodea canadensis within Lake Nordbytjern 400 m away from the lake outlet flowing into the stream Tveia. The rate of disappearance of E. canadensis eDNA was an order of magnitude loss over about 230 m in the lake and 1550 m in the stream. The time series study was performed monthly from May to October in lake Steinsfjorden harbouring E. canadensis, showing that eDNA concentrations varied by up to three orders of magnitude, peaking during fall. In both studies, the presence of suspended clay or turbidity for some samples did not hamper eDNA analysis. This study shows how efficient eDNA tools may be for tracking aquatic plants in the environment and provides key spatial and temporal information on the fate of eDNA.

Catching the fish with the worm: a case study on eDNA detection of the monogenean parasite Gyrodactylus salaris and two of its hosts, Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss).

BACKGROUND: Environmental DNA (eDNA) monitoring is growing increasingly popular in aquatic systems as a valuable complementary method to conventional monitoring. However, such tools have not yet been extensively applied for metazoan fish parasite monitoring. The fish ectoparasite Gyrodactylus salaris, introduced into Norway in 1975, has caused severe damage to Atlantic salmon populations and fisheries. Successful eradication of the parasite has been carried out in several river systems in Norway, and Atlantic salmon remain infected in only seven rivers, including three in the Drammen region. In this particular infection region, a prerequisite for treatment is to establish whether G. salaris is also present on rainbow trout upstream of the salmon migration barrier. Here, we developed and tested eDNA approaches to complement conventional surveillance methods. METHODS: Water samples (2 × 5 l) were filtered on-site through glass fibre filters from nine locations in the Drammen watercourse, and DNA was extracted with a CTAB protocol. We developed a qPCR assay for G. salaris targeting the nuclear ribosomal ITS1 region, and we implemented published assays targeting the mitochondrial cytochrome-b and NADH-regions for Atlantic salmon and rainbow trout, respectively. All assays were transferred successfully to droplet digital PCR (ddPCR). RESULTS: All qPCR/ddPCR assays performed well both on tissue samples and on field samples, demonstrating the applicability of eDNA detection for G. salaris, rainbow trout and Atlantic salmon in natural water systems. With ddPCR we eliminated a low cross-amplification of Gyrodactylus derjavinoides observed using qPCR, thus increasing specificity and sensitivity substantially. Duplex ddPCR for G. salaris and Atlantic salmon was successfully implemented and can be used as a method in future surveillance programs. The presence of G. salaris eDNA in the infected River Lierelva was documented, while not elsewhere. Rainbow trout eDNA was only detected at localities where the positives could be attributed to eDNA release from upstream land-based rainbow trout farms. Electrofishing supported the absence of rainbow trout in all of the localities. CONCLUSIONS: We provide a reliable field and laboratory protocol for eDNA detection of G. salaris, Atlantic salmon and rainbow trout, that can complement conventional surveillance programs and substantially reduce the sacrifice of live fish. We also show that ddPCR outperforms qPCR with respect to the specific detection of G. salaris.

Molecular detection and genotyping of Aphanomyces astaci directly from preserved crayfish samples uncovers the Norwegian crayfish plague disease history.

Aphanomyces astaci causes crayfish plague in European freshwater crayfish, but most historical epizootics lack agent isolation and identification. Although declared as crayfish plague outbreaks by the Norwegian Competent Authorities, only presumptive diagnoses without agent isolation exist from Norwegian epizootics until 2005. Molecular methods now allow both A. astaci detection and genotype determination from preserved samples. We therefore aimed to (1) investigate molecularly if A. astaci was involved in a selection of mass-mortality events in Norwegian noble crayfish populations from 1971 to 2004, and (2) determine the eventually involved A. astaci genotype groups both from these historical and also more recent mass-mortality events. DNA was extracted directly from presumptively infected crayfish tissues, and screened by A. astaci specific qPCR. A representative selection of positive samples was confirmed by ITS-sequencing. Finally, genotype determination was performed with microsatellite markers that distinguish all known A. astaci genotype groups. The molecular examination detected A. astaci in crayfish materials from all examined mass-mortality events. The first event in 1971-1974 was caused by the A. astaci genotype group A, presumably the first genotype group that entered Europe more than 150 years ago. All later outbreaks were caused by the A. astaci genotype group B which was introduced to Europe by importation of signal crayfish in the 1960s. The results suggest that molecular methods can verify the involvement of A. astaci in the vast majority of observed crayfish mass mortalities in Europe whenever preserved materials exist. Moreover, microsatellite genotyping can reveal at least parts of the underlying epidemiology.

Monitoring the spore dynamics of Aphanomyces astaci in the ambient water of latent carrier crayfish.

The specialized crayfish parasite Aphanomyces astaci causes the devastating crayfish plague in European crayfish. Even though A. astaci sporulation has been thoroughly studied under pure culture conditions, little is known about the sporulation dynamic from its live host. Our purpose was to investigate the A. astaci spore dynamic in its native parasite-host relationship by monitoring the sporulation from carrier crayfish into the ambient water using agent specific qPCR. American signal crayfish (Pacifastacus leniusculus) with known positive carrier status were housed individually and communally in two experimental set-ups using multiple replicates and different temperatures. Water samples were collected weekly, and spore numbers were quantified. We demonstrate here that live latent carrier crayfish continuously released a moderate number of A. astaci spores (~2700 spores per crayfish/week) in the absence of death and moulting events. In contrast, a pronounced sporulation increase was seen already one week prior to death in moribund crayfish, suggesting a crayfish plague-like condition developing in weakened or stressed individuals. Significantly more spores were produced at 18°C compared to 4°C, while a negative correlation was detected between spore numbers and temperatures rising from 17 to 23°C. This study is the first attempt to quantify the spore release from carrier crayfish on the basis of qPCR applied on water samples, and demonstrate that the approach successfully unravel A. astaci sporulation patterns. The results emphasize that carrier crayfish pose a constant infection risk to highly susceptible crayfish species regardless of crayfish life cycle state.

Detection and quantification of the crayfish plague agent in natural waters: direct monitoring approach for aquatic environments.

Aphanomyces astaci, a specialised parasite of North American freshwater crayfish, is the disease agent of crayfish plague that is lethal to European freshwater crayfish. The life cycle of A. astaci has been inferred from experimental laboratory studies, but less is known about its natural sustainability and ecology. To address such questions, tools for monitoring of A. astaci directly in aquatic environments are needed. Here, we present an approach for detecting and quantifying A. astaci directly from water samples using species-specific TaqMan minor groove binder real-time PCR. Samples of a 10-fold dilution series from approximately 10(4) to approximately 1 spore of A. astaci were repeatedly tested, and reliable detection down to 1 spore was demonstrated. Further, to simulate real-life samples from natural water bodies, water samples from lakes of various water qualities were spiked with spores. The results demonstrated that co-extracted humic acids inhibit detection significantly. However, use of bovine serum albumin or the TaqMan Environmental Master Mix largely removes this problem. The practical application of the approach was successfully demonstrated on real-life water samples from crayfish farms in Finland hosting infected North American signal crayfish Pacifastacus leniusculus. Direct monitoring of A. astaci from aquatic environments may find application in the management of wild noble crayfish Astacus astacus stocks, improved aquaculture practices and more targeted conservation actions. The approach will further facilitate studies of A. astaci spore dynamics during plague outbreaks and in carrier crayfish populations, which will broaden our knowledge of the biology of this devastating crayfish pathogen.