The Experience of a District General Hospital with a Large Outdoor Music Festival in England.

OBJECTIVES: To assess the impact of the Parklife annual music festival on the local hospital, North Manchester General. METHODS: Data was obtained retrospectively by analysis of emergency department records during the weekend of Parklife 2015. RESULTS: 32 patients were identified, 56% reported taking drugs. 34% were admitted for overnight observation. 4 patients presented with methaemoglobinaemia following oral ingestion of amyl nitrate. One patient had a methaemoglobin fraction of 90.6%, which is amongst the most extreme recorded in literature. CONCLUSION: Music festivals can impose a burden on local health services. Organisers should operate an efficient surveillance system in order to prevent the sale and use of recreational drugs, providing adequate on-site health services and working in collaboration with local emergency services.

Epidemiology of Bonamia in the UK, 1982 to 2012.

Bonamiasis, caused by Bonamia ostreae, was confirmed in native flat oysters Ostrea edulis L. in England in 1982. Hudson & Hill (1991; Aquaculture 93:279-285) documented investigations into the initial spread of the disease in wild and cultivated stocks of native oysters in the UK. They also described the controls that were initially applied to prevent the further spread of the pathogen. This paper reports on subsequent controls and associated monitoring applied in the UK and reports on the epidemiology of the disease in the 30 yr from 1982 to 2012. Bonamiasis remained confined to the zones in England as documented by Hudson & Hill (1991) until 2005, when it was confirmed in Lough Foyle, Northern Ireland. In 2006 it was found in 2 new areas, one in Wales and one in Scotland. Subsequent further spread to additional areas in all parts of the UK has resulted in 9 zones being currently designated as infected with the disease. In addition, a single oyster from one area has tested positive for the closely related B. exitiosa. In general, analysis of the results of the monitoring programme in England and Wales shows no clear trend in infection levels over time, although there has been an apparent decrease in the level of infection in some fishery areas. In an autumn sampling programme the highest levels of infection were detected in October.

Virus-like particles associated with mortalities of the Manila clam Ruditapes philippinarum in England.

Mortalities of the Manila clam Ruditapes philippinarum (Adams & Reeve, 1850) were reported in southern England (Kent and Poole Harbour) during late spring of 2008. In response to these reported mortalities, samples were collected from 5 sites across the south coast of England. Clams were sampled for both histology and electron microscopy. Transmission electron microscopy (TEM) revealed unenveloped virus-like particles within the connective tissue of the gills and surrounding the tubules in the digestive gland. The virus-like particles appeared to be free within the cytoplasm or associated with endoplasmic reticulum membranes and cytoplasmic vesicles. Particles were icosahedral in shape, with a diameter of 25 to 30 nm. The location, size and morphology of the virus-like particles suggest that they belong to the Picornaviridae family. This is the first report of this virus infection in wild and farmed R. philippinarum within the UK.

Multi-centre testing and validation of current protocols for the identification of Gyrodactylus salaris (Monogenea).

Despite routine screening requirements for the notifiable fish pathogen Gyrodactylus salaris, no standard operating procedure exists for its rapid identification and discrimination from other species of Gyrodactylus. This study assessed screening and identification efficiencies under real-world conditions for the most commonly employed identification methodologies: visual, morphometric and molecular analyses. Obtained data were used to design a best-practice processing and decision-making protocol allowing rapid specimen throughput and maximal classification accuracy. True specimen identities were established using a consensus from all three identification methods, coupled with the use of host and location information. The most experienced salmonid gyrodactylid expert correctly identified 95.1% of G. salaris specimens. Statistical methods of classification identified 66.7% of the G. salaris, demonstrating the need for much wider training. Molecular techniques (internal transcribed spacer region-restriction fragment length polymorphism (ITS-RFLP)/cytochrome c oxidase I (COI) sequencing) conducted in the diagnostic laboratory most experienced in the analysis of gyrodactylid material, identified 100% of the true G. salaris specimens. Taking into account causes of potential specimen loss, the probabilities of a specimen being accurately identified were 95%, 87% and 92% for visual, morphometric and molecular techniques, respectively, and the probabilities of correctly identifying a specimen of G. salaris by each method were 81%, 58% and 92%. Inter-analyst agreement for 189 gyrodactylids assessed by all three methods using Fleiss' Kappa suggested substantial agreement in identification between the methods. During routine surveillance periods when low numbers of specimens are analysed, we recommend that specimens be analysed using the ITS-RFLP approach followed by sequencing of specimens with a "G. salaris-like" (i.e. G. salaris, Gyrodactylus thymalli) banding pattern. During periods of suspected outbreaks, where a high volume of specimens is expected, we recommended that specimens be identified using visual identification, as the fastest processing method, to select "G. salaris-like" specimens, which are subsequently identified by molecular-based techniques.

An assessment of the variation in the prevalence of renal myxosporidiosis and hepatitis in wild brown trout, Salmo trutta L., within and between rivers in South-West England.

The prevalence of renal myxosporidiosis in wild brown trout, Salmo trutta, in seven river catchments in South-West England was investigated. Three hundred and twenty-seven fish were sampled from 16 sites, of which 54 (16.5%) were found, by histological examination of the kidney, to be infected with Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease. No T. bryosalmonae infected fish were found in one river catchment, in other catchments the prevalence ranged from 2.5% to 36%. Hepatitis was strongly associated with the presence of T. bryosalmonae (odds ratio = 20.2, P < 0.001). Chloromyxum schurovi was found in 25% of fish and in six of seven river catchments, where the prevalence ranged from 2.4% to 63%. There was a strong negative association between the presence of T. bryosalmonae and C. schurovi (odds ratio = 0.10, P < 0.001). A hierarchical binomal model of the variance indicated that for T. bryosalmonae most of the variance existed at the site level, whereas for C. schurovi most variance existed at the river catchment level, suggesting that prevalence of T. bryosalmonae infection is determined largely by site level factors (e.g. presence of alternate host). The intraclass correlation coefficients (ICC) were 0.2 and 0.4 for T. bryosalmonae and C. schurovi, respectively, indicating the latter has higher effective transmission because of a higher level of infectiousness and/or abundance of alternate oligochaete hosts. These values can be used in future studies to estimate the sample sizes required to generate prevalence estimates with the required precision.

Laser-assisted microdissection: a new tool for aquatic molecular parasitology.

Laser-assisted microdissection (LMD) has been developed to isolate distinct cell populations from heterogeneous tissue sections, cytological preparations, or live cell samples. Downstream applications typically include gene expression studies using real-time PCR and array platforms, diagnostic PCR, and protein expression studies. LMD techniques are now commonplace in mainstream biological research and clearly have suitable applications in the field of aquatic pathology and parasitology. The present study used LMD to isolate 2 dinoflagellate parasites (Hematodinium spp.) from formalin-fixed paraffin-embedded tissue sections from 2 crustacean hosts, Cancer pagurus and Portunus trituberculatus. DNA was isolated from LMD parasite preparations, and partial regions (up to 300 bp) of the small subunit and the first internal transcribed spacer region of the rRNA gene complex from the Hematodinium spp. were PCR amplified using diagnostic primers. The amplification products were sequenced to confirm the identity of the targeted regions. The techniques, applications, and limitations of LMD to address questions in aquatic molecular pathology and parasitology are discussed.

Enterospora canceri n. gen., n. sp., intranuclear within the hepatopancreatocytes of the European edible crab Cancer pagurus.

Only 1 genus (Nucleospora) within 1 family (Enterocytozoonidae) of the Microsporidia contains species that are parasitic within the nuclei of their host cells; to date, all described intranuclear Nucleospora spp. parasitise fish. This study describes the first intranuclear microsporidian parasite of an invertebrate, the European edible crab Cancer pagurus L. (Decapoda: Cancridae). Infected crabs displayed no obvious external signs, and maximum apparent prevalence of infection within a monthly sample was 3.45%. Infected hepatopancreatic tubules were characterised by varying numbers of hypertrophic and eosinophilic nuclei within epithelial cells. Parasite stages appeared as eosinophilic granular accumulations causing margination of host chromatin. In advanced cases, the tubule epithelia degenerated, with parasites and sloughed epithelial cells appearing in tubule lumens. All life stages of the parasite were observed within host nuclei. Uninucleate meronts were not detected, although binucleate stages were observed. Multinucleate plasmodia (sporogonal plasmodia) contained up to 22 nuclei in section, and late-stage plasmodia contained multiple copies of apparatus resembling the polar filament and anchoring disk, apparently associated with individual plasmodial nuclei. As such, aggregation and early assembly of sporoblast components took place within the intact sporogonial plasmodium, a feature unique to the Enterocytozoonidae. Liberation of sporoblasts from plasmodia or the presence of liberated sporoblasts was not observed in this study. However, large numbers of maturing and mature spores (measuring 1.3 +/- 0.02 x 0.7 +/- 0.01 microm) were frequently observed in direct contact with the host nucleoplasm. Considering the shared features of this parasite with microsporidians of the family Enterocytozoonidae, and the unique presence of this parasite within the nucleoplasm of decapod crustacean hepatopancreatocytes, this parasite (Enterospora canceri) is proposed as the type species of a new genus (Enterospora) of microsporidian. Molecular taxonomic work is now required, comparing Enterospora to Enterocytozoon and Nucleospora, the 2 other genera within the Enterocytozoonidae.

Immunostaining of spores and plasmodia of disparate myxozoan genera with comments on the properties of the sporular mucus envelope.

Species of the phylum Myxozoa are common parasites of fish and can cause severe losses in cultured species. Although a number of myxozoan life-cycles have now been elucidated, little is known about the biology of these organisms in the fish host. Monoclonal antibody B4 raised to the myxozoan Tetracapsuloides bryosalmonae has been previously noted to react with a number of species infecting fish kidney. We present the results of a survey of 55 myxosporean species that determined that this antibody detects an antigen on the spore surface of 33 of these species in the genera Myxobolus, Sphaerospora and Thelohanellus. However, there appears to be no clear relationship between those spores that contain the MAb B4 reactive antigen and the host or organ in which they are detected. The antigen appears to be synthesized in the plasmodial cytoplasm and is intimately associated with the surface of the spore capsules and, where present, the mucus envelope. The nature of this envelope is further discussed in relation to its formation and distinctive properties.

Descriptions, development and pathogenicity of myxozoan (Myxozoa: Myxosporea) parasites of juvenile cyprinids (Pisces: Cyprinidae).

Approximately 5000 young of the year (0+) cyprinids comprising roach, chub, dace, minnow, bleak, bream, barbel and gudgeon were examined histologically for the presence of myxozoan infections. Thirteen myxozoans were identified to species, the majority being Myxobolus spp. In addition, two species of Myxidium and of Sphaerospora were recorded. All organs were examined, with the majority of infections being found in the gills, musculature and kidney. However, isolated spores were occasionally found in other tissues. Whilst roach contained the highest number of myxozoan species, it was chub that showed the greatest host response to sporogonic forms. Data are provided on spore morphology, pathogenic responses and tissue and host specificity of the myxozoans recorded.

Observations of Dermocystidium sp. infections in bullheads, Cottus gobio L., from a river in southern England.

Bullheads, Cottus gobio, with macroscopic external cysts on the skin and fins measuring up to 3 mm in diameter were detected in the River Allen and its tributaries in southern England between 1992 and 1998. The prevalence of these cysts was up to 50% at some sites. Examination of cyst contents revealed the presence of numerous spores, typical of the genus Dermocystidium, measuring 8 microm in diameter. The parasite developed within well-defined cysts, which were located in the hypodermal connective tissues of the host. No cysts were present on the fins of any of the fish examined. Histological examination revealed a cyst wall consisting of an inner layer of dense eosinophilic material similar to that reported for Dermocystidium spp. forming coenocytic hyphae. No evidence was found of systemic infection or hyphal formation. Spores contained a prominent refractile body, which gave a weakly positive reaction for polysaccharides with the periodic-acid Schiff reaction and was positively stained with acidic dyes. Several examples of ruptured cysts were seen in histological sections and in some of these cases the host epithelial layer was breached, allowing release of the spores to the environment. Morphological features of, and host response towards, the Dermocystidium sp. in bullheads are compared with similar infections in salmonids and other freshwater fish species.

Histopathological biomarkers in estuarine fish species for the assessment of biological effects of contaminants.

The increasing emphasis on the assessment and monitoring of estuarine ecosystems has highlighted the need to deploy appropriate biological indices for these locations. Fish diseases and histopathology, with a broad range of causes, are increasingly being used as indicators of environmental stress since they provide a definite biological end-point of historical exposure. This study reports on the histopathological alterations observed in selected organs and tissues of three species of estuarine fish (Platichthys flesus, Pomatoschistus minutus and Zoarces viviparus), captured from four British estuaries (the Tyne, Tees, Mersey and Alde), differently impacted by contaminants, including PAHs. A biannual sampling regime was used to identify the important seasonal variations that occur in terms of the observed biological effects. Inflammatory lesions and hepatocellular fibrillar inclusions attained their highest prevalence in P. flesus captured from the Tyne, Tees and Mersey. The presence of pre-neoplastic and neoplastic toxicopathic lesions was highest in P. flesus captured from these sites, when compared to fish from the Aide reference site. In particular, the prevalence of hepatic foci of cellular alteration (up to 43.3%) and hepatocellular adenoma (up to 10%) were highest in P. flesus captured from the Mersey estuary. Intersex (ovotestis) was only recorded in male P.flesus captured from the Mersey estuary (up to 8.3%) and from male Z. viviparous captured from the Tyne estuary (25%). Pathologies associated with the gill and the kidney were also most prevalent in fish captured from the Tyne, Tees and Mersey estuaries. This study has successfully applied histopathology to an estuarine monitoring program, both for the recording of toxicopathic lesions in the liver and other organs, and for the detection of the endpoint of endocrine disruption, intersex. As such, it provides a powerful integrative tool for the assessment of biological effects of contaminants in these environments.

Recent advances in our knowledge of the Myxozoa.

In the last few years two factors have helped to significantly advance our understanding of the Myxozoa. First, the phenomenal increase in fin fish aquaculture in the 1990s has lead to the increased importance of these parasites; in turn this has lead to intensified research efforts, which have increased knowledge of the development, diagnosis. and pathogenesis of myxozoans. The hallmark discovery in the 1980s that the life cycle of Myxobolus cerebralis requires development of an actinosporean stage in the oligochaete. Tubifex tubifex, led to the elucidation of the life cycles of several other myxozoans. Also, the life cycle and taxonomy of the enigmatic PKX myxozoan has been resolved: it is the alternate stage of the unusual myxozoan, Tetracapsula bryosalmonae, from bryozoans. The 18S rDNA gene of many species has been sequenced, and here we add 22 new sequences to the data set. Phylogenetic analyses using all these sequences indicate that: 1) the Myxozoa are closely related to Cnidaria (also supported by morphological data); 2) marine taxa at the genus level branch separately from genera that usually infect freshwater fishes; 3) taxa cluster more by development and tissue location than by spore morphology; 4) the tetracapsulids branched off early in myxozoan evolution, perhaps reflected by their having bryozoan, rather than annelid hosts; 5) the morphology of actinosporeans offers little information for determining their myxosporean counterparts (assuming that they exist); and 6) the marine actinosporeans from Australia appear to form a clade within the platysporinid myxosporeans. Ribosomal DNA sequences have also enabled development of diagnostic tests for myxozoans. PCR and in situ hybridisation tests based on rDNA sequences have been developed for Myxobolus cerebralis, Ceratomyxa shasta, Kudoa spp., and Tetracapsula bryosalmonae (PKX). Lectin-based and antibody tests have also been developed for certain myxozoans, such as PKX and C. shasta. We also review important diseases caused by myxozoans, which are emerging or re-emerging. Epizootics of whirling disease in wild rainbow trout (Oncorhynchus mykiss) have recently been reported throughout the Rocky Mountain states of the USA. With a dramatic increase in aquaculture of fishes using marine netpens, several marine myxozoans have been recognized or elevated in status as pathological agents. Kudoa thyrsites infections have caused severe post-harvest myoliquefaction in pen-reared Atlantic salmon (Salmo salar), and Ceratomyxa spp., Sphaerospora spp., and Myxidium leei cause disease in pen-reared sea bass (Dicentrarchus labrax) and sea bream species (family Sparidae) in Mediterranean countries.

Induction of proliferative kidney disease (PKD) in rainbow trout Oncorhynchus mykiss via the bryozoan Fredericella sultana infected with Tetracapsula bryosalmonae.

Proliferative kidney disease (PKD) is a serious infection of wild and farmed salmonids, affecting mainly the kidney and spleen but becoming systemic in most susceptible fish hosts. This report deals with the transmission of Tetracapsula bryosalmonae Canning, Curry, Feist, Longshaw & Okamura 1999 from naturally infected bryozoans Fredericella sultana Blumenbach 1779 to naive rainbow trout Oncorhynchus mykiss Walbaum 1792, thereby confirming the recent conclusion based on partial 18S rDNA sequence data that bryozoans are hosts of the myxozoan parasite T. bryosalmonae (formerly PKX organism) that causes the disease. Parasite transmission using T. bryosalmonae spores was successful by short-term exposure to disrupted bryozoans known to contain T. bryosalmonae spores and T bryosalmonae sacs liberated from the bryozoans, and by long-term cohabitation with infected bryozoan colonies. Infection was confirmed by examination of kidney imprints, detection of the parasite in stained tissue sections, PCR using T. bryosalmonae-specific primers, and comparison of amplified 18S rDNA sequences from the bryozoans and experimentally infected fish. Transmission was not apparent, nor was PKD induced, in fish challenged by intraperitoneal injection of spores isolated from F. sultana.

Patterns of occurrence and 18S rDNA sequence variation of PKX (Tetracapsula bryosalmonae), the causative agent of salmonid proliferative kidney disease.

Recent progress in understanding the etiology of proliferative kidney disease (PKD) includes the identification of freshwater bryozoans as the natural hosts of the myxozoan parasite that causes the disease in salmonid fish and formal description of the parasite as Tetracapsula bryosalmonae. This paper presents data on patterns of occurrence of T. bryosalmonae and sequence variation among isolates. T. bryosalmonae infects bryozoans that range from primitive to more derived genera within the Phylactolaemata and that differ in growth form and habits. Infected bryozoans have been collected in diverse habitats including cold, clear streams and warm, eutrophic lakes. Temporal surveys reveal intra- and interannual variation in infection levels, and spatial variation in incidence of infection is implicit by the apparent absence of T. bryosalmonae from many bryozoan populations. The significance of minor variation in partial sequences of 18S rDNA requires further investigation. The information presented here provides the first significant insights into the ecology of T. bryosalmonae.

A new class and order of myxozoans to accommodate parasites of bryozoans with ultrastructural observations on Tetracapsula bryosalmonae (PKX organism).

Tetracapsula bryosalmonae, formerly PKX organism, is a myxozoan parasite that causes proliferative kidney disease in salmonid fish. Its primary hosts, in which it undergoes a sexual phase, are phylactolaemate bryozoans. It develops in the bryozoan coelomic cavity as freely floating sacs which contain two types of cells, stellate cells and sporoplasmogenic cells, which become organised as spores. Eight stellate cells differentiate as four capsulogenic cells and four valve cells which surround a single sporoplasmogenic cell. The sporoplasmogenic cell undergoes meiosis and cytoplasmic fission to produce two sporoplasms with haploid nuclei. Sporoplasms contain secondary cells. The unusual development supports previously obtained data from 18S rDNA sequences, indicating that species of Tetracapsula form a clade. It diverged early in the evolution of the Myxozoa, before the radiation that gave rise to the better known genera belonging to the two orders in the single class Myxosporea. The genus Tetracapsula as seen in bryozoans shares some of the characters unique to the myxosporean phase and others typical of the actinosporean phase of genera belonging to the class Myxosporea. However, it exhibits other features which are not found in either phase. A new class Malacosporea and order Malacovalvulida are proposed to accommodate the family Saccosporidae and genus Tetracapsula. Special features of the new class are the sac-like proliferative body, valve cells not covering the exit point of the polar filament, lack of a stopper-like structure sealing the exit, maintenance of valve cell integrity even at spore maturity, absence of hardened spore walls and unique structure of sporoplasmosomes in the sporoplasms.

Pseudanthocotyloides heterocotyle (van Beneden, 1871) Euzet & Prost, 1969 (Monogenea: Polyopisthocotylea: Mazocraeidae), a parasite of herring Clupea harengus L. and sprat Sprattus sprattus L. (Teleostei: Clupeidae).

A total of 690 herring Clupea harengus L. and 88 sprat Sprattus sprattus L. caught off the west coast of Sweden, in the North Sea and off the west and south coasts of the United Kingdom, were examined for gill parasites. The monogenean Pseudanthocotyloides heterocotyle (van Beneden, 1871) Euzet & Prost, 1969 was found in 38 (5.5%) herring and one (1.1%) sprat. The parasite was significantly (P<0.05) more common off the west coast of Sweden than elsewhere and most specimens (62.5%) were found on the pseudobranchs. Only the smaller herring were infected. P. heterocotyle is redescribed and its taxonomy discussed, together with the possibility of host and parasite misidentification in previous reports.