A rapid phenotype change in the pathogen Perkinsus marinus was associated with a historically significant marine disease emergence in the eastern oyster.

The protozoan parasite Perkinsus marinus, which causes dermo disease in Crassostrea virginica, is one of the most ecologically important and economically destructive marine pathogens. The rapid and persistent intensification of dermo in the USA in the 1980s has long been enigmatic. Attributed originally to the effects of multi-year drought, climatic factors fail to fully explain the geographic extent of dermo's intensification or the persistence of its intensified activity. Here we show that emergence of a unique, hypervirulent P. marinus phenotype was associated with the increase in prevalence and intensity of this disease and associated mortality. Retrospective histopathology of 8355 archival oysters from 1960 to 2018 spanning Chesapeake Bay, South Carolina, and New Jersey revealed that a new parasite phenotype emerged between 1983 and 1990, concurrent with major historical dermo disease outbreaks. Phenotypic changes included a shortening of the parasite's life cycle and a tropism shift from deeper connective tissues to digestive epithelia. The changes are likely adaptive with regard to the reduced oyster abundance and longevity faced by P. marinus after rapid establishment of exotic pathogen Haplosporidium nelsoni in 1959. Our findings, we hypothesize, illustrate a novel ecosystem response to a marine parasite invasion: an increase in virulence in a native parasite.

Statewide Quality Improvement Initiative to Reduce Early Elective Deliveries and Improve Birth Registry Accuracy.

OBJECTIVE: To evaluate the success of a quality improvement initiative to reduce early elective deliveries at less than 39 weeks of gestation and improve birth registry data accuracy rapidly and at scale in Ohio. METHODS: Between February 2013 and March 2014, participating hospitals were involved in a quality improvement initiative to reduce early elective deliveries at less than 39 weeks of gestation and improve birth registry data. This initiative was designed as a learning collaborative model (group webinars and a single face-to-face meeting) and included individual quality improvement coaching. It was implemented using a stepped wedge design with hospitals divided into three balanced groups (waves) participating in the initiative sequentially. Birth registry data were used to assess hospital rates of nonmedically indicated inductions at less than 39 weeks of gestation. Comparisons were made between groups participating and those not participating in the initiative at two time points. To measure birth registry accuracy, hospitals conducted monthly audits comparing birth registry data with the medical record. Associations were assessed using generalized linear repeated measures models accounting for time effects. RESULTS: Seventy of 72 (97%) eligible hospitals participated. Based on birth registry data, nonmedically indicated inductions at less than 39 weeks of gestation declined in all groups with implementation (wave 1: 6.2-3.2%, P<.001; wave 2: 4.2-2.5%, P=.04; wave 3: 6.8-3.7%, P=.002). When waves 1 and 2 were participating in the initiative, they saw significant decreases in rates of early elective deliveries as compared with wave 3 (control; P=.018). All waves had significant improvement in birth registry accuracy (wave 1: 80-90%, P=.017; wave 2: 80-100%, P=.002; wave 3: 75-100%, P<.001). CONCLUSIONS: A quality improvement initiative enabled statewide spread of change strategies to decrease early elective deliveries and improve birth registry accuracy over 14 months and could be used for rapid dissemination of other evidence-based obstetric care practices across states or hospital systems.

Infectious diseases of marine molluscs and host responses as revealed by genomic tools.

More and more infectious diseases affect marine molluscs. Some diseases have impacted commercial species including MSX and Dermo of the eastern oyster, QPX of hard clams, withering syndrome of abalone and ostreid herpesvirus 1 (OsHV-1) infections of many molluscs. Although the exact transmission mechanisms are not well understood, human activities and associated environmental changes often correlate with increased disease prevalence. For instance, hatcheries and large-scale aquaculture create high host densities, which, along with increasing ocean temperature, might have contributed to OsHV-1 epizootics in scallops and oysters. A key to understanding linkages between the environment and disease is to understand how the environment affects the host immune system. Although we might be tempted to downplay the role of immunity in invertebrates, recent advances in genomics have provided insights into host and parasite genomes and revealed surprisingly sophisticated innate immune systems in molluscs. All major innate immune pathways are found in molluscs with many immune receptors, regulators and effectors expanded. The expanded gene families provide great diversity and complexity in innate immune response, which may be key to mollusc's defence against diverse pathogens in the absence of adaptive immunity. Further advances in host and parasite genomics should improve our understanding of genetic variation in parasite virulence and host disease resistance.

Transcriptome analysis reveals strong and complex antiviral response in a mollusc.

Viruses are highly abundant in the oceans, and how filter-feeding molluscs without adaptive immunity defend themselves against viruses is not well understood. We studied the response of a mollusc Crassostrea gigas to Ostreid herpesvirus 1 µVar (OsHV-1µVar) infections using transcriptome sequencing. OsHV-1µVar can replicate extremely rapidly after challenge of C. gigas as evidenced by explosive viral transcription and DNA synthesis, which peaked at 24 and 48 h post-inoculation, respectively, accompanied by heavy oyster mortalities. At 120 h post-injection, however, viral gene transcription and DNA load, and oyster mortality, were greatly reduced indicating an end of active infections and effective control of viral replication in surviving oysters. Transcriptome analysis of the host revealed strong and complex responses involving the activation of all major innate immune pathways that are equipped with expanded and often novel receptors and adaptors. Novel Toll-like receptor (TLR) and MyD88-like genes lacking essential domains were highly up-regulated in the oyster, possibly interfering with TLR signal transduction. RIG-1/MDA5 receptors for viral RNA, interferon-regulatory factors, tissue necrosis factors and interleukin-17 were highly activated and likely central to the oyster's antiviral response. Genes related to anti-apoptosis, oxidation, RNA and protein destruction were also highly up-regulated, while genes related to anti-oxidation were down-regulated. The oxidative burst induced by the up-regulation of oxidases and severe down-regulation of anti-oxidant genes may be important for the destruction of viral components, but may also exacerbate oyster mortality. This study provides unprecedented insights into antiviral response in a mollusc. The mobilization and complex regulation of expanded innate immune-gene families highlights the oyster genome's adaptation to a virus-rich marine environment.

Climate change influences on marine infectious diseases: implications for management and society.

Infectious diseases are common in marine environments, but the effects of a changing climate on marine pathogens are not well understood. Here we review current knowledge about how the climate drives host-pathogen interactions and infectious disease outbreaks. Climate-related impacts on marine diseases are being documented in corals, shellfish, finfish, and humans; these impacts are less clearly linked for other organisms. Oceans and people are inextricably linked, and marine diseases can both directly and indirectly affect human health, livelihoods, and well-being. We recommend an adaptive management approach to better increase the resilience of ocean systems vulnerable to marine diseases in a changing climate. Land-based management methods of quarantining, culling, and vaccinating are not successful in the ocean; therefore, forecasting conditions that lead to outbreaks and designing tools/approaches to influence these conditions may be the best way to manage marine disease.

Quantitative PCR assay to determine prevalence and intensity of MSX (Haplosporidium nelsoni) in North Carolina and Rhode Island oysters Crassostrea virginica.

The continuing challenges to the management of both wild and cultured eastern oyster Crassostrea virginica populations resulting from protozoan parasites has stimulated interest in the development of molecular assays for their detection and quantification. For Haplosporidium nelsoni, the causative agent of multinucleated sphere unknown (MSX) disease, diagnostic evaluations depend extensively on traditional but laborious histological approaches and more recently on rapid and sensitive (but not quantitative) end-point polymerase chain reaction (PCR) assays. Here, we describe the development and application of a quantitative PCR (qPCR) assay for H. nelsoni using an Applied Biosystems TaqMan® assay designed with minor groove binder (MGB) probes. The assay was highly sensitive, detecting as few as 20 copies of cloned target DNA. Histologically evaluated parasite density was significantly correlated with the quantification cycle (Cq), regardless of whether quantification was categorical (r2 = 0.696, p < 0.0001) or quantitative (r2 = 0.797, p < 0.0001). Application in field studies conducted in North Carolina, USA (7 locations), revealed widespread occurrence of the parasite with moderate to high intensities noted in some locations. In Rhode Island, USA, application of the assay on oysters from 2 locations resulted in no positives.

Widespread survey finds no evidence of Haplosporidium nelsoni (MSX) in Gulf of Mexico oysters.

The advent of molecular detection assays has provided a set of very sensitive tools for the detection of pathogens in marine organisms, but it has also raised problems of how to interpret positive signals that are not accompanied by visual confirmation. PCR-positive results have recently been reported for Haplosporidium nelsoni (MSX), a pathogen of the oyster Crassostrea virginica in 31 of 40 oysters from 6 sites in the Gulf of Mexico and the Caribbean Sea. Histological confirmation of the PCR results was not undertaken, and no haplosporidian has been reported from the numerous histological studies and surveys of oysters in the region. To further investigate the possibility that H. nelsoni is present in this region, we sampled 210 oysters from 40 sites around the Gulf of Mexico and Puerto Rico using PCR and 180 of these using tissue-section histology also. None of the oysters showed evidence of H. nelsoni by PCR or of any haplosporidian by histology. We cannot, therefore, confirm that H. nelsoni is present and widespread in the Gulf of Mexico and the Caribbean Sea. Our results do not prove that H. nelsoni is absent from the region, but taken together with results from previous histological surveys, they suggest that for the purposes of controlling oyster importation, the region should continue to be considered free of the parasite.

Microarray analysis of gene expression in eastern oyster (Crassostrea virginica) reveals a novel combination of antimicrobial and oxidative stress host responses after dermo (Perkinsus marinus) challenge.

Dermo disease, caused by Perkinsus marinus, is one of the most severe diseases of eastern oysters, Crassostrea virginica. It causes serious mortalities in both wild and aquacultured oysters. Using existing expressed sequence tag (EST) resources, we developed a 12K in situ oligonucleotide microarray and used it for the analysis of gene expression profiles of oysters during the interactions between P. marinus and its oyster host. Significant gene expression regulation was found at day 30 post-challenge in the eastern oyster. Putative identities of the differentially expressed genes revealed a set of genes involved in several processes including putative antimicrobial defenses, pathogen recognition and uptake, anti-oxidation and apoptosis. Consistent with results obtained from previous, smaller-scale experiments, expression profiles revealed a large set of genes likely involved in an active mitigating response to oxidative stress and apoptosis induced by P. marinus. Additionally, a unique galectin from C. virginica, CvGal, which serves as a preferential receptor for P. marinus trophozoites, was found to be significantly down-regulated in gill tissue of oysters with both light and heavy infection, suggesting an attempt to control parasite uptake and proliferation in the later stages of infection. Potential histone-derived antimicrobial responses to P. marinus were also revealed in the gene expression profiles.

Minchinia mercenariae n. sp. (Haplosporidia) in the hard clam Mercenaria mercenaria: implications of a rare parasite in a commercially important host.

During routine histopathology of 180 juvenile hard clams, Mercenaria mercenaria, from a site in Virginia, USA, in 2007, we discovered a single individual heavily infected with a parasite resembling a haplosporidian, some members of which cause lethal bivalve diseases. Scanning electron microscopy of spores and sequencing of small subunit ribosomal DNA confirmed a new species: Minchinia mercenariae n. sp. Further sampling of clams at the site found prevalences up to 38% using polymerase chain reaction (PCR). No parasites were found in routine histological screening of the same individuals, but re-examination of clams judged positive by in situ hybridization (ISH) revealed very faintly staining plasmodia. No unusual mortalities have occurred among the sampled groups. Analysis of clams from Massachusetts to Florida by PCR failed to detect the parasite, but a haplosporidian found in a clam from New Jersey in 2001 was subsequently identified by ISH as M. mercenariae. No other haplosporidians have been reported in thousands of hard clams from the US east coast examined histologically since the mid-1980s. The discovery underscores critical questions about how to assess the risks associated with parasites in groups known to be lethal, but that themselves are not considered a problem.

Using bivalves as particle collectors with PCR detection to investigate the environmental distribution of Haplosporidium nelsoni.

Recently, PCR technology has been applied to search for marine microorganisms in environmental samples. Such sampling, however, has drawbacks, including the need to collect and filter large volumes of water, and the presence of substances in environmental samples that may destroy DNA or interfere with DNA isolation and amplification. We explored the possibility of using suspension-feeding bivalves in conjunction with PCR to investigate the environmental distribution of microparasites using the oyster pathogen Haplosporidium nelsoni (the MSX disease agent) with oysters and mussels in Delaware Bay, USA, as a model system. Delaware Bay oysters Crassostrea virginica have become very resistant to H. nelsoni infection development and rarely exhibit histologically detectable lesions. The ribbed mussel Geukensia demissa is also resistant. Infections were detected in only 6% of the histologically examined oysters in the present study, although PCR-positive signals for H. nelsoni were found in feces, gill or heart samples of up to 100% of oysters. Positive signals were found in the feces and gill (but not heart) samples of up to 50% of mussels. The temporal evolution of PCR signals suggested a wave-like pattern of putative infective particles, which mimicked a pattern documented in early mortality studies. The present study demonstrated that H. nelsoni persists throughout Delaware Bay, although it is rarely detected using standard histology. We propose that the use of suspension-feeding bivalves as particle collectors in combination with PCR could be a method for detecting the presence of marine microparasites and might help answer questions about factors that prevent outbreaks of epizootics in certain regions.

Repeated sampling of individual bivalve mollusks I: intraindividual variability and consequences for haemolymph constituents of the Manila clam, Ruditapes philippinarum.

Components of the haemolymph are understood to constitute the internal defense system of bivalve mollusks and their levels are often considered to be indicators of "health"; however, relatively little proof exists of the role that these elements play in the success or failure of defense against a pathogen. A change associated with infection may be the consequence of disease rather than a measure of the capacity to respond effectively to a pathogen. One way to assess whether haemocyte or serum-component concentrations are related to resistance to microbial infection is to sample individuals over time, both before and after they are experimentally or naturally infected. But sampling itself may alter the parameter being assessed. In addition, interindividual variation is large and the degree of intraindividual variation over time is largely unknown. To evaluate intra- vs interindividual variability measured over time and to assess the effects of repeated sampling, we subjected Manila clams, Ruditapes philippinarum, to multiple haemolymph samplings during both field and laboratory experiments, and measured four parameters: haemocyte density, protein concentration, and the activities of leucine amino peptidase and DOPA-oxidase. A repeated-measures ANOVA indicated that individuals with high or low levels at one sampling, tended to have high or low levels, respectively, at the other sampling times. Furthermore, the index of individuality, which is the ratio of intra- to interindividual variability, for these four parameters was comparable to that for human serum components. Repeated sampling had no measured effect on field-deployed clams, which were sampled at intervals of 1-3 months, but significantly depressed values in laboratory-held clams sampled at 1-month intervals. Results demonstrated relative intraindividual constancy in the measured variables and suggested that minimizing sample frequency and volume, and maintaining animals in a comparatively natural environment should all facilitate repeated sampling with minimum injury to experimental mollusks.

Effects of the pathogenic Vibrio tapetis on defence factors of susceptible and non-susceptible bivalve species: II. Cellular and biochemical changes following in vivo challenge.

This work compared the effect of challenge with Vibrio tapetis, the etiologic agent of brown ring disease (BRD) in clams, and other bacterial strains on defence-related factors in four bivalve species: Ruditapes philippinarum (highly susceptible to BRD), R. decussatus (slightly susceptible to BRD), Mercenaria mercenaria and Crassostrea virginica (both non-susceptible to BRD). Results show that bacterial challenge modulated defence-related factors, namely total and differential haemocyte counts, percentage of viable haemocytes, and lysozyme activity, both in haemolymph and extrapallial fluid. Injection with bacteria induced a response that was dependent upon the bacterial and bivalve species investigated, and upon the site of inoculation: external (pallial cavity), pseudo-internal (extrapallial space), or internal compartment (adductor muscle). The most conspicuous changes were systematically measured in R. philippinarum injected with V. tapetis, indicating a bacterial pathogenicity particular to the host in which it causes a specific disease syndrome. Alterations of defence-related factors were maximal in haemolymph of clams injected with V. tapetis in the muscle, and in the extrapallial fluid when the bacteria were injected into the pallial or the extrapallial cavity. Resistance to the development of the BRD symptom was not related to the extent of the haemocyte reaction measured following in vivo challenge.

Effects of the pathogenic Vibrio tapetis on defence factors of susceptible and non-susceptible bivalve species: I. Haemocyte changes following in vitro challenge.

In microbial infections, the interaction between microorganisms and phagocytic cells is a crucial determinant in the outcome of the disease process. We used flow cytometry to study the in vitro interactions between Vibrio tapetis, the bacterium responsible for Brown Ring Disease (BRD) in the Manila clam Ruditapes philippinarum, and haemocytes from three bivalve species: the Manila clam (susceptible to BRD), the hard clam Mercenaria mercenaria and the eastern oyster Crassostrea virginica (both non-susceptible to BRD). Results demonstrated that V. tapetis cells and extracellular products elicit major changes in the haemocytes of R. philippinarum, including decreased viability and phagocytic activity, and altered size and internal structure. V. tapetis was able to kill haemocytes from M. mercenaria and C. virginica but to a far lesser extent than those of R. philippinarum. These results suggest that disease resistance is not solely dependent on a host activity against the pathogen, but is also a function and magnitude of the injury to the host cell by a given pathogen.

Discovery of genes expressed in response to Perkinsus marinus challenge in Eastern (Crassostrea virginica) and Pacific (C. gigas) oysters.

The protozoan pathogen Perkinsus marinus is the causative agent of Dermo, a lethal disease of the eastern oyster Crassostrea virginica, but not the Pacific oyster Crassostrea gigas. To understand the response of these two oysters to parasite exposure, a suppression subtractive hybridization (SSH) method was employed to characterize genes up-regulated during parasite challenge in both hemocytes and gills. The number of differentially expressed gene sequences obtained was 107 for C. virginica and 69 for C. gigas, including 46 and 37 sequences, respectively, that matched known genes in GenBank. Most of the sequences have not been characterized in other molluscs. Nineteen genes involved in immune system and cell communication, protein regulation and transcription, cell cycle, respiratory chain and cytoskeleton were selected for expression analysis by semi-quantitative PCR. Although varying in magnitude and timing post exposure, all genes screened showed over-expression in challenged oysters in both species, validating the SSH method. Results of this study highlighted some differences in gene expression between the two oysters in response to P. marinus infection, providing candidate genes and pathways for further analysis.

Comparison of in vitro-cultured and wild-type Perkinsus marinus. I. Pathogen virulence.

Perkinsus marinus is a highly contagious pathogen of the eastern oyster Crassostrea virginica. Until recently, transmission studies have employed wild-type parasites isolated directly from infected oysters. Newly developed methods to propagate P. marinus in vitro have led to using cultured parasites for infection studies, but results suggest that cultured parasites are less virulent than wild-type parasites In this paper, we report results of experiments designed to quantify differences between wild-type and cultured P. marinus virulence and to test the following hypotheses: (1) in vitro-cultured parasites are less virulent than wild-type parasites; (2) virulence decreases gradually during in vitro culture; (3) virulence of in vitro cultures can be restored by in vivo passage; (4) virulence changes with culture phase. Our results demonstrate that parasites freshly isolated from infected hosts are much more virulent than those propagated in culture, indicating a potential deficiency in the culture medium used. Virulence was lost immediately in culture and, for that reason, the practice of repassing cultured cells through the host to restore virulence does not work for P. marinus. Virulence was also associated with culture phase: log-phase parasites were significantly more virulent than those obtained from lag- or stationary-phase cultures.

Comparison of in vitro-cultured and wild-type Perkinsus marinus. II. Dosing methods and host response.

Endoparasites must breach host barriers to establish infection and then must survive host internal defenses to cause disease. Such barriers may frustrate attempts to experimentally transmit parasites by 'natural' methods. In addition, the host's condition may affect a study's outcome. The experiments reported here examined the effect of dosing method and host metabolic condition on measures of virulence for the oyster parasite Perkinsus marinus. Oysters, Crassostrea virginica, were challenged with wild-type and cultured forms of P. marinus via feeding, shell-cavity injection, gut intubation and adductor-muscle injection. For both parasite types, adductor-muscle injections produced the heaviest infections followed by shell-cavity injection, gut intubation, and feeding. There was no difference in parasite burdens between oysters fed cultured cells by acute vs chronic dosing, and parasite loads stabilized over time, suggesting a dynamic equilibrium between invasion and elimination. P. marinus distribution among tissues of challenged oysters indicated that parasites invaded the mantle and gill, as well as the gut, which has been considered the primary portal of entry. Frequency distributions of P. marinus in oysters challenged with 3 different culture phases indicated an aggregated distribution among hosts and suggested that stationary-phase parasites were easiest for the oyster to control or eliminate and log-phase parasites were the most difficult. Host metabolic condition also affected experimental outcomes, as indicated by increased infection levels in oysters undergoing spawning and/or exposed to low oxygen stress.

Comparison of in vitro-cultured and wild-type Perkinsus marinus. III. Fecal elimination and its role in transmission.

Perkinsus marinus, a pathogen of the eastern oyster Crassostrea virginica, is transmitted directly among oysters. Previous studies found viable P. marinus parasites in the feces and pseudofeces of oysters within hours of injection with parasites, suggesting that the parasite may be voided from live oysters and subsequently dispersed in the water column. The experiments described here were designed to quantify P. marinus shed in the feces and pseudofeces of experimentally infected oysters. The results indicated that parasites were shed in 2 phases. A 'decreasing' phase occurred within 2 wk of challenge and before net parasite proliferation began in the host. An 'increasing' phase occurred after P. marinus had begun replicating. The quantity of P. marinus recovered in the feces and pseudofeces of exposed oysters was only about 5 % of the dose administered. In vitro-cultured P. marinus were eliminated at a greater rate than wild-type P. marinus and the fraction discharged was not associated with culture phase. Oysters that were continuously dosed with P. marinus in their food gradually lost the ability to discard the parasite in pseudofeces. The quantity of P. marinus shed in feces of infected oysters was correlated with both the P. marinus body burden and subsequent survival time, suggesting that noninvasive fecal counts could predict infection intensity and survival. The results indicate that in an epizootic, shedding of P. marinus via feces is relatively small compared to the potential number released by cadavers of heavily infected oysters, but that fecal discharge may be important in transmission before infections become lethal.

Pathogenicity of Vibrio tapetis, the etiological agent of brown ring disease in clams.

Brown ring disease (BRD) causes high mortalities in the introduced Manila clam Ruditapes philippinarum cultured in western Europe. The etiological agent of BRD, Vibrio tapetis, adheres to and disrupts the production of the periostracal lamina, causing the anomalous deposition of periostracum around the inner shell. Because the primary sign of BRD is found outside the soft tissues, the processes leading to death are not as obvious as those for internal pathogens. This study was designed to evaluate the pathogenicity of V. tapetis, in an attempt to help explain the mechanisms of mortality. We found high mortalities (up to 100%) for clams following the inoculation of V. tapetis into the extrapallial space (between mantle and inner shell) or the posterior adductor muscle of healthy R. philippinarum. Microscopy and immunological detection methods showed that the pathogen was rapidly eliminated from tissues and hemolymph of animals that survived the inoculation. In clams that died, the bacteria were found to have proliferated, resulting in severe tissue disruption. Bacteria were able to penetrate into tissues from the extrapallial space through the external epithelium of the mantle. In contrast, no mortalities were observed following injection of V. tapetis in the native European clam Ruditapes decussatus, which is resistant to BRD. This clam rapidly eliminated the bacterium from hemolymph and soft tissues. Clam mortality associated with BRD in the field is likely to result from the penetration of V. tapetis into the clam's extrapallial space through the disrupted periostracal lamina and from there into the soft tissues through the irritated mantle epithelium. Some bacteria also penetrate through the digestive epithelia. In either case, bacteria proliferate rapidly in the soft tissues, causing severe damage and subsequent death.