STUDIES ON MYXOZOAN PARASITES OF FRESHWATER FISH AND INVERTEBRATE HOSTS

A study of myxozoan parasites has been investigated in hosts from freshwater environments in the UK. Over 17,000 oligochaetes, almost 5,000 juvenile cyprinids representing 7 species and over 60 invertebrate species have been examined for the presence of myxozoan parasites. In addition, studies on the lifecycle of Tetracapsuloides bryosalmonae (the causative agent of salmonid proliferative kidney disease, PKD) and of selected cyprinid myxozoans were conducted. A total of 21 actinospore types in seven collective groups were isolated and described from oligochaetes collected from seven different river systems in England and Wales. Twelve of the actinospores isolated appear to be new to science. Differences were noted in types of actinospores released at different sites and between seasons. Most actinospores were released from oligochaetes in spring and summer with prevalence of release ranging from 0.11% up to 5.83%. The most common actinospores were members of the collective group Echinactinomyxon with seven types identified, followed by the collective group Triactinomyxon, of which 6 types were identified. Five actinospores types were each encountered only once during the study. In juvenile cyprinid fish, 14 identifiable species of myxozoans in the genera Myxidium, Myxobolus and Sphaerospora plus three developmental stages were detected by histological examination. The most common myxozoans in cyprinids were Myxobolus pseudodispar and Myxobolus pfeifferi. Roach contained the most number of myxozoan species. Only seven myxozoan species were found in chub, but pathological responses and intensity of infections, particularly with M. pseudodispar, M. pfeifferi and Myxobolus buckei were greater when compared to other cyprinids examined. Juvenile cyprinids only appear to mount a pathological response to myxozoans once sporogony is initiated and some of those responses were considered severe enough to be detrimental to host survival. Mathematical models were produced using parasite data and incorporating a variety of data, including fish length, year class strength and environmental data to attempt to demonstrate a population level effect of disease. Many of the models developed clearly show that parasitism by Myxobolus spp. and Bucephalus polymorphus in juvenile fish is strongly correlated with population success in selected UK rivers. Laboratory experiments to transmit Myxobolus spp., Myxidium spp. and Sphaerospora spp. from selected cyprinid hosts to oligochaetes were unsuccessful. The most likely explanation is that the genetic strain of Tubifex tubifex used in the trials was not susceptible to infections by the myxospores selected. Specific DNA primers for Tetracapsuloides bryosalmonae were used on samples of over 60 invertebrate species collected from sites enzootic for PKD and on all 21 actinospore types isolated during the current study. All PCR reactions were negative for the presence of T. bryosalmonae DNA. Naive rainbow trout exposed to T. bryosalmonae spores from naturally infected bryozoans by bath challenge for 10 minutes developed PKD. Intraperitoneal injection of spores failed to induce the disease. The favoured route of entry by the parasite appears to be through mucous cells in the skin epithelium.The Centre for Environment, Fisheries and Aquaculture Science, Weymouth, Dorse

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

The effect of octopaminergic compounds on the behaviour and transmission of Gyrodactylus

Background: The high transmission potential of species belonging to the monogenean parasite genus Gyrodactylus, coupled with their high fecundity, allows them to rapidly colonise new hosts and to increase in number. One gyrodactylid, Gyrodactylus salaris Malmberg, 1957, has been responsible for devastation of Altantic salmon (Salmo salar L.) populations in a number of Norwegian rivers. Current methods of eradicating G. salaris from river systems centre around the use of non-specific biocides, such as rotenone and aluminium sulphate. Although transmission routes in gyrodactylids have been studied extensively, the behaviour of individual parasites has received little attention. Specimens of Gyrodactylus gasterostei Gläser, 1974 and G. arcuatus Bychowsky, 1933, were collected from the skin of their host, the three-spined stickleback (Gasterosteus aculeatus L.), and permitted to attach to the substrate. The movements of individual parasites were recorded and analysed. Results: The behaviour patterns of the two species were similar and parasites were more active in red light and darkness than in white light. Four octopaminergic compounds were tested and all four inhibited the movements of parasites. Treatment ultimately led to death at low concentrations (0.2 μM), although prolonged exposure was necessary in some instances. Conclusions: Octopaminergic compounds may affect the parasite's ability to locate and remain on its host and these or related compounds might provide alternative or supplementary treatments for the control of G. salaris infections. With more research there is potential for use of octopaminergic compounds, which have minimal effects on the host or its environment, as parasite-specific treatments against G. salaris infections

Gyrodactylus quadratidigitus n. sp. (Monogenea: Gyrodactylidae), a parasite of the leopard-spotted goby Thorogobius ephippiatus (Lowe) from the south-western coast of the UK

Gyrodactylus quadratidigitus n. sp. (Monogenea, Gyrodactylidae) is described from the gills, fins and skin of Thorogobius ephippiatus (Lowe) (leopard-spotted goby) from Portland Castle Bay, Dorset, UK. G. quadratidigitus n. sp. most closely resembles the species of Gyrodactylus previously described by Geets from Pomatoschistus microps(Krøyer) in the approximate shape and size of the attachment hooks but can be readily distinguished from other species of the genus by the distinctive square shape of the toe region of the marginal hook sickle, the anteriorly positioned cirrus bulb and the unusually short intestinal crura which extend only as far as the level of the testes

The accidental transfer of Gyrodactylus (Monogenea) during Short duration fish transportation

The potential of parasite transfer to an alternative host during short periods of cohabitation was explored. The work described in this paper suggests that either the field sampling process itself or the subsequent transportation of multiple fish species in the same container, may affect the correct allocation of parasites to hosts, and the diagnosis, management and control of gyrodactylosis in a variety of fish. Gyrodactylus infected specimens of three-spine sticklebacks Gasterosteus aculeatus, minnows Phoxinus phoxinus and stone loach Barbatula barbatula from one Scottish river were cohabited with one another in small volumes of water for 3 h. The study found that a small number of Gyrodactylus spp. transfer to atypical hosts. This study indicates that personnel involved in fish disease surveillance programmes should be aware of the possible consequences, in terms of inter-host transfer, of transporting multiple species in the same transport vessel. Diagnosticians should be aware of the facts that fish may act as temporary/paratenic hosts and that the apparent gyrodactylid fauna present following transport may not reflect that encountered under normal circumstances

Zoonotic Disease Pathogens in Fish Used for Pedicure

“Doctor” fish might not be such good doctors after all. These fish are used for the increasingly popular spa treatment called fish pedicures. During these sessions, spa patrons immerse their feet in water, allowing the live fish to feed on dead skin, mainly for cosmetic reasons. However, examinations of doctor fish destined for these spas found that they can carry harmful bacteria. Thus, although reports of human infection after fish pedicures are few, there may be some risks. Spa patrons who have underlying medical conditions (such as diabetes, immunosuppression, or even simple breaks in the skin) are already discouraged from taking such treatments. However, spas that offer fish pedicures should also consider using only disease-free fish reared in controlled facilities under high standards of husbandry and welfare

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

Safety and immunogenicity of seven COVID-19 vaccines as a third dose (booster) following two doses of ChAdOx1 nCov-19 or BNT162b2 in the UK (COV-BOOST): a blinded, multicentre, randomised, controlled, phase 2 trial

Background: Few data exist on the comparative safety and immunogenicity of different COVID-19 vaccines given as a third (booster) dose. To generate data to optimise selection of booster vaccines, we investigated the reactogenicity and immunogenicity of seven different COVID-19 vaccines as a third dose after two doses of ChAdOx1 nCov-19 (Oxford–AstraZeneca; hereafter referred to as ChAd) or BNT162b2 (Pfizer–BioNtech, hearafter referred to as BNT). Methods: COV-BOOST is a multicentre, randomised, controlled, phase 2 trial of third dose booster vaccination against COVID-19. Participants were aged older than 30 years, and were at least 70 days post two doses of ChAd or at least 84 days post two doses of BNT primary COVID-19 immunisation course, with no history of laboratory-confirmed SARS-CoV-2 infection. 18 sites were split into three groups (A, B, and C). Within each site group (A, B, or C), participants were randomly assigned to an experimental vaccine or control. Group A received NVX-CoV2373 (Novavax; hereafter referred to as NVX), a half dose of NVX, ChAd, or quadrivalent meningococcal conjugate vaccine (MenACWY) control (1:1:1:1). Group B received BNT, VLA2001 (Valneva; hereafter referred to as VLA), a half dose of VLA, Ad26.COV2.S (Janssen; hereafter referred to as Ad26) or MenACWY (1:1:1:1:1). Group C received mRNA1273 (Moderna; hereafter referred to as m1273), CVnCov (CureVac; hereafter referred to as CVn), a half dose of BNT, or MenACWY (1:1:1:1). Participants and all investigatory staff were blinded to treatment allocation. Coprimary outcomes were safety and reactogenicity and immunogenicity of anti-spike IgG measured by ELISA. The primary analysis for immunogenicity was on a modified intention-to-treat basis; safety and reactogenicity were assessed in the intention-to-treat population. Secondary outcomes included assessment of viral neutralisation and cellular responses. This trial is registered with ISRCTN, number 73765130. Findings: Between June 1 and June 30, 2021, 3498 people were screened. 2878 participants met eligibility criteria and received COVID-19 vaccine or control. The median ages of ChAd/ChAd-primed participants were 53 years (IQR 44–61) in the younger age group and 76 years (73–78) in the older age group. In the BNT/BNT-primed participants, the median ages were 51 years (41–59) in the younger age group and 78 years (75–82) in the older age group. In the ChAd/ChAD-primed group, 676 (46·7%) participants were female and 1380 (95·4%) were White, and in the BNT/BNT-primed group 770 (53·6%) participants were female and 1321 (91·9%) were White. Three vaccines showed overall increased reactogenicity: m1273 after ChAd/ChAd or BNT/BNT; and ChAd and Ad26 after BNT/BNT. For ChAd/ChAd-primed individuals, spike IgG geometric mean ratios (GMRs) between study vaccines and controls ranged from 1·8 (99% CI 1·5–2·3) in the half VLA group to 32·3 (24·8–42·0) in the m1273 group. GMRs for wild-type cellular responses compared with controls ranged from 1·1 (95% CI 0·7–1·6) for ChAd to 3·6 (2·4–5·5) for m1273. For BNT/BNT-primed individuals, spike IgG GMRs ranged from 1·3 (99% CI 1·0–1·5) in the half VLA group to 11·5 (9·4–14·1) in the m1273 group. GMRs for wild-type cellular responses compared with controls ranged from 1·0 (95% CI 0·7–1·6) for half VLA to 4·7 (3·1–7·1) for m1273. The results were similar between those aged 30–69 years and those aged 70 years and older. Fatigue and pain were the most common solicited local and systemic adverse events, experienced more in people aged 30–69 years than those aged 70 years or older. Serious adverse events were uncommon, similar in active vaccine and control groups. In total, there were 24 serious adverse events: five in the control group (two in control group A, three in control group B, and zero in control group C), two in Ad26, five in VLA, one in VLA-half, one in BNT, two in BNT-half, two in ChAd, one in CVn, two in NVX, two in NVX-half, and one in m1273. Interpretation: All study vaccines boosted antibody and neutralising responses after ChAd/ChAd initial course and all except one after BNT/BNT, with no safety concerns. Substantial differences in humoral and cellular responses, and vaccine availability will influence policy choices for booster vaccination. Funding: UK Vaccine Taskforce and National Institute for Health Research