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.

Discovery of the parasite Marteilia cocosarum sp. nov. In common cockle (Cerastoderma edule) fisheries in Wales, UK and its comparison with Marteilia cochillia.

Diseases of bivalve molluscs caused by paramyxid parasites of the genus Marteilia have been linked to mass mortalities and the collapse of commercially important shellfish populations. Until recently, no Marteilia spp. have been detected in common cockle (Cerastoderma edule) populations in the British Isles. Molecular screening of cockles from ten sites on the Welsh coast indicates that a Marteilia parasite is widespread in Welsh C. edule populations, including major fisheries. Phylogenetic analysis of ribosomal DNA (rDNA) gene sequences from this parasite indicates that it is a closely related but different species to Marteilia cochillia, a parasite linked to mass mortality of C. edule fisheries in Spain, and that both are related to Marteilia octospora, for which we provide new rDNA sequence data. Preliminary light and transmission electron microscope (TEM) observations support this conclusion, indicating that the parasite from Wales is located primarily within areas of inflammation in the gills and the connective tissue of the digestive gland, whereas M. cochillia is found mainly within the epithelium of the digestive gland. The impact of infection by the new species, here described as Marteilia cocosarum n. sp., upon Welsh fisheries is currently unknown.

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.

First Detection of Francisella halioticida Infecting a Wild Population of Blue Mussels Mytilus edulis in the United Kingdom.

In the last decade, declines in the population of wild blue mussels Mytilus edulis in the Tamar estuary (United Kingdom) have been noted. In archived samples collected from 2013 to 2019, between 7% (in 2013) and 18% (in 2019) showed large granulocytoma and haemocytic infiltration in the interstitial tissue of the digestive gland. Four samples were selected for 16S rRNA gene Nanopore sequencing. A consensus sequence of 1449 bp showed nucleotide similarities between 99.93-100% with published sequences of Francisella halioticida. In situ hybridisation (ISH) confirmed the presence of F. halioticida DNA within individual granulocytes of granulocytomas and also in prokaryotic-like inclusion bodies within the digestive epithelial cells. The design of diagnostic tests for surveillance of F. halioticida, including more specific ISH probes and sequencing the genome of the isolates infecting mussels, will shed more light on the pathogenicity and spread of this pathogen.

Txikispora philomaios n. sp., n. g., a micro-eukaryotic pathogen of amphipods, reveals parasitism and hidden diversity in Class Filasterea.

This study provides a morphological, ultrastructural, and phylogenetic characterization of a novel micro-eukaryotic parasite (2.3-2.6 µm) infecting amphipod genera Echinogammarus and Orchestia. Longitudinal studies across two years revealed that infection prevalence peaked in late April and May, reaching 64% in Echinogammarus sp. and 15% in Orchestia sp., but was seldom detected during the rest of the year. The parasite infected predominantly hemolymph, connective tissue, tegument, and gonad, although hepatopancreas and nervous tissue were affected in heavier infections, eliciting melanization and granuloma formation. Cell division occurred inside walled parasitic cysts, often within host hemocytes, resulting in hemolymph congestion. Small subunit (18S) rRNA gene phylogenies including related environmental sequences placed the novel parasite as a highly divergent lineage within Class Filasterea, which together with Choanoflagellatea represent the closest protistan relatives of Metazoa. We describe the new parasite as Txikispora philomaios n. sp. n. g., the first confirmed parasitic filasterean lineage, which otherwise comprises four free-living flagellates and a rarely observed endosymbiont of snails. Lineage-specific PCR probing of other hosts and surrounding environments only detected T. philomaios in the platyhelminth Procerodes sp. We expand the known diversity of Filasterea by targeted searches of metagenomic datasets, resulting in 13 previously unknown lineages from environmental samples.

Parasites, pathogens, and other symbionts of copepods.

There is a large diversity of eukaryotic symbionts of copepods, dominated by epizootic protists such as ciliates, and metazoan parasites. Eukaryotic endoparasites, copepod-associated bacteria, and viruses are less well known, partly due to technical limitations. However, new molecular techniques, combined with a range of other approaches, provide a complementary toolkit for understanding the complete symbiome of copepods and how the symbiome relates to their ecological roles, relationships with other biota, and responses to environmental change. In this review we provide the most complete overview of the copepod symbiome to date, including microeukaryotes, metazoan parasites, bacteria, and viruses, and provide extensive literature databases to inform future studies.

Cosmopolitan Distribution of Endozoicomonas-Like Organisms and Other Intracellular Microcolonies of Bacteria Causing Infection in Marine Mollusks.

Intracellular microcolonies of bacteria (IMC), in some cases developing large extracellular cysts (bacterial aggregates), infecting primarily gill and digestive gland, have been historically reported in a wide diversity of economically important mollusk species worldwide, sometimes associated with severe lesions and mass mortality events. As an effort to characterize those organisms, traditionally named as Rickettsia or Chlamydia-like organisms, 1950 specimens comprising 22 mollusk species were collected over 10 countries and after histology examination, a selection of 99 samples involving 20 species were subjected to 16S rRNA gene amplicon sequencing. Phylogenetic analysis showed Endozoicomonadaceae sequences in all the mollusk species analyzed. Geographical differences in the distribution of Operational Taxonomic Units (OTUs) and a particular OTU associated with pathology in king scallop (OTU_2) were observed. The presence of Endozoicomonadaceae sequences in the IMC was visually confirmed by in situ hybridization (ISH) in eight selected samples. Sequencing data also indicated other symbiotic bacteria. Subsequent phylogenetic analysis of those OTUs revealed a novel microbial diversity associated with molluskan IMC infection distributed among different taxa, including the phylum Spirochetes, the families Anaplasmataceae and Simkaniaceae, the genera Mycoplasma and Francisella, and sulfur-oxidizing endosymbionts. Sequences like Francisella halioticida/philomiragia and Candidatus Brownia rhizoecola were also obtained, however, in the absence of ISH studies, the association between those organisms and the IMCs were not confirmed. The sequences identified in this study will allow for further molecular characterization of the microbial community associated with IMC infection in marine mollusks and their correlation with severity of the lesions to clarify their role as endosymbionts, commensals or true pathogens.

Diagnosis and prevalence of two new species of haplosporidians infecting shore crabs Carcinus maenas: Haplosporidium carcini n. sp., and H. cranc n. sp.

This study provides a morphological and phylogenetic characterization of two novel species of the order Haplosporida (Haplosporidium carcini n. sp., and H. cranc n. sp.) infecting the common shore crab Carcinus maenas collected at one location in Swansea Bay, South Wales, UK. Both parasites were observed in the haemolymph, gills and hepatopancreas. The prevalence of clinical infections (i.e. parasites seen directly in fresh haemolymph preparations) was low, at ~1%, whereas subclinical levels, detected by polymerase chain reaction, were slightly higher at ~2%. Although no spores were found in any of the infected crabs examined histologically (n = 334), the morphology of monokaryotic and dikaryotic unicellular stages of the parasites enabled differentiation between the two new species. Phylogenetic analyses of the new species based on the small subunit (SSU) rDNA gene placed H. cranc in a clade of otherwise uncharacterized environmental sequences from marine samples, and H. carcini in a clade with other crustacean-associated lineages.

Ultrastructure, phylogeny and histopathology of two novel haplosporidians parasitising amphipods, and importance of crustaceans as hosts.

This study provides morphological, ultrastructural and phylogenetic characterization of 2 novel species of Haplosporidia (Haplosporidium echinogammari n. sp. and H. orchestiae n. sp.) infecting amphipods of the genera Echinogammarus and Orchestia collected in southwestern England. Both parasites infect the connective tissues associated with the digestive gland and the tegument, and eventually infect other organs causing disruption of host tissues with associated motor impairment and fitness reduction. Prevalence of infection varied with host species, provenance and season, being as high as 75% for individuals of E. marinus infected with H. echinogammari in June (n = 50). Although no spores were found in any of the infected amphipods examined (n = 82), the morphology of monokaryotic and dikaryotic unicellular stages of the parasites enabled differentiation between the 2 new species. Phylogenetic analysis of the new species based on the small subunit (SSU) rDNA gene placed H. echinogammari close to H. diporeiae in haplosporidian lineage C, and H. orchestiae in a novel branch within Haplosporidium. Genetic diversity of the haplosporidians infecting these and other amphipod species was evaluated and compared to morphological and ultrastructural changes to host tissues. The phylogenetic relationship of haplosporidian infections in other crustacean hosts is discussed after inclusion into the analysis of 25 novel SSU rDNA sequences obtained from crabs, isopods and crayfish.

Detection and characterisation of haplosporidian parasites of the blue mussel Mytilus edulis, including description of the novel parasite Minchinia mytili n. sp.

The edible mussel Mytilus edulis is a major aquaculture commodity in Europe, with 168000 t produced in 2015. A number of abundant, well characterised parasites of the species are known, though none are considered to cause significant mortality. Haplosporida (Rhizaria, Endomyxa) is an order of protistan parasites of aquatic invertebrates, the best studied of which are the oyster pathogens Haplosporidium nelsoni and Bonamia ostreae. While these species are well characterised within their hosts, the diversity, life-cycle and modes of transmission of haplosporidians are very poorly understood. Haplosporidian parasites have previously been reported from Mytilus spp., however the majority of these remain uncharacterised, and no molecular data exist for any species. In this study, we identified 2 novel haplosporidian parasites of M. edulis present in the UK. The first of these, observed by light microscopy and in situ hybridisation infecting the gills, mantle, gonadal tubules and digestive connective tissues of mussels in the Tamar estuary, England, we describe as Minchinia mytili on the basis of 18S sequence data. The second, observed infecting a single archive specimen collected in Loch Spelve, Mull, Scotland, infects the foot muscle, gills and connective tissue of the digestive gland. Sequence data places this parasite in an uncharacterised clade of sequences amplified from tropical bivalve guts and water samples, sister to H. nelsoni. Screening of water and sediment samples collected at the sample site in the Tamar estuary revealed the presence of both sequence types in the water column, suggesting host-free or planktonic life stages.

Environmental Sequencing Fills the Gap Between Parasitic Haplosporidians and Free-living Giant Amoebae.

Class Ascetosporea (Rhizaria; Endomyxa) comprises many parasites of invertebrates. Within this group, recent group-specific environmental DNA (eDNA) studies have contributed to the establishment of the new order Mikrocytida, a new phylogeny and characterization of Paramyxida, and illuminated the diversity and distribution of haplosporidians. Here, we use general and lineage-specific PCR primers to investigate the phylogenetic "gap" between haplosporidians and their closest known free-living relatives, the testate amoeba Gromia and reticulate amoeba Filoreta. Within this gap are Paradinium spp. parasites of copepods, which we show to be highly diverse and widely distributed in planktonic and benthic samples. We reveal a robustly supported radiation of parasites, ENDO-3, comprised of Paradinium and three further clades (ENDO-3a, ENDO-3b and SPP). A further environmental group, ENDO-2, perhaps comprising several clades, branches between this radiation and the free-living amoebae. Early diverging haplosporidians were also amplified, often associated with bivalves or deep-sea samples. The general primer approach amplified an overlapping set of novel lineages within ENDO-3 and Haplosporida, whereas the group-specific primer strategy, targeted to amplify from the earliest known divergent haplosporidians to Gromia, generated greater sequence diversity across part of this phylogenetic range.

A new phylogeny and environmental DNA insight into paramyxids: an increasingly important but enigmatic clade of protistan parasites of marine invertebrates.

Paramyxida is an order of rhizarian protists that parasitise marine molluscs, annelids and crustaceans. They include notifiable pathogens (Marteilia spp.) of bivalves and other taxa of economic significance for shellfish production. The diversity of paramyxids is poorly known, particularly outside of commercially important hosts, and their phylogenetic position is unclear due to their extremely divergent 18S rDNA sequences. However, novel paramyxean lineages are increasingly being detected in a wide range of invertebrate hosts, and interest in the group is growing, marked by the first 'Paramyxean Working Group' Meeting held in Spain in February 2015. We review the diversity, host affiliations, and geographical ranges of all known paramyxids, present a comprehensive phylogeny of the order and clarify its taxonomy. Our phylogenetic analyses confirm the separate status of four genera: Paramarteilia, Marteilioides, Paramyxa and Marteilia. Further, as including M. granula in Marteilia would make the genus paraphyletic we suggest transferring this species to a new genus, Eomarteilia. We present sequence data for Paramyxa nephtys comb. n., a parasite of polychaete worms, providing morphological data for a clade of otherwise environmental sequences, sister to Marteilioides. Light and electron microscopy analyses show strong similarities with both Paramyxa and Paramyxoides, and we further discuss the validity of those two genera. We provide histological and electron microscopic data for Paramarteilia orchestiae, the type species of that genus originally described from the amphipod Orchestia; in situ hybridisation shows that Paramarteilia also infects crab species. We present, to our knowledge, the first known results of a paramyxid-specific environmental DNA survey of environmental (filtered water, sediment, etc.) and organismally-derived samples, revealing new lineages and showing that paramyxids are associated with a wider range of hosts and habitat types than previously known. On the basis of our new phylogeny we propose phylogenetic hypotheses for evolution of lifecycle and infectivity traits observed in different paramyxid genera.

Mikrocytids are a broadly distributed and divergent radiation of parasites in aquatic invertebrates.

Microcell parasites have independently evolved in several eukaryotic lineages and are increasingly recognized as important and emerging pathogens of diverse hosts, including species of economic importance subject to international legislation concerning the trading of aquatic animals [1-3]. The microcell Mikrocytos mackini causes Denman Island disease of oysters and represents one of the most genetically divergent eukaryotes known. Mikrocytos has remained an isolated lineage with a limited distribution. We investigated two emerging diseases of juvenile crabs and oysters from the UK using massively parallel sequencing and targeted primer approaches to reveal that their causative agents are highly divergent lineages related to M. mackini (Paramikrocytos canceri n. gen. et n. sp. and M. mimicus sp. nov., respectively). We demonstrate a major new globally distributed parasite radiation (Mikrocytida ord. nov.) with phylogenetic affinities to the commercially important haplosporidian parasites of invertebrates. Mikrocytids have eluded detection because of their small size, intracellular habit, and extreme sequence divergence. P. canceri was frequently detected in a range of shoreline invertebrates, demonstrating that these newly recognized parasites are in fact common, diverse, and widespread and should be considered when assessing the risks of aquaculture activities, invasive species spread, and movements of ballast water and sediments with associated invertebrates.