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.

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.

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.

Molecular detection of the oyster parasite Mikrocytos mackini, and a preliminary phylogenetic analysis.

The protistan parasite Mikrocytos mackini, the causative agent of Denman Island disease in the oyster Crassostrea gigas in British Columbia, Canada, is of wide concern because it can infect other oyster species and because its life cycle, mode of transmission, and origins are unknown. PCR and fluorescent in situ hybridization (FISH) assays were developed for M. mackini, the PCR assay was validated against standard histopathological diagnosis, and a preliminary phylogenetic analysis of the M. mackini small-subunit ribosomal RNA gene (SSU rDNA) was undertaken. A PCR designed specifically not to amplify host DNA generated a 544 bp SSU rDNA fragment from M. mackini-infected oysters and enriched M. mackini cell isolates, but not from uninfected control oysters. This fragment was confirmed by FISH to be M. mackini SSU rDNA. A M. mackini-specific PCR was then designed which detected 3 to 4x more M. mackini infections in 1056 wild oysters from Denman Island, British Columbia, than standard histopathology. Mikrocytos mackini prevalence estimates based on both PCR and histopathology increased (PCR from 4.4 to 7.4%, histopathology from 1.2 to 2.1%) when gross lesions were processed in addition to standard samples (i.e. transverse sections for histopathology, left outer palp DNA for PCR). The use of histopathology and tissue imprints plus PCR, and standard samples plus observed gross lesions, represented a 'total evidence' approach that provided the most realistic estimates of the true prevalence of M. mackini. Maximum parsimony and evolutionary distance phylogenetic analyses suggested that M. mackini may be a basal eukaryote, although it is not closely related to other known protistan taxa.

Transcriptome analysis of neoplastic hemocytes in soft-shell clams Mya arenaria: Focus on cell cycle molecular mechanism.

In North America, a high mortality of soft-shell clams Mya arenaria was found to be related to the disease known as disseminated neoplasia (DN). Disseminated neoplasia is commonly recognized as a tetraploid disorder related to a disruption of the cell cycle. However, the molecular mechanisms by which hemocytes of clams are transformed in the course of DN remain by far unknown. This study aims at identifying the transcripts related to DN in soft shell clams' hemocytes using next generation of sequencing (Illumina HiSeq2000). This study mainly focuses on transcripts and molecular mechanisms involved in cell cycle. Using Illumina next generation of sequencing, more than 95,399,159 reads count with an average length of 45 bp was generated from three groups of hemocytes: (1) a healthy group with less than 10% of tetraploid cells; (2) an intermediate group with tetraploid hemocytes ranging between 10% and 50% and (3) a diseased group with more than 50% of tetraploid cells. After the reads were cleaned by removing the adapters, de novo assembly was performed on the sequences and more than 73,696 contigs were generated with a mean contig length estimated at 585 bp ranging from 189 bp to 14,773 bp. Once a Blastx search against NCBI Non Redundant database was performed and the duplicates removed, 18,378 annotated sequences matched known sequences, 3078 were hypothetical and 9002 were uncharacterized sequences. Fifty percent and 41% of known sequences match sequences from Mollusca and Gastropoda respectively. Among the bivalvia, 33%, 17%, 17% and 15% of the contigs match sequences from Ostreoida, Veneroida, Pectinoida and Mytiloida respectively. Gene ontology analysis showed that metabolic, cellular, transport, cell communication and cell cycle represent 33%, 15%, 9%, 8.5% and 7% respectively of the total biological process. Approximately 70% of the component process is related to intracellular process and 15% is linked to protein and ribonucleoprotein complex. Catalytic activities and binding molecular processes represent 39% and 33% of the total molecular functions. Interestingly, nucleic acid binding represents more than 18% of the total protein class. Transcripts involved in the molecular mechanisms of cell cycle are discussed providing new avenues for future investigations.

Induction of transposase and polyprotein RNA levels in disseminated neoplastic hemocytes of soft-shell clams: Mya arenaria.

In Prince Edward Island, a high mortality of soft-shell clams Mya arenaria was found to be related to the disease known as disseminated neoplasia (DN). However, the molecular mechanisms by which hemocytes of clams are transformed in the course of DN remain by far unknown. This study aims at identifying the transcripts involved in the development of the disease. Four subtractive cDNA sequence libraries were generated and more than 200,000 reads were obtained. Following similarity searches in genome databases, the transcripts were assigned to cellular functions including mitochondrial respiration, structural proteins, cytoskeleton, nucleic acid regulation, general metabolism, signal transduction, apoptosis, cell cycle regulation, as well as virus transcripts. The expression levels of transposase and polyprotein genes were evaluated in clams with various percentages of tetraploid hemocytes. Data have shown that expression levels were significantly higher in clams with a high percentage of tetraploid hemocytes. These results reinforce the hypothesis of endogenous retrotransposon involvement in the etiology of the disease. Further investigations are needed, however, to elucidate the role of transposase and polyprotein in the disease development.

Differential gene expression of gamma-actin, Toll-like receptor 2 (TLR-2) and interleukin-1 receptor-associated kinase 4 (IRAK-4) in Mya arenaria haemocytes induced by in vivo infections with two Vibrio splendidus strains.

Immune function gene expression in Mya arenaria haemocytes was evaluated following in vivo infection with Vibrio splendidus LGP32-GFP and 7SHRW. Elongation factor 1alpha (EF-1alpha) with 2 (EF-2), after challenge with LGP32-GFP, and EF-1alpha with the ribosomal protein S-18, after challenge with 7SHRW, were found to be the most stable housekeeping genes. Using these internal controls and comparing the regulation induced by both strains, up-regulation of gamma-actin, down-regulation of TLR-2 and up-regulation of IRAK-4 was significantly higher after challenge with LGP32-GFP (p<0.001, p=0.001 and p<0.05, respectively). These results suggest specific responses at a molecular level modulated by the bacterial strains. LGP32-GFP induced marked responses which coincide with a similar trend previously found on phenotypic responses under our experimental model.

Potential link between exposure to fungicides chlorothalonil and mancozeb and haemic neoplasia development in the soft-shell clam Mya arenaria: a laboratory experiment.

The aetiology of haemic neoplasia (HN) is unknown, so far but many causative factors are suggested such as viral, pollution and genetics. The aim of this study was to determine if, under chronic exposure, two major pesticides (chlorothalonil and mancozeb) which are used in potato production could induce HN in soft-shell clams (Mya arenaria). Short-term experiments with acute exposure were also performed. Clams were collected from an epizootic site (North River, PEI) and from a site free of the disease (Magdalen Islands, Quebec). The tetraploid level of haemocytes was assessed by flow cytometry for each clam to determine the HN status. The bioaccumulation of pesticides in tissues was quantified by gas chromatography/mass spectrometry (GC/MS) for chlorothalonil while mancozeb and manganese were quantified by inductively coupled plasma-mass spectrometer (ICP/MS). Long term exposure to fungicide Bravo 500((R)) did not induce high tetraploid levels on negative calm from North River and the analysis of the digestive gland and the mantle did not reveal any detectable level of chlorothalonil. In the Manzate 200 DF((R)), some clams revealed high level of tetraploid cells but no difference were observed between the treatments and the control. The analysis of the digestive gland and the mantle for manganese did not highlight any significant difference in tissue concentration (p=0.05). For the acute exposure, chlorothalonil analysis showed that the active ingredient is distributed between four chlorinated compounds: 99.5% for chlorothalonil isomers, 0.4% for pentachlorothalonil and 0.1% for trichlorothalonil isomers. For a 72 h experiment, the accumulation was within 4h; the higher tissue concentration of chlorothalonil was 59.2 microg g(-1) in the mantle after 48 h, following by a decrease to an undetectable level at the end. For the manganese, the accumulation was detected after 4h; the higher tissue concentration was 48.8 microg g(-1) in the mantle after 24h and, over the following 48 h, the accumulation decreased until the end of the trial. Based on the data, the accumulation of these fungicides seems to be transitory. Chlorothalonil and mancozeb are both oxidative-stress promoters and could have induced cell dysfunction while in the tissue. Study on the effect of these fungicides on the p53 protein system is an example of strategy that would provide information on cellular events promoting neoplasia.