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.

Regulation of the molecular repertoires of oxidative stress response in the gills and olfactory organ of Atlantic salmon following infection and treatment of the parasite Neoparameoba perurans.

The present study investigated the involvement of key molecular regulators of oxidative stress in amoebic gill disease (AGD), a parasitic infestation in Atlantic salmon. In addition, the study evaluated how these molecular biomarkers responded when AGD-affected fish were exposed to a candidate chemotherapeutic peracetic acid (PAA). Atlantic salmon were experimentally infected with the parasite Neoparameoba perurans, the causative agent of AGD, by bath exposure and after 2 weeks, the fish were treated with three commercial PAA products (i.e., Perfectoxid, AquaDes and ADDIAqua) at a dose of 5 ppm. Two exposure durations were evaluated - 30 min and 60 min. Sampling was performed 24 h and 2 weeks after PAA treatment (equivalent to 2- and 4-weeks post infection). At each sampling point, the following parameters were evaluated: gross gill pathology, gill parasitic load, plasma reactive oxygen species (ROS) and total antioxidant capacity (TAC), histopathology and gene expression profiling of genes with key involvement in oxidative stress in the gills and olfactory organ. AGD did not result in systemic oxidative stress as ROS and TAC levels remained unchanged. There were no clear patterns of AGD-mediated regulation of the oxidative stress biomarkers in both the gills and olfactory organ; significant changes in the expression were mostly related to time rather than infection status. However, the expression profiles of the oxidative stress biomarkers in AGD-affected salmon, following treatment with PAA, revealed that gills and olfactory organ responded differently - upregulation was prominent in the gills while downregulation was more frequent in the olfactory organ. The expression of catalase, glutathione S-transferase and thioredoxin reductase 2 was significantly affected by the treatments, both in the gills and olfactory organ, and these alterations were influenced by the duration of exposure and PAA product type. Parasitic load in the gills did significantly increase after treatment regardless of the product and exposure duration; the parasite was undetectable in some fish treated with AquaDes for 30 mins. However, PAA treated groups for 30 min showed lower macroscopic gill scores than the infected-untreated fish. Histology disclosed the classic pathological findings such as multifocal hyperplasia and increased number of mucous cells in AGD-affected fish. Microscopic scoring of gill injuries showed that AGD-infected-PAA-treated fish had lower scores, however, an overall trend could not be established. The morphology and structural integrity of the olfactory organ were not significantly altered by parasitism or PAA treatment. Collectively, the results indicate that AGD did not affect the systemic and mucosal oxidative status of Atlantic salmon. However, such a striking profile was changed when AGD-affected fish were exposed to oxidative chemotherapeutics. Moreover, the gills and olfactory organ demonstrated distinct patterns of gene expression of oxidative stress biomarkers in AGD-infected-PAA-treated fish. Lastly, PAA treatment did not fully resolve the infection, but appeared not to worsen the mucosal health either.

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. .

qPCR screening for Yersinia ruckeri clonal complex 1 against a background of putatively avirulent strains in Norwegian aquaculture.

Although a number of genetically diverse Yersinia ruckeri strains are present in Norwegian aquaculture environments, most if not all outbreaks of yersiniosis in Atlantic salmon in Norway are associated with a single specific genetic lineage of serotype O1, termed clonal complex 1. To investigate the presence and spread of virulent and putatively avirulent strains in Norwegian salmon farms, PCR assays specific for Y. ruckeri (species level) and Y. ruckeri clonal complex 1 were developed. Following extensive screening of water and biofilm, the widespread prevalence of putatively avirulent Y. ruckeri strains was confirmed in freshwater salmon hatcheries, while Y. ruckeri clonal complex 1 was found in fewer farms. The formalin-killed bacterin yersiniosis vaccine was detected in environmental samples by both PCR assays for several weeks post-vaccination. It is thus important to interpret results from recently vaccinated fish with great care. Moreover, field studies and laboratory trials confirmed that stressful management procedures may result in increased shedding of Y. ruckeri by sub-clinically infected fish. Analysis of sea water sampled throughout thermal delousing procedures proved effective for detection of Y. ruckeri in sub-clinically infected populations.

The circulating plasma metabolome of Neoparamoeba perurans-infected Atlantic salmon (Salmo salar).

Metabolomics can provide insights into the dynamic small-molecule fluctuations occurring in response to infection and has become a valuable tool in studying the pathophysiology of diseases in recent years. However, its application in fish disease research is limited. Here, we report the circulating plasma metabolome of Atlantic salmon (Salmo salar) experimentally infected with Neoparamoeba perurans-the causative agent of amoebic gill disease (AGD). Plasma samples were collected from fish with varying degrees of infection inferred from an external gross morphological score of gill pathology (i.e., gill score [GS] 1 -- GS3), where a higher GS indicates advanced infection stage. Uninfected fish (GS0) served as the control. Typical pathologies associated with AGD infection, such as hyperplastic lesions and lamellar fusion, were evident in infected gill samples. Plasma metabolites were identified by ultra-performance liquid chromatography coupled with a high-resolution quadrupole-orbitrap mass spectrometer. Identification of compounds were performed at four levels of certainty, where level 1 provided the most accurate compound identity. A total of 900 compounds were detected in the samples of which 143 were annotated at level 3, 68 on level 2b, 74 on level 2a, and 66 on level 1. Versus GS0, GS1 showed the highest number of significantly affected metabolites (104), which decreased with a higher GS. Adrenaline and adenosine were the two Level 1 compounds significantly affected by AGD regardless of GS, with the former increasing and the latter decreasing in infected fish. Hippuric acid significantly increased in GS1 and GS2, while the tryptophan metabolite indole-3-lactic acid decreased in response to the initial stage of infection but returned to basal levels at a higher GS. There were ten significantly affected metabolic pathways: Eight of which were significantly downregulated while two were downregulated in GS1 relative to GS0. The super-pathway of purine nucleotide salvage was enriched both within the upregulated metabolites in GS1vsGS0 and the down-regulated metabolites in GS3vsGS1. This is the first report on the circulating plasma metabolome of AGD infected salmon, and the results show that low infection levels resulted in a more dramatic metabolomic dysregulation than advanced infection stages. The metabolites identified are potential biological markers for the systemic physiological impact of AGD.

Corrigendum: Metabolic Profiling of a CB2 Agonist, AM9338, Using LC-MS and Microcoil-NMR: Identification of a Novel Dihydroxy Adamantyl Metabolite.

[This corrects the article DOI: 10.3389/fphar.2020.575691.].

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.

Metabolic Profiling of a CB2 Agonist, AM9338, Using LC-MS and Microcoil-NMR: Identification of a Novel Dihydroxy Adamantyl Metabolite.

Adamantyl groups are key structural subunit commonly used in many marketed drugs targeting diseases ranging from viral infections to neurological disorders. The metabolic disposition of adamantyl compounds has been mostly studied using LC-MS based approaches. However, metabolite quantities isolated from biological preparations are often insufficient for unambiguous structural characterization by NMR. In this work, we utilized microcoil NMR in conjunction with LC-MS to characterize liver microsomal metabolites of an adamantyl based CB2 agonist AM9338, 1-(3-(1H-1,2,3-triazol-1-yl) propyl)-N-(adamantan-1-yl)-1H-indazole-3-carboxamide, a candidate compound for potential multiple sclerosis treatment. We have identified a total of 9 oxidative metabolites of AM9338 whereas mono- or di-hydroxylation of the adamantyl moiety is the primary metabolic pathway. While it is generally believed that the tertiary adamantyl carbons are the preferred sites of CYP450 oxidation, both the mono- and di-hydroxyl metabolites of AM9338 show that the primary oxidative sites are located on the secondary adamantyl carbons. To our knowledge this di-hydroxylated metabolite is a novel adamantyl metabolite that has not been reported before. Further, the stereochemistry of both mono- and di-hydroxyl adamantyl metabolites has been determined using NOE correlations. Furthermore, docking of AM9338 into the CYP3A4 metabolic enzyme corroborates with our experimental findings, and the modelling results also provide a possible mechanism for the unusual susceptibility of adamantyl secondary carbons to metabolic oxidations. The novel dihydroxylated AM9338 metabolite identified in this study, along with the previously known adamantyl metabolites, gives a more complete picture of the metabolic disposition for adamantyl compounds.

Mammary-type Myofibroblastoma of the Pre-sacral Space: A Rare Neoplasm.

INTRODUCTION: Mammary-type myofibroblastoma is a very rare, benign, mesenchymal neoplasm that is histologically identical to a myofibroblastoma of the breast but located in an extra-mammary location. To our knowledge, there have been about 160 cases of extra-mammary myofibroblastoma reported to date. Our report describes a mammary-type myofibroblastoma located retro-rectally in the pre-sacral space. CASE REPORT: Our patient is a 55-year-old male that presented via referral for evaluation of a pelvic mass. He noted having a few loose stools since the mass had become apparent but did not report any other associated symptoms. An MRI of the pelvis revealed a 9 cm, fat containing, solid, retro-rectal mass within the pre-sacral space, which did not appear to be contiguous with the rectum, ureters, or pelvic sidewall. He elected to have the mass surgical removed. The mass was removed as a single specimen that measured 9.5 x 7.5 x 7.0 cm. By immunohistochemistry, the neoplastic cells show co-expression of desmin, CD34, estrogen receptor, and loss of RB1 expression, which is consistent with the diagnosis of mammary-type myofibroblastoma. DISCUSSION: Mammary-type myofibroblastoma is a very rare, benign, soft tissue neoplasm. These neoplasms most often present as a painless slow growing mass in a middle-aged male. Although exceedingly rare, mammary-type myofibroblastoma should be on the differential diagnosis of patients presenting with a mass that was found incidentally or one that is producing mass-effect symptoms. When found, these tumors should be investigated to rule out other more serious pathologies and removed due to their high curability with surgical resection.

Microcystins in European Noble Crayfish Astacus astacus in Lake Steinsfjorden, a Planktothrix-Dominated Lake.

Lake Steinsfjorden, an important Norwegian location for noble crayfish (Astacus astacus), is often affected by cyanobacterial blooms caused by microcystin (MC)-producing Planktothrix spp. The impact of MCs on noble crayfish as a food source and crayfish health is largely unknown. We investigated the quantities and correlations of MCs in noble crayfish and lake water during and after a cyanobacterial bloom peaking in June-July 2015. Noble crayfish and water samples were collected monthly from June to October 2015 and in October 2016. The content of MCs was analysed by ELISA from tail muscle, intestine, stomach and hepatopancreas. PCR analysis for Planktothrix gene markers was performed on crayfish stomach content. Water samples were analysed for phytoplankton composition, biomass and MCs. PCR-positive stomach contents indicated Planktothrix to be part of the noble crayfish diet. Concentrations of MCs were highest in the hepatopancreas, stomach and intestine, peaking in August-September. Tail muscle contained low concentrations of MCs. Similar levels of MCs were found in crayfish from 2016. Except in September 2015, a normal portion of boiled noble crayfish tails was below the tolerable daily intake (TDI) for MCs for humans. Removing the intestine more than halved the content of MCs and seems a reasonable precautionary measure for noble crayfish consumers.

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.

Development of a qPCR assay to detect and quantify ichthyotoxic flagellates along the Norwegian coast, and the first Norwegian record of Fibrocapsa japonica (Raphidophyceae).

Blooms of ichthyotoxic microalgae pose a great challenge to the aquaculture industry world-wide, and there is a need for fast and specific methods for their detection and quantification in monitoring programs. In this study, quantitative real-time PCR (qPCR) assays for the detection and enumeration of three ichthyotoxic flagellates: the dinoflagellate Karenia mikimotoi (Miyake & Kominami ex Oda) Hansen & Moestrup and the two raphidophytes Heterosigma akashiwo (Hada) Hada ex Hara & Chihara and Fibrocapsa japonica Toriumi & Takano were developed. Further, a previously published qPCR assay for the dinoflagellate Karlodinium veneficum (Ballantine) Larsen was used. Monthly samples collected for three years (Aug 2009-Jun 2012) in outer Oslofjorden, Norway were analysed, and the results compared with light microscopy cell counts. The results indicate a higher sensitivity and a lower detection limit (down to 1 cell L-1) for both qPCR assays. Qualitative and semi-quantitative results were further compared with those obtained by environmental 454 high throughput sequencing (HTS, metabarcoding) and scanning electron microscopy (SEM) examination from the same samplings. All four species were detected by qPCR and HTS and/or SEM in outer Oslofjorden (Aug 2009-Jun 2012); Karlodinium veneficum was present year-round, whereas Karenia mikimotoi, Heterosigma akashiwo and Fibrocapsa japonica appeared mainly during the autumn in all three years. This is the first observation of Fibrocapsa japonica in Norwegian coastal waters. This species has previously been recorded off the Swedish west coast and German Bight, which may suggest a northward dispersal.

Monitoring of noble, signal and narrow-clawed crayfish using environmental DNA from freshwater samples.

For several hundred years freshwater crayfish (Crustacea-Decapoda-Astacidea) have played an important ecological, cultural and culinary role in Scandinavia. However, many native populations of noble crayfish Astacus astacus have faced major declines during the last century, largely resulting from human assisted expansion of non-indigenous signal crayfish Pacifastacus leniusculus that carry and transmit the crayfish plague pathogen. In Denmark, also the non-indigenous narrow-clawed crayfish Astacus leptodactylus has expanded due to anthropogenic activities. Knowledge about crayfish distribution and early detection of non-indigenous and invasive species are crucial elements in successful conservation of indigenous crayfish. The use of environmental DNA (eDNA) extracted from water samples is a promising new tool for early and non-invasive detection of species in aquatic environments. In the present study, we have developed and tested quantitative PCR (qPCR) assays for species-specific detection and quantification of the three above mentioned crayfish species on the basis of mitochondrial cytochrome oxidase 1 (mtDNA-CO1), including separate assays for two clades of A. leptodactylus. The limit of detection (LOD) was experimentally established as 5 copies/PCR with two different approaches, and the limit of quantification (LOQ) were determined to 5 and 10 copies/PCR, respectively, depending on chosen approach. The assays detected crayfish in natural freshwater ecosystems with known populations of all three species, and show promising potentials for future monitoring of A. astacus, P. leniusculus and A. leptodactylus. However, the assays need further validation with data 1) comparing traditional and eDNA based estimates of abundance, and 2) representing a broader geographical range for the involved crayfish species.

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.

Zoning in on parents' needs: understanding parents' perspectives in order to provide person-centered care.

PURPOSE: In order to develop a theoretical framework for person-centered care models for children with epilepsy and their parents, we conducted a qualitative study to explore and understand parents' needs, values, and preferences to ultimately reduce barriers that may be impeding parents from accessing and obtaining help for their children's co-occurring problems. METHODS: A qualitative grounded theory study design was utilized to understand parents' perspectives. The participants were 22 parents of children with epilepsy whose age ranged from 31 to 53 years. Interviews were conducted using open-ended semistructured questions to facilitate conversation. Transcripts were analyzed using grounded theory guidelines. RESULTS: In order to understand the different perspectives parents had about their child, we devised a theory composed of three zones (Zones 1, 2, and 3) that can be used to conceptualize parents' viewpoints. Zone location was based on a parent's perspectives on their child's comorbidities in the context of epilepsy. These zones were developed to help identify distinctions between parents' perspectives and to provide a framework within which to understand parents' readiness to access and implement interventions to address the child's struggles. These zones of understanding describe a parent's perspectives on their children's struggles at a particular point in time. This is the perspective from which parents address their child's needs. This theoretical perspective provides a structure in which to discuss a parent's perspectives on conceptualizing or comprehending the child's struggles in the context of epilepsy. The zones are based on how the parents describe (a) their concerns about the child's struggles and (b) their understanding of the struggles and (c) the parent's view of the child's future. CONCLUSIONS: Clinicians working with individuals and families with epilepsy are aware that epilepsy is a complex and unpredictable disorder. The zones help clinicians conceptualize and build a framework within which to understand how parents view their child's struggles, which influences the parents' ability to understand and act on clinician feedback and recommendations. Zones allow for increased understanding of the parent at a particular time and provide a structure within which a clinician can provide guidance and feedback to meet parents' needs, values, and preferences. This theory allows clinicians to meet the parents where they are and address their needs in a way that benefits the parents, family, and child.

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.

Potent infection reservoir of crayfish plague now permanently established in Norway.

Noble crayfish Astacus astacus is threatened in Europe due to invasive crayfish carrying the crayfish plague agent Aphanomyces astaci. Norway is among the last countries in which the introduction of non-indigenous crayfish has been limited through strict legislation practices. However, North American signal crayfish Pacifastacus leniusculus were recently discovered in a water-course that has been repeatedly hit by the plague. We mapped the distribution and relative density (catch per unit effort) of signal crayfish within this lake, and performed agent-specific real-time PCR to estimate the prevalence of A. astaci in the population. The resulting length frequencies and relative density estimates clearly demonstrate a well-established signal crayfish population, in which 86.4% of the analysed individuals were confirmed carriers. The success of detection was significantly higher (84.1%) in the crayfish tailfan (i.e. uropods) than in the soft abdominal cuticle (38.4%), which is commonly used in prevalence studies. We therefore propose tailfan (uropods and telson) as the preferred tissue for studying A. astaci prevalence in signal crayfish populations. The likelihood of detecting an A. astaci-positive signal crayfish increased significantly with increasing crayfish length. Further, large female crayfish expressed significantly higher PCR-forming units values than large males. In surveys primarily exploring the presence of A. astaci-positive individuals in a population, large females should be selected for molecular analyses. Our study demonstrates that a potent crayfish plague infection reservoir, evidently originating from the illegal human introduction of signal crayfish, has permanently been established in Norway.