Realising a global One Health disease surveillance approach: insights from wastewater and beyond.

One Health is a recognition of the shared environment inhabited by humans, animals and plants, and the impact of their interactions on the health of all organisms. The COVID-19 pandemic highlighted the need for a framework of pathogen surveillance in a tractable One Health paradigm to allow timely detection and response to threats to human and animal health. We present case studies centered around the recent global approach to tackle antimicrobial resistance and the current interest in wastewater testing, with the concept of "one sample many analyses" to be further explored as the most appropriate means of initiating this endeavor.

Infection of Norway lobster (Nephrops norvegicus) by the parasite Hematodinium sp.: insights from 30 years of field observations.

The Norway lobster, Nephrops norvegicus, is an important representative of the benthos and also supports valuable fisheries across Europe. Nephrops are susceptible to infection by Hematodinium sp., an endoparasitic dinoflagellate that causes morbidity and mortality. From an epizootiological perspective, the Clyde Sea Area (CSA; west of Scotland) is the best-studied Hematodinium-Nephrops pathosystem, with historical data available between 1988 and 2008. We have revisited this pathosystem by curating and updating prevalence values, differentiating host traits associated with disease exposure and progression, and comparing Hematodinium sp. disease dynamics in the CSA to other locations and to other decapod hosts (Cancer pagurus, Carcinus maenas). Prevalence from a 2018/2019 survey (involving 1739 lobsters) revealed Hematodinium sp. still mounts a synchronized patent infection in the CSA; hence this pathogen can be considered as enzootic in this location. We highlight for the first time that Nephrops size is associated with high severity infection, while females are more exposed to Hematodinium sp. More generally, regardless of the host (Norway lobster, brown and shore crabs) or the geographical area (Ireland, Wales, Scotland), Hematodinium sp. patent infections peak in spring/summer and reach their nadir during autumn. We contend that Hematodinium must be considered one of the most important pathogens of decapod crustaceans in temperate waters.

Salinity and temperature affect the symbiont profile and host condition of Florida USA blue crabs Callinectes sapidus.

Subtropical Florida blue crabs, Callinectes sapidus, exhibit differing life history traits compared to their temperate counterparts, likely influencing symbiont infection dynamics. Little information exists for Florida C. sapidus symbiont profiles, their distribution among various habitats, and influence on crab condition. Using histopathology, genomics, and transmission electron microscopy, we describe the first symbiont profiles for Florida C. sapidus occupying freshwater to marine habitats. Twelve symbiont groups were identified from 409 crabs including ciliophorans, digenean, microsporidian, Haplosporidia, Hematodinium sp., Nematoda, filamentous bacteria, gregarine, Callinectes sapidus nudivirus, Octolasmis sp., Cambarincola sp., and putative microcell. Overall, 78% of C. sapidus were documented with one or more symbiont groups demonstrating high infection rates in wild populations. Environmental variables water temperature and salinity explained 48% of the variation in symbiont groups among Florida habitats, and salinity was positively correlated with C. sapidus symbiont diversity. This suggests freshwater C. sapidus possess fewer symbionts and represent healthier individuals compared to saltwater populations. Crab condition was examined using the reflex action mortality predictor (RAMP) to determine if reflex impairment could be linked to symbiont prevalence. Symbionts were found positively correlated with crab condition, and impaired crabs were more likely to host symbionts, demonstrating symbiont inclusion may boost predictive ability of the RAMP application. The microsporidian symbiont group had a particularly strong effect on C. sapidus reflex response, and impairment was on average 1.57 times higher compared to all other symbiont groups. Our findings demonstrate the importance of considering full symbiont profiles and their associations with a spatially and temporally variable environment to fully assess C. sapidus population health.

Environmental DNA/RNA for pathogen and parasite detection, surveillance, and ecology.

Detection of pathogens, parasites, and other symbionts in environmental samples via eDNA/eRNA (collectively eNA) is an increasingly important source of information about their occurrence and activity. There is great potential for using such detections as a proxy for infection of host organisms in connected habitats, for pathogen monitoring and surveillance, and for early warning systems for disease. However, many factors require consideration, and appropriate methods developed and verified, in order that eNA detections can be reliably interpreted and adopted for surveillance and assessment of disease risk, and potentially inclusion in international standards, such as the World Organisation for Animal Health guidelines. Disease manifestation results from host-symbiont-environment interactions between hosts, demanding a multifactorial approach to interpretation of eNA signals.

Pseudohepatospora borealis n. gen. n. sp. (Microsporidia: Enterocytozoonida): A microsporidian pathogen of the Jonah crab (Cancer borealis).

The microsporidian diversity catalogued so far has resulted in the development of several taxonomic groups, one of which is the Enterocytozoonida - a group of generalist 'ultimate opportunists', which infect many fished and aquacultured animals, as well as a broad suite of host taxa, including humans. In this study, we provide phylogenetic, ultrastructural, developmental, and pathological evidence for the creation of a new genus and species to hold a microsporidian parasite of the Jonah crab, Cancer borealis. Cancer borealis represents a species of commercial interest and has become the target of a recently developed fishery on the USA and Canadian Atlantic coast. This species was found to harbour a microsporidian parasite that develops in the cytoplasm of alpha and beta cells of the hepatopancreas. We retrieved a 937 bp fragment of the parasite SSU region, alongside developmental and ultrastructural data that suggests this species is âˆ¼ 87 % similar to Parahepatospora carcini and develops in a similar manner in direct association with the host cell cytoplasm. The mature spores are ovoid in shape and measure 1.48 ± 0.15 µm (SD) in length and 1.00 ± 0.11 µm (SD) in width. Phylogenetically, the new parasite clades in the Enterocytozoonida on the same branch as P. carcini. We provide a new genus and species to hold the parasite: Pseudohepatospora borealis n. gen. n. sp. (Microsporidia: Enterocytozoonida) and explore the likelihood that this species may fit into the Hepatoporidae family.

Microsporidia: a new taxonomic, evolutionary, and ecological synthesis.

Microsporidian diversity is vast. There is a renewed drive to understand how microsporidian pathological, genomic, and ecological traits relate to their phylogeny. We comprehensively sample and phylogenetically analyse 125 microsporidian genera for which sequence data are available. Comparing these results with existing phylogenomic analyses, we suggest an updated taxonomic framework to replace the inconsistent clade numbering system, using informal taxonomic names: Glugeida (previously clades 5/3), Nosematida (4a), Enterocytozoonida (4b), Amblyosporida (3/5), Neopereziida (1), and Ovavesiculida (2). Cellular, parasitological, and ecological traits for 281 well-defined species are compared with identify clade-specific patterns across long-branch Microsporidia. We suggest that future taxonomic circumscriptions of Microsporidia should involve additional markers (SSU/ITS/LSU), and that a comprehensive suite of phenotypic and ecological traits help to predict broad microsporidian functional and lineage diversity.

Propionigenium and Vibrio species identified as possible component causes of shrimp white feces syndrome (WFS) associated with the microsporidian Enterocytozoon hepatopenaei.

White feces syndrome (WFS) in cultivated shrimp is characterized by white shrimp midguts (intestines) and white fecal strings that float as mats on pond surfaces. The etiology of WFS is complex, but one type called EHP-WFS is associated with the microsporidian Enterocytozoon hepatopenaei (EHP). The hepatopancreas (HP), midgut and fecal strings of EHP-WFS shrimp exhibit massive quantities of EHP spores together with mixed, unidentified bacteria. In EHP-WFS ponds, some EHP-infected shrimp show white midguts (WG) and produce white feces while other EHP-infected shrimp in the same pond show grossly normal midguts (NG) and produce no white feces. We hypothesized that comparison of the microbial flora between WG and NG shrimp would reveal probable combinations of microbes significantly associated with EHP-WFS. To test this, we selected a Penaeus vannamei cultivation pond exhibiting severe WFS and used microscopic and microbial profiling analyses to compare WG and NG samples. Histologically, EHP was confirmed in the HP and midgut of both WG and NG shrimp, but EHP burdens were higher and EHP tissue damage was more severe in WG shrimp. Further, intestinal microbiomes in WG shrimp were less diverse and had higher abundance of bacteria from the genera Vibrio and Propionigenium. Propionigenium burden in the HP of WG shrimp (9364 copies/100 ng DNA) was significantly higher (P = 1.1 × 10-5) than in NG shrimp (12 copies/100 ng DNA). These findings supported our hypothesis by revealing two candidate bacterial genera that should be tested in combination with EHP as potential component causes of EHP-WFS in P. vannamei.

Microsporidian Pathogens of Aquatic Animals.

Around 57.1% of microsporidia occupy aquatic environments, excluding a further 25.7% that utilise both terrestrial and aquatic systems. The aquatic microsporidia therefore compose the most diverse elements of the Microsporidia phylum, boasting unique structural features, variable transmission pathways, and significant ecological influence. From deep oceans to tropical rivers, these parasites are present in most aquatic environments and have been shown to infect hosts from across the Protozoa and Animalia. The consequences of infection range from mortality to intricate behavioural change, and their presence in aquatic communities often alters the overall functioning of the ecosystem.In this chapter, we explore aquatic microsporidian diversity from the perspective of aquatic animal health. Examples of microsporidian parasitism of importance to an aquacultural ('One Health') context and ecosystem context are focussed upon. These include infection of commercially important penaeid shrimp by Enterocytozoon hepatopenaei and interesting hyperparasitic microsporidians of wild host groups.Out of ~1500 suggested microsporidian species, 202 have been adequately taxonomically described using a combination of ultrastructural and genetic techniques from aquatic and semi-aquatic hosts. These species are our primary focus, and we suggest that the remaining diversity have additional genetic or morphological data collected to formalise their underlying systematics.

Histopathological survey for parasite groups in Gammarus varsoviensis (Amphipoda).

Invasive non-native amphipods (Crustacea) are becoming a model system in which to explore the impact and diversity of invasive parasites-parasites that are carried along an invasion route with their hosts. Gammarus varsoviensis is a freshwater amphipod species that has a recently explored invasion history. We provide a histopathological survey for a putatively invasive non-native population of this amphipod, identifying 8 symbiotic groups: Acanthocephala, Rotifera, Digenea, ciliated protozoa, Haplosporidia, Microsporidia, 'Candidatus Aquirickettsiella', and a putative nudivirus, at various prevalence. Our survey indicates that the parasites have no sex bias and that each has the potential to be carried in either sex along an invasion route. We discuss the pathology and prevalence of the above symbiotic groups and whether those that are parasitic may pose a risk if G. varsoviensis were to carry them to novel locations.

Naidispora caidianensis n. gen. n. sp. infecting coelomocytes of oligochaete Branchiura sowerbyi (Oligochaeta: Naididae) in China.

A new microsporidian parasite, Naidispora caidianensis n. gen. n. sp. was found in coelomocytes of oligochaete Branchiura sowerbyi Beddard, 1892 from Wuhan city, Hubei Province, China. Opaque, hypertrophied coelomocytes (0.17-0.24 mm in diameter) depicted clinical signs of infection. Electron microscopy revealed a microsporidian with monokaryotic life stages. Rounded uninucleate meronts subsequently transformed into multinucleate merogonial plasmodia with masses of electron-dense projections on their plasma membrane. Electron-dense sporogonial plasmodia separated into uninucleate sporonts through rosette-like budding, and further developed into sporoblasts, enclosed by a sporophorous vesicle. Uninucleate mature spores were pyriform, measured 4.1 ± 0.1 (3.9-4.3) µm × 2.1 ± 0.07 (1.9-2.2) µm (average ± SE, range, n = 50), and contained a mushroom-like anchoring disk, bipartite polaroplast, electron-lucent posterior vacuole, trilaminar spore wall and 12-14 turns of an isofilar polar filament arranged in 2-3 ranks. The 1542 bp sequence from B. soweryi showed the highest similarity (below 80 %) with Nematocenator marisprofundi (accession number: JX463178). SSU rRNA gene-based phylogenetic analysis demonstrated that the novel taxon formed an independent clade from known microsporidian parasites. Based on the ultrastructural features and SSU rRNA gene sequence, we propose the establishment of a novel genus (Naidispora n. gen.) and species (Naidispora caidianensis n. sp.) to contain this parasite.

High prevalence of Paramarteilia canceri infecting velvet swimming crabs Necora puber in Ireland.

The velvet swimming crab Necora puber has been fished in Ireland since the early 1980s and contributes significant income to smaller fishing vessels. From 2016 onwards, reduced landings have been reported. We undertook a full pathological investigation of crabs from fishing grounds at 3 sites on the west (Galway), southwest (Castletownbere) and east (Howth) coasts of Ireland. Histopathology, transmission electron microscopy and molecular taxonomic and phylogenetic analyses showed high prevalence and infection level of Paramarteilia canceri, previously only reported from the edible crab Cancer pagurus. This study provides the first molecular data for P. canceri, and shows its phylogenetic position in the order Paramyxida (Rhizaria). Other parasites and symbionts detected in the crabs were also noted, including widespread but low co-infection with Hematodinium sp. and a microsporidian consistent with the Ameson and Nadelspora genera. This is the first histological record of Hematodinium sp. in velvet crabs from Ireland. Four N. puber individuals across 2 sites were co-infected by P. canceri and Hematodinium sp. At one site, 3 velvet crabs infected with P. canceri were co-infected with the first microsporidian recorded from this host; the microsporidian 18S sequence was almost identical to Ameson pulvis, known to infect European shore crabs Carcinus maenas. The study provides a comprehensive phylogenetic analysis of this and all other available Ameson and Nadelspora 18S sequences. Together, these findings provide a baseline for further investigations of N. puber populations along the coast of Ireland.

Resistance to white spot syndrome virus in the European shore crab is associated with suppressed virion trafficking and heightened immune responses.

Introduction: All decapod crustaceans are considered potentially susceptible to White Spot Syndrome Virus (WSSV) infection, but the degree of White Spot Disease (WSD) susceptibility varies widely between species. The European shore crab Carcinus maenas can be infected with the virus for long periods of time without signs of disease. Given the high mortality rate of susceptible species, the differential susceptibility of these resistant hosts offers an opportunity to investigate mechanisms of disease resistance. Methods: Here, the temporal transcriptional responses (mRNA and miRNA) of C. maenas following WSSV injection were analysed and compared to a previously published dataset for the highly WSSV susceptible Penaeus vannamei to identify key genes, processes and pathways contributing to increased WSD resistance. Results: We show that, in contrast to P. vannamei, the transcriptional response during the first 2 days following WSSV injection in C. maenas is limited. During the later time points (7 days onwards), two groups of crabs were identified, a recalcitrant group where no replication of the virus occurred, and a group where significant viral replication occurred, with the transcriptional profiles of the latter group resembling those of WSSV-susceptible species. We identify key differences in the molecular responses of these groups to WSSV injection. Discussion: We propose that increased WSD resistance in C. maenas may result from impaired WSSV endocytosis due to the inhibition of internal vesicle budding by dynamin-1, and a delay in movement to the nucleus caused by the downregulation of cytoskeletal transcripts required for WSSV cytoskeleton docking, during early stages of the infection. This response allows resistant hosts greater time to fine-tune immune responses associated with miRNA expression, apoptosis and the melanisation cascade to defend against, and clear, invading WSSV. These findings suggest that the initial stages of infection are key to resistance to WSSV in the crab and highlight possible pathways that could be targeted in farmed crustacean to enhance resistance to WSD.

Identification and Full Characterisation of Two Novel Crustacean Infecting Members of the Family Nudiviridae Provides Support for Two Subfamilies.

Multiple enveloped viruses with rod-shaped nucleocapsids have been described, infecting the epithelial cell nuclei within the hepatopancreas tubules of crustaceans. These bacilliform viruses share the ultrastructural characteristics of nudiviruses, a specific clade of viruses infecting arthropods. Using histology, electron microscopy and high throughput sequencing, we characterise two further bacilliform viruses from aquatic hosts, the brown shrimp (Crangon crangon) and the European shore crab (Carcinus maenas). We assembled the full double stranded, circular DNA genome sequences of these viruses (~113 and 132 kbp, respectively). Comparative genomics and phylogenetic analyses confirm that both belong within the family Nudiviridae but in separate clades representing nudiviruses found in freshwater and marine environments. We show that the three thymidine kinase (tk) genes present in all sequenced nudivirus genomes, thus far, were absent in the Crangon crangon nudivirus, suggesting there are twenty-eight core genes shared by all nudiviruses. Furthermore, the phylogenetic data no longer support the subdivision of the family Nudiviridae into four genera (Alphanudivirus to Deltanudivirus), as recently adopted by the International Committee on Taxonomy of Viruses (ICTV), but rather shows two main branches of the family that are further subdivided. Our data support a recent proposal to create two subfamilies within the family Nudiviridae, each subdivided into several genera.

Panopeispora mellora n. gen. n. sp. (microsporidia) infecting Say's crab (Dyspanopeus sayi) from the Atlantic shoreline of Canada.

Say's mud crab, Dyspanopeus sayi (Brachyura: Panopeidae) is a native shallow subtidal and inter-tidal inhabitant of the Atlantic coastline of North America and an invasive species in the Mediterranean and Black Seas. Little is known about the microparasites of this host and the broader Panopeidae. We describe a novel microsporidian parasite infecting the musculature of D. sayi from Malagash, Nova Scotia (Canada), at a prevalence of 7%. Histopathology and molecular diagnostics were used to describe pathology and parasite phylogenetics, respectively. Based on SSU rDNA gene sequencing we propose that the microsporidian requires establishment of a new genus (Panopeispora n. gen.) and species (Panopeispora mellora n. sp.), due to significant differences to closest known taxa (e.g. Facilispora margolisi [81% similarity] and Thelohania butleri [80% similarity]), residing in Clade V of the Microsporidia. Archived, wax-embedded histological material was re-processed for transmission electron microscopy to obtain preliminary details of its intracellular development cycle and ultrastructure within the host musculature. The discovery of this pathogen is discussed with relevance to microsporidian taxonomy and the potential for achieving ultrastructural data from archived material.

Global mRNA and miRNA Analysis Reveal Key Processes in the Initial Response to Infection with WSSV in the Pacific Whiteleg Shrimp.

White Spot Disease (WSD) presents a major barrier to penaeid shrimp production. Mechanisms underlying White Spot Syndrome Virus (WSSV) susceptibility in penaeids are poorly understood due to limited information related to early infection. We investigated mRNA and miRNA transcription in Penaeus vannamei over 36 h following infection. Over this time course, 6192 transcripts and 27 miRNAs were differentially expressed-with limited differential expression from 3-12 h post injection (hpi) and a more significant transcriptional response associated with the onset of disease symptoms (24 hpi). During early infection, regulated processes included cytoskeletal remodelling and alterations in phagocytic activity that may assist WSSV entry and translocation, novel miRNA-induced metabolic shifts, and the downregulation of ATP-dependent proton transporter subunits that may impair cellular recycling. During later infection, uncoupling of the electron transport chain may drive cellular dysfunction and lead to high mortalities in infected penaeids. We propose that post-transcriptional silencing of the immune priming gene Dscam (downregulated following infections) by a novel shrimp miRNA (Pva-pmiR-78; upregulated) as a potential mechanism preventing future recognition of WSSV that may be suppressed in surviving shrimp. Our findings improve our understanding of WSD pathogenesis in P. vannamei and provide potential avenues for future development of prophylactics and treatments.

Patterns of infection in a native and an invasive crayfish across the UK.

Invasive crayfish and the introduction of non-native diseases pose a significant risk for the conservation of endangered, white-clawed crayfish (Austropotamobius pallipes). Continued pollution of waterways is also of concern for native species and may be linked with crayfish disease dynamics. We explore whether crayfish species or environmental quality are predictors of infection presence and prevalence in native A. pallipes and invasive signal crayfish (Pacifastacus leniusculus). We use a seven-year dataset of histology records, and a field survey comparing the presence and prevalence of infectious agents in three isolated A. pallipes populations; three isolated P. leniusculus populations, and three populations where the two species had overlapped in the past. We note a lower diversity of parasites (Simpson's Index) in P. leniusculus ('Pacifastacus leniusculus Bacilliform Virus' - PlBV) (n = 1 parasite) relative to native A. pallipes (n = 4 parasites), which host Thelohania contejeani, 'Austropotamobius pallipes bacilliform virus' (ApBV), Psorospermium haeckeli and Branchiobdella astaci, at the sites studied. The infectious group present in both species was an intranuclear bacilliform virus of the hepatopancreas. The prevalence of A. astaci in A. pallipes populations was higher in more polluted water bodies, which may reflect an effect of water quality, or may be due to increased chance of transmission from nearby P. leniusculus, a species commonly found in poor quality habitats.

Understanding the role of the shrimp gut microbiome in health and disease.

With rapid increases in the global shrimp aquaculture sector, a focus on animal health during production becomes ever more important. Animal productivity is intimately linked to health, and the gut microbiome is becoming increasingly recognised as an important driver of cultivation success. The microbes that colonise the gut, commonly referred to as the gut microbiota or the gut microbiome, interact with their host and contribute to a number of key host processes, including digestion and immunity. Gut microbiome manipulation therefore represents an attractive proposition for aquaculture and has been suggested as a possible alternative to the use of broad-spectrum antibiotics in the management of disease, which is a major limitation of growth in this sector. Microbiota supplementation has also demonstrated positive effects on growth and survival of several different commercial species, including shrimp. Development of appropriate gut supplements, however, requires prior knowledge of the host microbiome. Little is known about the gut microbiota of the aquatic invertebrates, but penaeid shrimp are perhaps more studied than most. Here, we review current knowledge of information reported on the shrimp gut microbiota, highlighting the most frequently observed taxa and emphasizing the dominance of Proteobacteria within this community. We discuss involvement of the microbiome in the regulation of shrimp health and disease and describe how the gut microbiota changes with the introduction of several economically important shrimp pathogens. Finally, we explore evidence of microbiome supplementation and consider its role in the future of penaeid shrimp production.

The shrimp microsporidian Enterocytozoon hepatopenaei (EHP): Biology, pathology, diagnostics and control.

Disease is a major limiting factor in the global production of cultivated shrimp. The microsporidian parasite Enterocytozoon hepatopenaei (EHP) was formally characterized in 2009 as a rare infection of the black tiger shrimp Penaeus monodon. It remained relatively unstudied until mid-2010, after which infection with EHP became increasingly common in the Pacific whiteleg shrimp Penaeus vannamei, by then the most common shrimp species farmed in Asia. EHP infects the hepatopancreas of its host, causing hepatopancreatic microsporidiosis (HPM), a condition that has been associated with slow growth of the host in aquaculture settings. Unlike other infectious disease agents that have caused economic losses in global shrimp aquaculture, EHP has proven more challenging because too little is still known about its environmental reservoirs and modes of transmission during the industrial shrimp production process. This review summarizes our current knowledge of the EHP life cycle and the molecular strategies that it employs as an obligate intracellular parasite. It also provides an analysis of available and new methodologies for diagnosis since most of the current literature on EHP focuses on that topic. We summarize current knowledge of EHP infection and transmission dynamics and currently recommended, practical control measures that are being applied to limit its negative impact on shrimp cultivation. We also point out the major gaps in knowledge that urgently need to be bridged in order to improve control measures.