The urgent need for robust coral disease diagnostics

Coral disease has emerged over recent decades as a significant threat to coral reef ecosystems, with declines in coral cover and diversity of Caribbean reefs providing an example of the potential impacts of disease at regional scales. If similar trends are to be mitigated or avoided on reefs worldwide, a deeper understanding of the factors underlying the origin and spread of coral diseases and the steps that can be taken to prevent, control, or reduce their impacts is required. In recent years, an increased focus on coral microbiology and the application of classic culture techniques and emerging molecular technologies has revealed several coral pathogens that could serve as targets for novel coral disease diagnostic tools. The ability to detect and quantify microbial agents identified as indicators of coral disease will aid in the elucidation of disease causation and facilitate coral disease detection and diagnosis, pathogen monitoring in individuals and ecosystems, and identification of pathogen sources, vectors, and reservoirs. This information will advance the field of coral disease research and contribute knowledge necessary for effective coral reef management. This paper establishes the need for sensitive and specific molecular-based coral pathogen detection, outlines the emerging technologies that could serve as the basis of a new generation of coral disease diagnostic assays, and addresses the unique challenges inherent to the application of these techniques to environmentally derived coral samples.     

海产贝类尼氏单抱子虫病害的研究进展

目前,我国形成规模养殖的经济贝类有近20种,贝类增养殖已经成为沿海海水养殖业的支柱产业。资料显示,2004年全国海水贝类产量为1024万吨,占海水养殖总产量的77．82％。但是目前,由于气候变化、海洋环境污染、外来生物入侵等因素导致我国海产贝类病害越来越重,寄生虫就是主要病原之一,其中尼氏单孢子虫就是其中的一种原生动物寄生虫,寄生于很多种海产贝类体内。这种病害在很多地区都有暴发,国外对其研究较多,国内梁玉波等时这一病害进行了研究。因此,系统阐述国外贝类尼氏单孢子虫病害的研究现状与进展,对我国海产贝类病害的研究具有现实意义。本文时尼氏单孢子虫的分类、病害的流行情况、尼氏单孢子虫的形态学,病害的主要症状。尼氏单孢子虫的检测方法,尼氏单孢子虫与寄主之间的交互作用,环境因素时病害流行的影响等方面进行了论述,为我国贝类病害的研究和防治提供参考。     

Loop-mediated isothermal amplification (LAMP) assays for detection and identification of aquaculture pathogens: current state and perspectives

Since its invention in 2000, loop-mediated isothermal amplification (LAMP) assay has been one of the most extensively used molecular diagnostic tools in bio-medical fields due to the rapidity, accuracy, and cost-effectiveness of the technique. This technique has also earned popularity in aquaculture disease diagnosis. Aquaculture, as a result of its rapid intensification and expansion, experiences increased infectious disease occurrences. For maintenance of economic viability, rapid, sensitive and efficient diagnosis of disease causing agents is an important step prior to undertaking effective prevention and control measures in aquaculture. Constraints on time and expertise required for conventional biochemical, serological and polymerase chain reaction (PCR)-based techniques offer avenues in adoption of the LAMP by the aquaculturists at field conditions. This assay has been successfully applied in detection of several bacterial, viral and parasitic pathogens causing serious diseases in aquaculture. In this review, we endeavored to accommodate the LAMP methodology with its different recent improvements and an overview of its application for the detection of aquaculture-associated pathogens.     

Essential diagnostics--the role of the Department of Biomedical Research of the Royal Tropical Institute in the 21st century

In the light of emerging and overlooked infectious diseases and widespread drug resistance, diagnostics have become increasingly important in supporting surveillance, disease control and outbreak management programs. In many low-income countries the diagnostic service has been a neglected part of health care, often lacking quantity and quality or even non-existing at all. High-income countries have exploited few of their advanced technical abilities for the much-needed development of low-cost, rapid diagnostic tests to improve the accuracy of diagnosis and accelerate the start of appropriate treatment. As is now also recognized by World Health Organization, investment in the development of affordable diagnostic tools is urgently needed to further our ability to control a variety of diseases that form a major threat to humanity. The Royal Tropical Institute's Department of Biomedical Research aims to contribute to the health of people living in the tropics. To this end, its multidisciplinary group of experts focuses on the diagnosis of diseases that are major health problems in low-income countries. In partnership we develop, improve and evaluate simple and cheap diagnostic tests, and perform epidemiological studies. Moreover, we advice and support others--especially those in developing countries--in their efforts to diagnose infectious diseases.     

A Box Model for Ecosystem-Level Management of Mussel Culture Carrying Capacity in a Coastal Bay

The carrying capacity of shellfish aquaculture is determined by the interaction of cultured species with the ecosystem, particularly food availability to suspension feeders. A multiple box dynamic ecosystem model was constructed to examine the carrying capacity for mussel (Mytilus edulis) aquaculture in Tracadie Bay, Prince of Edward Island, Canada. Criteria for carrying capacity were based on chlorophyll concentration. The model was run in two different years (1998 and 1999) in which time series for three points inside the bay and a point outside the bay were available. This data set allows spatial validation of the ecosystem model and assessment of its sensitivity to changes in boundary conditions. The model validation process indicated that the differential equations and parameters used in the simulation provided robust prediction of the ecological dynamics within the bay. Results verified that mussel biomass exerts top-down control of phytoplankton populations. The model indicates that conditions observed during 1999 are more sensitive to grazing pressure from aquaculture than was observed during 1998, highlighting the importance of inter-annual variability in carrying capacity of the bay. This result is important from a management perspective because it emphasizes application of a precautionary policy and prediction in regulation of aquaculture activity in the bay. Retrospective scenarios showed that although the bay could yield greater mussel biomass production, stress on the environment would lead the ecosystem outside of its natural range of variation. Despite the spatial simplicity employed in the present model, it provides substantial management capability as well as an ecosystem-oriented approach to shellfish aquaculture.     

A research agenda for malaria eradication: diagnoses and diagnostics

Many of malaria's signs and symptoms are indistinguishable from those of other febrile diseases. Detection of the presence of Plasmodium parasites is essential, therefore, to guide case management. Improved diagnostic tools are required to enable targeted treatment of infected individuals. In addition, field-ready diagnostic tools for mass screening and surveillance that can detect asymptomatic infections of very low parasite densities are needed to monitor transmission reduction and ensure elimination. Antibody-based tests for infection and novel methods based on biomarkers need further development and validation, as do methods for the detection and treatment of Plasmodium vivax. Current rapid diagnostic tests targeting P. vivax are generally less effective than those targeting Plasmodium falciparum. Moreover, because current drugs for radical cure may cause serious side effects in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency, more information is needed on the distribution of G6PD-deficiency variants as well as tests to identify at-risk individuals. Finally, in an environment of very low or absent malaria transmission, sustaining interest in elimination and maintaining resources will become increasingly important. Thus, research is required into the context in which malaria diagnostic tests are used, into diagnostics for other febrile diseases, and into the integration of these tests into health systems.     

Has the Time Come for Big Science in Wildlife Health?

The consequences of wildlife emerging diseases are global and profound with increased burden on the public health system, negative impacts on the global economy, declines and extinctions of wildlife species, and subsequent loss of ecological integrity. Examples of health threats to wildlife include Batrachochytrium dendrobatidis, which causes a cutaneous fungal infection of amphibians and is linked to declines of amphibians globally; and the recently discovered Pseudogymnoascus (Geomyces) destructans, the etiologic agent of white nose syndrome which has caused precipitous declines of North American bat species. Of particular concern are the novel pathogens that have emerged as they are particularly devastating and challenging to manage. A big science approach to wildlife health research is needed if we are to make significant and enduring progress in managing these diseases. The advent of new analytical models and bench assays will provide us with the mathematical and molecular tools to identify and anticipate threats to wildlife, and understand the ecology and epidemiology of these diseases. Specifically, new molecular diagnostic techniques have opened up avenues for pathogen discovery, and the application of spatially referenced databases allows for risk assessments that can assist in targeting surveillance. Long-term, systematic collection of data for wildlife health and integration with other datasets is also essential. Multidisciplinary research programs should be expanded to increase our understanding of the drivers of emerging diseases and allow for the development of better disease prevention and management tools, such as vaccines. Finally, we need to create a National Fish and Wildlife Health Network that provides the operational framework (governance, policies, procedures, etc.) by which entities with a stake in wildlife health cooperate and collaborate to achieve optimal outcomes for human, animal, and ecosystem health.     

Diagnostic tests and their application in the management of soil‐ and water‐borne oomycete pathogen species

Oomycete diseases cause significant losses across a broad range of crop and aquaculture commodities worldwide. These losses can be greatly reduced by disease management practices steered by accurate and early diagnoses of pathogen presence. Determinations of disease potential can help guide optimal crop rotation regimes, varietal selections, targeted control measures, harvest timings and crop post‐harvest handling. Pathogen detection prior to infection can also reduce the incidence of disease epidemics. Classical methods for the isolation of oomycete pathogens are normally deployed only after disease symptom appearance. These processes are often time consuming, relying on culturing the putative pathogen(s) and the availability of expert taxonomic skills for accurate identification, a situation that frequently results in either delayed application, or routine ‘blanket’ over‐application of control measures. Increasing concerns about pesticides in the environment and the food chain, removal or restriction of their usage combined with rising costs have focussed interest in the development and improvement of disease management systems. To be effective, these require timely, accurate and preferably quantitative diagnoses. A wide range of rapid diagnostic tools, from point of care immunodiagnostic kits to next generation nucleotide sequencing have potential application in oomycete disease management. Here we review currently available as well as promising new technologies in the context of commercial agricultural production systems, considering the impacts of specific biotic and abiotic and other important factors such as speed and ease of access to information and cost effectiveness.     

A real‐time PCR assay for detection and quantification of Lactococcus garvieae

Aims: To develop a rapid, sensitive, specific tool for the detection and quantification of Lactococcus garvieae in food and environmental samples. Methods and Results: A real‐time quantitative PCR (qPCR) assay with primers for CAU12F and CAU12R based on the 16S rRNA gene of L. garvieae was successfully established. The limit of detection for L. garvieae genomic DNA was 1 ng DNA in conventional PCR and 32 fg with a mean C_T value of 36·75 in qPCR. Quantification of L. garvieae vegetative cells was linear (R² = 0·99) over a 7‐log‐unit dynamic range down to ten L. garvieae cells. Conclusions: This method is highly specific, sensitive and reproducible for the detection of L. garvieae compared to gel‐based conventional PCR assays, thus providing precise quantification of L. garvieae in food and natural environments. Significance and Impact of the Study: This work provides efficient diagnostic and monitoring tools for the rapid identification of L. garvieae, an emerging pathogen in aquaculture and an occasional human pathogen from other members of the genus Lactobacillus.     

Biofouling in marine aquaculture: a review of recent research and developments

Biofouling in marine aquaculture is one of the main barriers to efficient and sustainable production. Owing to the growth of aquaculture globally, it is pertinent to update previous reviews to inform management and guide future research. Here, the authors highlight recent research and developments on the impacts, prevention and control of biofouling in shellfish, finfish and seaweed aquaculture, and the significant gaps that still exist in aquaculturalists’ capacity to manage it. Antifouling methods are being explored and developed; these are centred on harnessing naturally occurring antifouling properties, culturing fouling-resistant genotypes, and improving farming strategies by adopting more sensitive and informative monitoring and modelling capabilities together with novel cleaning equipment. While no simple, quick-fix solutions to biofouling management in existing aquaculture industry situations have been developed, the expectation is that effective methods are likely to evolve as aquaculture develops into emerging culture scenarios, which will undoubtedly influence the path for future solutions.     

Development of a Multiplexed Bead-Based Suspension Array for the Detection and Discrimination of Pospiviroid Plant Pathogens

Efficient and reliable diagnostic tools for the routine indexing and certification of clean propagating material are essential for the management of pospiviroid diseases in horticultural crops. This study describes the development of a true multiplexed diagnostic method for the detection and identification of all nine currently recognized pospiviroid species in one assay using Luminex bead-based suspension array technology. In addition, a new data-driven, statistical method is presented for establishing thresholds for positivity for individual assays within multiplexed arrays. When applied to the multiplexed array data generated in this study, the new method was shown to have better control of false positives and false negative results than two other commonly used approaches for setting thresholds. The 11-plex Luminex MagPlex-TAG pospiviroid array described here has a unique hierarchical assay design, incorporating a near-universal assay in addition to nine species-specific assays, and a co-amplified plant internal control assay for quality assurance purposes. All assays of the multiplexed array were shown to be 100% specific, sensitive and reproducible. The multiplexed array described herein is robust, easy to use, displays unambiguous results and has strong potential for use in routine pospiviroid indexing to improve disease management strategies.     

Production, characterization, and application of antibodies against heat-labile type-I toxin for detection of enterotoxigenic Escherichia coli

Strains of enterotoxigenic Escherichia coli (ETEC) are responsible for significant rates of morbidity and mortality among children, particularly in developing countries. The majority of clinical and public health laboratories are capable of isolating and identifying Salmonella, Shigella, Campylobacter, and Escherichia coli O157:H7 from stool samples, but ETEC cannot be identified by routine methods. The method most often used to identify ETEC is polymerase chain reaction for heat-stable and heat-labile enterotoxin genes, and subsequent serotyping, but most clinical and public health laboratories do not have the capacity or resources to perform these tests. In this study, polyclonal rabbit and monoclonal mouse IgG2b antibodies against ETEC heat-labile toxin-I (LT) were characterized and the potential applicability of a capture assay was analyzed. IgG-enriched fractions from rabbit polyclonal and the IgG2b monoclonal antibodies recognized LT in a conformational shape and they were excellent tools for detection of LT-producing strains. These findings indicate that the capture immunoassay could be used as a diagnostic assay of ETEC LT-producing strains in routine diagnosis and in epidemiological studies of diarrhea in developing countries as enzyme linked immunosorbent assay techniques remain as effective and economical choice for the detection of specific pathogen antigens in cultures.     

The use of probiotics in shrimp aquaculture

Shrimp aquaculture, as well as other industries, constantly requires new techniques in order to increase production yield. Modern technologies and other sciences such as biotechnology and microbiology are important tools that could lead to a higher quality and greater quantity of products. Feeding and new practices in farming usually play an important role in aquaculture, and the addition of various additives to a balanced feed formula to achieve better growth is a common practice of many fish and shrimp feed manufacturers and farmers. Probiotics, as 'bio-friendly agents' such as lactic acid bacteria and Bacillus spp., can be introduced into the culture environment to control and compete with pathogenic bacteria as well as to promote the growth of the cultured organisms. In addition, probiotics are nonpathogenic and nontoxic microorganisms without undesirable side-effects when administered to aquatic organisms. These strains of bacteria have many other positive effects, which are described in this article.     

DNA-mounted self-assembly: new approaches for genomic analysis and SNP detection

This article presents an overview of new emerging approaches for nucleic acid detection via hybridization techniques that can potentially be applied to genomic analysis and SNP identification in clinical diagnostics. Despite the availability of a diverse variety of SNP genotyping technologies on the diagnostic market, none has truly succeeded in dominating its competitors thus far. Having been designed for specific diagnostic purposes or clinical applications, each of the existing bio-assay systems (briefly outlined here) is usually limited to a relatively narrow aspect or format of nucleic acid detection, and thus cannot entirely satisfy all the varieties of commercial requirements and clinical demands. This drives the diagnostic sector to pursue novel, cost-effective approaches to ensure rapid and reliable identification of pathogenic or hereditary human diseases. Hence, the purpose of this review is to highlight some new strategic directions in DNA detection technologies in order to inspire development of novel molecular diagnostic tools and bio-assay systems with superior reliability, reproducibility, robustness, accuracy and sensitivity at lower assay cost. One approach to improving the sensitivity of an assay to confidently discriminate between single point mutations is based on the use of target assembled, split-probe systems, which constitutes the main focus of this review.     

水产品中创伤弧菌DNA环介导恒温扩增快速检测方法的建立及初步应用

创伤弧菌是一种重要的食源性致病菌,主要存在于河口和海洋环境中,严重危害水产养殖业的发展和人类健康。建立快速、准确、易操作的检测方法对防控创伤弧菌的传染,保障水产养殖业发展和增强食品安全意义重大。基于创伤弧菌vvHA基因,利用一种新型的核酸扩增技术-环介导恒温扩增(loop-mediated isothermal amplification,LAMP),建立了创伤弧菌LAMP快速检测方法。对11种共46株细菌进行扩增,仅创伤弧菌为LAMP阳性结果,说明LAMP方法具有高度特异性。灵敏度试验结果表明,对创伤弧菌纯培养菌的检测灵敏度为15CFU/ml,对污染食品中创伤弧菌的检测灵敏度为24CFU/g。此法40~60min内即可完成检测,检验检疫实践证明:LAMP方法操作简便、特异性强、灵敏度高且成本低廉,具有良好的应用前景。     

The mouse bioassay for diarrhetic shellfish poisoning: a gross misuse of laboratory animals and of scientific methodology

The UK shellfish industry has recently been affected by the statutory closure of several cockle beds, following the detection of samples causing rapid and severe reactions in the regulatory approved test for diarrhetic shellfish poisoning (DSP) toxins, the mouse bioassay (MBA). It is contended that these so-called atypical results are due to procedural artefacts of the MBA; so far, several studies have failed to identify their cause. This paper critically assesses the development, regulatory use and methodological deficiencies of the MBA. It also discusses how testing for DSP toxins could and should have been improved and made more humane by applying the Three Rs concept of Reduction, Refinement and Replacement, and by the proper validation of the test method used. It is concluded that the MBA should not have been developed for the routine screening of shellfish samples, as it has a substantially severe endpoint and is not used as part of a tiered-testing strategy with non-animal methods. Moreover, during the UK monitoring programme for DSP toxins, the assay has been used without an optimised and universal protocol, and apparently without due regard to the principles of basic scientific methodology. In view of this, the atypical results obtained for cockle samples cannot be relied on as evidence of a human health hazard. It is recommended that the use of the MBA should be discontinued as soon as possible, in favour of other methods, especially those involving non-animal techniques. In the short-term, these methods should be based on analytical chemical detection systems and the essential availability of the relevant pure toxin standards. The lack of any known toxins in samples should be taken as evidence of lack of contamination. The suitability of the existing non-animal methods needs to be assessed as a matter of urgency. It is crucial that all new methods should be properly validated, and that their acceptability for their stated purposes should be endorsed by recognised criteria and validation centres, before being recommended to, or required by, regulatory agencies. In this way, the possibility that scientifically unsuitable methods will once again be used for monitoring for the contamination of shellfish with toxins can be avoided. This gross misuse of laboratory animals and ill-judged application of science should never be allowed to occur again.     

Distribution,occurrence and biotoxin composition of the main shellfish toxin producing microalgae within European waters: A comparison of methods of analysis

Harmful algal blooms (HABs) are a natural global phenomena emerging in severity and extent. Incidents have many economic, ecological and human health impacts. Monitoring and providing early warning of toxic HABs are critical for protecting public health. Current monitoring programmes include measuring the number of toxic phytoplankton cells in the water and biotoxin levels in shellfish tissue. As these efforts are demanding and labour intensive, methods which improve the efficiency are essential. This study compares the utilisation of a multitoxin surface plasmon resonance (multitoxin SPR) biosensor with enzyme-linked immunosorbent assay (ELISA) and analytical methods such as high performance liquid chromatography with fluorescence detection (HPLC-FLD) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) for toxic HAB monitoring efforts in Europe. Seawater samples (n = 256) from European waters, collected 2009–2011, were analysed for biotoxins: saxitoxin and analogues, okadaic acid and dinophysistoxins 1/2 (DTX1/DTX2) and domoic acid responsible for paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP) and amnesic shellfish poisoning (ASP), respectively. Biotoxins were detected mainly in samples from Spain and Ireland. France and Norway appeared to have the lowest number of toxic samples. Both the multitoxin SPR biosensor and the RNA microarray were more sensitive at detecting toxic HABs than standard light microscopy phytoplankton monitoring. Correlations between each of the detection methods were performed with the overall agreement, based on statistical 2 × 2 comparison tables, between each testing platform ranging between 32% and 74% for all three toxin families illustrating that one individual testing method may not be an ideal solution. An efficient early warning monitoring system for the detection of toxic HABs could therefore be achieved by combining both the multitoxin SPR biosensor and RNA microarray.     

Use of two-dimensional gel electrophoresis to differentiate morphospecies of Alexandrium minutum, a paralytic shellfish poisoning toxin-producing dinoflagellate of harmful algal blooms

Contamination of shellfish with paralytic shellfish poisoning toxins (PST) produced by toxic harmful algal blooms (HABs) have been negatively affecting the shellfish and aquaculture industries worldwide. Therefore, accurate and early identification of toxic phytoplankton species is crucial in HABs surveillance programs that allow fish-farmers to take appropriate preventive measures in shellfish harvesting and other aquaculture activities to overcome the negative impacts of HABs on human health. The identification of toxic dinoflagellates present in the water is currently a time-consuming operation since it requires skillful taxonomists and toxicologists equipped with optical and scanning electron microscopes as well as sophisticated equipment, for example, high-performance liquid chromotography-fluorescence detection. In this paper, a two-dimensional gel electrophoresis (2-DE)-based proteomic approach was applied to discriminate between toxic and nontoxic strains of Alexandrium minutum. Variation in morphological features between toxic and nontoxic strains was minimal and not significant. Also, variation in 2-DE protein patterns within either toxic or nontoxic strains was low, but pronounced differences were detected between toxic and nontoxic strains. The most notable differences between these strains were several abundant proteins with pIs ranging from 4.8 to 5.3 and apparent molecular masses between 17.5 and 21.5 kDa. Groups of proteins, namely NT1, NT2, NT3, and NT4, were consistently found in all nontoxic strains, while T1 and T2 were prominent in the toxic strains. These specific protein spots characteristic for toxic and nontoxic strains remained clearly distinguishable irrespective of the various growth conditions tested. Therefore, they have the potential to serve as "taxonomic markers" to distinguish toxic and nontoxic strains within A. minutum. Initial studies revealed that the expression pattern of T1 was tightly correlated to toxin biosynthesis in the examined alga and may be used to serve as a potential toxin indicator.     

Surface Immuno-Functionalisation for the Capture and Detection of Vibrio Species in the Marine Environment: A New Management Tool for Industrial Facilities

Bacteria from the genus Vibrio are a common and environmentally important group of bacteria within coastal environments and include species pathogenic to aquaculture organisms. Their distribution and abundance are linked to specific environmental parameters, including temperature, salinity and nutrient enrichment. Accurate and efficient detection of Vibrios in environmental samples provides a potential important indicator of overall ecosystem health while also allowing rapid management responses for species pathogenic to humans or species implicated in disease of economically important aquacultured fish and invertebrates. In this study, we developed a surface immuno-functionalisation protocol, based on an avidin-biotin type covalent binding strategy, allowing specific sandwich-type detection of bacteria from the Vibrio genus. The assay was optimized on 12 diverse Vibrio strains, including species that have implications for aquaculture industries, reaching detection limits between 7×10³ to 3×10⁴ cells mL⁻¹. Current techniques for the detection of total Vibrios rely on laborious or inefficient analyses resulting in delayed management decisions. This work represents a novel approach for a rapid, accurate, sensitive and robust tool for quantifying Vibrios directly in industrial systems and in the environment, thereby facilitating rapid management responses.