Risk assessment in fish welfare, applications and limitations.

The Treaty of Amsterdam, in force since 1 May 1999, has established new ground rules for the actions of the European Union (EU) on animal welfare. It recognizes that animals are sentient beings and obliges the European Institutions to pay full regard to the welfare requirements of animals when formulating and implementing Community legislation. In order to properly address welfare issues, these need to be assessed in a scientific and transparent way. The principles of risk assessment in terms of transparency and use of available scientific data are probably well suited for this area. The application of risk assessment for terrestrial and aquatic animal welfare is a relatively new area. This paper describes the work developed in the context of the European Food Safety Authority (EFSA) opinions on the application of a risk assessment methodology to fish welfare. Risk assessment is a scientifically based process that seeks to determine the likelihood and consequences of an adverse event, which is referred to as a hazard. It generally consists of the following steps: (i) hazard identification, (ii) hazard characterisation, (iii) exposure assessment and (iv) risk characterisation. Different approaches can be used for risk assessments, such as qualitative, semi-quantitative and quantitative approaches. These are discussed in the context of fish welfare, using examples from assessments done to aquaculture husbandry systems and stunning/killing methods for farmed fish. A critical review of the applications and limitations of the risk methodology in fish welfare is given. There is a need to develop appropriate indicators of fish welfare. Yet, risk assessment methodology provides a transparent approach to identify significant hazards and support recommendations for improved welfare.

Effects of pesticide compounds (chlorothalonil and mancozeb) and benzo[a]pyrene mixture on aryl hydrocarbon receptor, p53 and ubiquitin gene expression levels in haemocytes of soft-shell clams (Mya arenaria).

The aim of this study is to investigate the effects of the pesticides/polycyclic aromatic hydrocarbon mixture on aryl hydrocarbon receptor (AhR), p53 and ubiquitin mRNA level in haemocytes of Mya arenaria exposed to a mixture of chlorothalonil, mancozeb and benzo[a]pyrene (BaP) for 48 and 72 h. AhR, p53 and ubiquitin gene expression levels were quantified using quantitative Real-time PCR. For robust and accurate quantification of transcripts, suitable housekeeping genes were selected from four sets of ribosomal and elongation factors transcripts previously sequenced from Mya arenaria using geNorm open source software. Quantitative Real-time PCR data exhibited a significantly high expression of AhR after 72 h of exposure (P ≤ 0.05). p53 gene expression seems to be up-regulated by the mixture after 48 h, however not significantly; but the level of p53 mRNA is down-regulated by the xenobiotics between 48 and 72 h after exposure. This study postulates that AhR mRNA levels could be used as an indicator of the exposure of clams' haemocytes to a mixture of xenobiotics such as chlorothalonil, mancozeb and BaP. However, further studies have to be pursued in order to unravel the molecular mechanisms involved in the p53 signaling pathway.

Applying a One Health Approach in Global Health and Medicine: Enhancing Involvement of Medical Schools and Global Health Centers.

Background: Multidisciplinary and multisectoral approaches such as One Health and related concepts (e.g., Planetary Health, EcoHealth) offer opportunities for synergistic expertise to address complex health threats. The connections between humans, animals, and the environment necessitate collaboration among sectors to comprehensively understand and reduce risks and consequences on health and wellbeing. One Health approaches are increasingly emphasized for national and international plans and strategies related to zoonotic diseases, food safety, antimicrobial resistance, and climate change, but to date, the possible applications in clinical practice and benefits impacting human health are largely missing. Methods: In 2018 the "Application of the One Health Approach to Global Health Centers" conference held at the Albert Einstein College of Medicine convened experts involved in One Health policy and practice. The conference examined issues relevant to One Health approaches, sharing examples of challenges and successes to guide application to medical school curricula and clinical practice for human health. This paper presents a synthesis of conference proceedings, framed around objectives identified from presentations and audience feedback. Findings and Recommendations: The following objectives provide opportunities for One Health involvement and benefits for medical schools and global health centers by: 1) Improving One Health resource sharing in global health and medical education; 2) Creating pathways for information flow in clinical medicine and global health practice; 3) Developing innovative partnerships for improved health sector outcomes; and 4) Informing and empowering health through public outreach. These objectives can leverage existing resources to deliver value to additional settings and stakeholders through resource efficiency, more holistic and effective service delivery, and greater ability to manage determinants of poor health status. We encourage medical and global health educators, practitioners, and students to explore entry points where One Health can add value to their work from local to global scale.

Identification and expression of immune-related genes in hemocytes of soft-shell clams, Mya arenaria, challenged with Vibrio splendidus.

Although the mollusc immune system has been studied at the cellular level, the response to pathogens at gene expression level has not been thoroughly investigated. This study aimed to investigate the early molecular response of hemocytes of soft-shell clams, Mya arenaria, to Vibrio splendidus strain LGP32 by identification of transcripts involved in immune defense. The Suppression Subtractive Hybridization (SSH) was used to selectively identify differentially expressed genes in hemocytes exposed to V. splendidus at a ratio 1:1 for 2 h. Both forward and reverse subtracted cDNA were constructed and a total of 16,000 reads were obtained and analyzed. Identity searches in genome databases were performed using BlastX program and transcripts were clustered to cellular functions including structural proteins, immunity, stress proteins, apoptosis, cell process, metabolism and signal transduction. Among the differentially expressed immune associated genes were ficolin, killer cell lectin-like receptor, natural resistance-associated macrophage protein 1 (Nramp-1), mitogen-activated protein kinases (MAPK), ferritin, heat shock proteins 90 (HSP90) and cathepsin and their expressions were quantified using Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) at 1, 2 and 3 h post-Vibrio challenge. These genes showed similar expression patterns, up-regulation at 1 h, followed by a down-regulation at 2 and 3 h. These data corroborates our previous observations of cell rounding, reduced phagocytosis and respiratory burst activity. To our knowledge, this is the first study to demonstrate an effect of V. splendidus on expression of genes related to immune system in soft-shell clams M. arenaria. However, further investigations are needed to unravel the molecular mechanisms of hemocytes subjected to V. splendidus.

Differential in vivo response of soft-shell clam hemocytes against two strains of Vibrio splendidus: changes in cell structure, numbers and adherence.

Host-pathogen interaction models in aquatic species are useful tools for understanding the pathogenicity of diseases in cultured and wild populations. In this study we report the differential in vivo response of soft-shell clam (Mya arenaria) hemocytes against two strains of Vibrio splendidus. Responses were measured 24h after injecting into the posterior adductor muscle either an endemic wild-type strain (7SHRW) or a strain associated with oyster mortalities (LGP32-GFP). Changes in hemocyte structure (percentage of rounded cells) were assessed microscopically. Changes in adherence and hemocyte numbers were analyzed by flow-cytometric cell counting. Increased percentages of rounded cells were found in response to both strains. However, values from the group infected with LGP32-GFP were significantly higher (p<0.01) than with 7SHRW. The cell adherence was markedly diminished (p<0.001) by LGP32-GFP whereas 7SHRW did not change it significantly. Increased numbers of hemocytes (p<0.001) were induced by LGP32-GFP, while no significant changes were found after infection with 7SHRW. These results show the regulatory capacity of soft-shell clams hemocytes to perform specific responses against different strains of V. splendidus.

Changes induced by two strains of Vibrio splendidus in haemocyte subpopulations of Mya arenaria, detected by flow cytometry with LysoTracker.

Flow-cytometric characterisation of bivalve haemocytes is usually performed by light-scatter profiles based on size and complexity of the cells. Additional means of characterisation such as specific fluorescent dyes are not commonly used to discriminate cell subpopulations in challenged and unchallenged haemocytes. In the present study, we characterise the changes in haemocyte subpopulations of soft-shell clam Mya arenaria induced by in vivo challenge with 2 strains of Vibrio splendidus by using a fluorescent probe. Responses were measured 24 h after infection with either a local wild strain (7SHRW) or a modification (LGP32-GFP) of a strain associated with oyster mortalities in France (LGP32). Changes in haemocyte subpopulations were analysed using flow cytometry based on 2-parameter scatter profiles and lysosomal content reflected by LysoTracker staining. Forward and side-scatter profiles revealed 2 haemocyte subpopulations: hyalinocytes and granulocytes. Granulocytes exhibited significantly higher levels of lysosomal staining (p < 0.01). Following infection with LGP32-GFP, both subpopulations merged into a single continuous group and their lysosomal content significantly decreased (p < 0.05). Independent modifications after infection were observed in the proportions of subpopulations established by their lysosomal content. While the subpopulation of hyalinocytes had lower levels of lysosomal content after infection, especially with LGP32-GFP (p < 0.001), the subpopulation of granulocytes had similar levels of lysosomes after infection with 7SHRW and significantly decreased levels after infection with LGP32-GFP (p = 0.001). Our data suggest specific modulation of bivalve responses against pathogenic bacteria that would include degranulation.

Immunophenotyping of Mya arenaria neoplastic hemocytes using propidium iodide and a specific monoclonal antibody by flow cytometry.

Disseminated neoplasia (DN) is a disorder referred to as hemic neoplasia (HN) in the soft-shell clam Mya arenaria. Traditionally, diagnosis is performed by hematocytology or histology. The intensity of the disease is generally given as the percentage of transformed neoplastic cells out of total number of hemocytes. Flow cytometry techniques have found a field of application in diagnosis of HN with analysis of ploidy. Hemocytes of the soft-shell clams with HN display tetraploid DNA content, as shown by propidium iodide staining. This feature makes difficult HN diagnosis in the soft-shell clam, especially for early stages of the condition, since the percentage of normal circulating cells undergoing mitosis, which also are tetraploid, remains unknown in molluscs. Use of specific monoclonal antibodies in a flow cytometry assay was foreseen as a way to overcome the difficulty. The purpose of this study was to develop a double staining protocol using propidium iodide for hemocyte cycle analysis and the MAb 1E10 for staining of HN cells. Our results showed a correlation between tetraploid and MAb 1E10-stained hemocytes in a single clam with moderate HN. This protocol offers some potential for further investigation of this cell disorder. However, a validation step will be necessary to confirm our preliminary results.

Assessment of haemic neoplasia in different soft shell clam Mya arenaria populations from eastern Canada by flow cytometry.

Diagnosis of haemic neoplasia (HN) in the soft shell clam, Mya arenaria, is often achieved by hematocytology and histology. Since neoplastic cells display tetraploid DNA contents, haemocyte cell cycle analysis was developed for use as a diagnosis tool. The aim of this study was to assess the application of a flow cytometry procedure of cell cycle analysis established for the common cockle, to clams and to evaluate different thresholds of value for the percentage of tetraploid cells for establishing HN disease status of individual clams and clam populations. HN status of six clam populations from eastern Canada was determined. Results of the present study demonstrate a flow cytometry procedure to be useful for HN diagnosis in clams. Individual clams were considered to be affected by HN when presenting at least 20% of haemocytes in S-4N phase; and negative when presenting less that 5% of haemocytes in S-4N phase. As discussed in this paper, intermediate cases represent uncertain diagnoses including either false-negative or false-positive clams, which are difficult to discriminate. At a population level, an additional threshold of 15% for the mean intensity of the disease is proposed, which means having in the population several individual clams presenting more than 20% of their haemocytes in S-4N phase. Based on these thresholds of value, only one population was considered as free of HN disease, and one population was unequivocally affected by HN. For the four other clam populations, further investigations are needed toward development and use of specific and objective biomarkers of HN.

Selection and evaluation of housekeeping genes for haemocytes of soft-shell clams (Mya arenaria) challenged with Vibrio splendidus.

Gene expression studies have opened a tremendous field of investigation in biological research over the last decades. Expression of genes is most frequently quantified by real time PCR (RT-qPCR), as this method has proven to be highly sensitive. One of the critical steps, however, in comparing transcription profiles is the availability of selected housekeeping genes. Expression of these genes should be steadily stable across the conditions under study so that they provide a baseline for gene expression comparison. Such a baseline is best established using a set of few housekeeping genes. Usually, those genes are involved in maintaining homeostasis and cell viability. In our study, nine candidate genes were used, including some commonly used housekeeping genes, such as ribosomal RNA (18S, S-15, S-18 and L-37), beta actin, ubiquitin, receptor activated C kinase (RACK) and elongation factor 1 and 2, in order to determine the most stable housekeeping genes, after haemocytes of Mya arenaria were exposed to Vibrio splendidus for 2 h. Our results showed that EF-1, S-18 and ubiquitin appear to be the most stable genes for this experimental condition. On the other hand, both 18S and beta actin, the most widely used housekeeping genes, turned out to be the least stable. This demonstrates the absolute need for preliminary assessment of housekeeping genes in gene expression studies.

Institutionalizing One Health: From Assessment to Action.

A One Health approach is critical to strengthening health security at country, regional, and global levels. However, operationally its uptake remains limited. Recent momentum in assessing capacity to effectively prevent, detect, and respond to disease threats has resulted in identification of gaps that require dedicated action. This article highlights relevant tools, standards, and guidance to assist countries and institutions in meeting the collective vision articulated at the 2018 Prince Mahidol Award Conference on "Making the World Safe from the Threats of Emerging Infectious Diseases." Taking stock of assessment findings, resources, priorities, and implementation initiatives across human and animal health, environment and disaster risk reduction sectors can help expand participation in global health security, target risk drivers, and form synergies for collective action and shared gains for both emerging and endemic disease challenges. In addition to health security gains, a multisectoral, One Health approach can drive benefits for wider health sector and global development goals.

Associations between animal welfare indicators and Campylobacter spp. in broiler chickens under commercial settings: A case study.

Few studies have previously investigated how poor animal welfare might be associated with infection of zoonotic pathogens in humans. This paper assesses the predictive value of the presence of Campylobacter spp. in broiler chicken flocks when animal-based measures related to footpad dermatitis, hock burns, body lesions and arthritis are identified under commercial conditions (high density). The study population included 32 flocks analysed on farm and at slaughter, slaughtered between April and August 2008 in six different slaughter plants in Brittany, France. Welfare and health indicators are those indicated by the European legislation and sampling was carried out in the framework of the European baseline survey on the prevalence of Campylobacter in broiler chicken. Caecal contents, sampled both on farm and at slaughter, and carcass skin samples from the neck and breast at slaughter, were investigated for the presence of Campylobacter spp. Logistic models/classification trees were used to estimate the probability of the presence (or absence) of a specific foodborne pathogen in a flock based on specific animal-based measures (or combinations of measures) in order to study the potential relationship between welfare indicators and foodborne pathogen prevalence/incidence levels. On farm, flocks with more than 25% animals with severe lesions on between 25 and 50% of the footpad are predicted to be Campylobacter-positive whereas flocks where less than 13 individuals have arthritis are predicted to be Campylobacter-negative. The error rate on farm and at slaughter was 10 and 4% respectively indicating good predicting abilities. A poor welfare environment may result in stress, which reduces chicken immunocompetence making them more susceptible to Campylobacter spp. An infection with Campylobacter spp may lead to impaired defence and susceptibility to other pathogens which may result in greater intestinal excretion. Poor welfare and high growing rate lead to digestive troubles that lead to litter humidity. Litter humidity that, among other things, causes footpad dermatitis may also influence the horizontal transmission of the Campylobacter spp. infection due to the normal coprophagic behaviour of poultry. Reducing welfare problems by a better management of rearing conditions would not only improve broiler welfare, but it would also decrease the risks of Campylobacter contamination, of carcass condemnations and of economic loss for the poultry industry.

One Health Economics to confront disease threats.

Global economic impacts of epidemics suggest high return on investment in prevention and One Health capacity. However, such investments remain limited, contributing to persistent endemic diseases and vulnerability to emerging ones. An interdisciplinary workshop explored methods for country-level analysis of added value of One Health approaches to disease control. Key recommendations include: 1. systems thinking to identify risks and mitigation options for decision-making under uncertainty; 2. multisectoral economic impact assessment to identify wider relevance and possible resource-sharing, and 3. consistent integration of environmental considerations. Economic analysis offers a congruent measure of value complementing diverse impact metrics among sectors and contexts.

Viral gametocytic hypertrophy of Crassostrea gigas in France: from occasional records to disease emergence?

Viral gametocytic hypertrophy was reported for the first time in 2001 in Pacific oyster Crassostrea gigas in France. Since this date, the number of reported cases and the distribution area have increased every year; however, the cases are not associated with macroscopic signs or increased mortality rates. Both male and female gametes were hypertrophied and basophilic inclusions were observed in gamete nuclei. Transmission electron microscopy revealed the presence of viral particles in these intranuclear basophilic inclusions. These particles had characteristics similar to those of the Papillomaviridae and Polyoma viridae families: they were small, non-enveloped, icosahedral, and 44 to 56 nm in diameter. The viral particles were found in male, female and hermaphrodite oysters and no significant difference in viral infection was observed between those groups. The frequency of detection and the intensity of infection were low and no host defence reaction was recognised, suggesting that the viral particles had a weak impact on C. gigas. The viral particles described in the present study seem to be similar to these described in C. virginica in the USA and Canada and in C. gigas in Korea, but further studies are required to confirm their identity. The issue of a possible emergence of this infection is discussed.

Molecular characterisation of an Australian isolate of Bonamia exitiosa.

An Australian (New South Wales) isolate of Bonamia was characterised at the molecular level by sequencing the 18S-ITS-1 region of the small subunit rRNA operon obtained from flat oysters Ostrea angasi shown to be infected by histological examination. Sequence data alignment with homologous genes from 2 other isolates of Bonamia (New Zealand and France) revealed high levels of nucleotide identity with both isolates. However, the Australian Bonamia is shown to be more closely related to the New Zealand isolate, suggesting the existence of an oceanic subgroup of Bonamia.

First report on histology and ultrastructure of an intrahemocytic paramyxean parasite (IPP) from tunicate Halocynthia roretzi in Korea.

In 2004, epizootiological studies were conducted on mass mortalities of tunicates Halocynthia roretzi in Goje, Korea. The clinical characteristics of infected H. roretzi were weakness of the tunic, loss of elasticity, and finally death involving a rupture of the tunic. Histological studies revealed severe hemocyte infiltration in the connective tissue surrounding the intestine and mantle of infected H. roretzi. Hypertrophied eosinophilic hemocytes containing several cytoplasmic vacuoles were observed in the connective tissue surrounding the intestine, gill and mantle. Ultrastructural examination revealed the presence of a parasite in the cytoplasm of hemocytes. Secondary cells were observed in the primary cell of the parasite. Spore formation within primary cells suggests that the parasite may be an intrahemocytic paramyxean parasite (IPP) and may cause mass mortality of H. roretzi.

Development of a TaqMan PCR assay for the detection of Bonamia species.

The development of molecular diagnostic assays with increased sensitivity compared with conventional histological techniques is highly desirable for effective management of bonamiosis in cultured oyster stocks and wild populations. A real-time TaqMan PCR assay was developed for the specific detection of Bonamia species in infected oyster tissues. The TaqMan assay was shown to be significantly more sensitive than histopathology. Although a real-time TaqMan PCR assay is comparable with conventional PCR in terms of sensitivity, it offers the advantages that it is a rapid test and has a very low risk of sample cross-contamination. Furthermore, it can be optimised to quantify the parasite load in samples. The assay detected Bonamia isolates from Australia, New Zealand, Europe, Canada, Chile and the USA and therefore demonstrated genus specificity as tested in this study.

Drivers for emerging issues in animal and plant health.

The history of agriculture includes many animal and plant disease events that have had major consequences for the sector, as well as for humans. At the same time, human activities beyond agriculture have often driven the emergence of diseases. The more that humans expand the footprint of the global population, encroach into natural habitats, alter these habitats to extract resources and intensify food production, as well as move animals, people and commodities along with the pathogens they carry, the greater the potential for pathogens and pests to spread and for infection to emerge or re-emerge. While essential to human well-being, producing food also plays a major role in disease dynamics. The risk of emergence of pests and pathogens has increased as a consequence of global changes in the way food is produced, moved and consumed. Climate change is likely to increase pressure on the availability of food and provide newly suitable conditions for invasive pests and pathogens. Human population displacements due to economic, political and humanitarian crises represent another set of potential drivers for emerging issues. The overlapping drivers of plant, animal and human disease emergence and environmental changes point towards the concept of 'One Health'. This paradigm underlines the urgent need to understand the influence of human behaviour and incorporate this understanding into our approach to emerging risks. For this, we face two major challenges. One is cultural; the second is methodological. We have to look at systems not under the narrow view of specific hazards but with a wider approach to system dynamics, and consider a broad spectrum of potential outcomes in terms of risk. In addition, we have to make sense of the vast amounts of data that are available in the modern age. This paper aims to help in preparing for the cultural and methodological shifts needed in our approach to emerging risks.

[Characterization of pathogenic bacteria of the cupped oyster Crassostrea gigas]. / Caractérisation de bactéries pathogènes de naissain d'huître creuse Crassostrea gigas.

The French mollusc production is mainly based on the Pacific cupped oyster, Crassostrea gigas. Since 1991, outbreaks of mass mortality of juveniles are reported during the summer period. These outbreaks are a major concern of oyster industry. Several studies have established given bacterial strains to be pathogenic for bivalve species, including oysters. Here we present a study of mortality outbreaks of C. gigas, as initiated in 1995. In a first step, bacterial strains were isolated during mass mortality outbreak and were biochemically characterised. Among the isolated strains, some strains of Vibrio splendidus biovar II were found to be pathogenic by means of experimental challenge of oyster juveniles. In the second step, a genotypical identification of the pathogenic strain was undertaken, based on 16S RNA sequences and phylogenetic analysis. It confirmed that the pathogenetic strain belonged to Vibrio splendidus biovar II.

Screening of Vibrio isolates to develop an experimental infection model in the Pacific oyster Crassostrea gigas.

In an attempt to develop a reproducible experimental model of bacterial infection in Crassostrea gigas, oysters taken from very localised sub-populations suffering natural mortality outbreaks were used in cohabitation trials under laboratory conditions. From these trials, a collection of Vibrio strains was isolated from moribund and healthy oysters. In a second step, strains were experimentally tested for virulence by means of injection into healthy oysters. This screening revealed a span of virulence among isolated strains from none to medium. When pooling injected strains, results suggest increased virulence. Vibrio strains may have additive/synergistic action leading to higher C. gigas mortality rates in experimental challenges. Although the study initially aimed to develop a simple experimental model, a complex of interactions emerged between several bacterial strains during the pathogenic process in their molluscan host. Selected strains provide a suitable model of experimental disease for further studies and better understanding of bacterial interaction and pathogenesis in C. gigas.

Prevalence and infection intensity of the ovarian parasite Marteilioides chungmuensis during an annual reproductive cycle of the oyster Crassostrea gigas.

The occurrence of Marteilioides chungmuensis, a protozoan paramyxean parasite in the reproductive system of the Pacific oyster Crassostrea gigas, was observed at Gosung Bay, Korea. Seasonal variation in gonad development was investigated in a suspended cultured oyster population. Gametogenesis began in February and first-spawning was observed between mid and late June when surface water temperature reached 22 to 25 degrees C. Spawning activity extended from mid June to late September, with 2 marked spawning peaks in June and August. Histological examination indicated that gonad development paralleled seasonal fluctuations in water temperature. Spawning in late June was partly associated with a sudden drop in salinity due to large freshwater inputs to the Bay with the summer monsoon. M. chungmuensis occurred in developing and fully mature eggs of spawning oysters in late June to January, but were not observed from February to May. Monthly mean infection intensity was high in late June when most oysters had their first spawning period. The infection level was also relatively high in late August and November, when oysters were spawning or had completed spawning. Several oysters collected in November (11.4%) and December (16.3%) carried a large quantity of ripe but M. chungmuensis-infected eggs, suggesting that infection also causes spawning failure by delaying spawning and destroying ripe oocytes.