Hepatic microsporidiosis of juvenile grey mullet, Chelon labrosus (Risso), due to Microgemma hepaticus gen.nov. sp.nov.

Abstract. Microgemma hepaticus gen.nov. sp.nov. is described from the liver of juvenile grey mullet, Chelon labrosus (Risso). Development occurs within xenomas (diameter 500μm) which have microvillar surfaces, encircling bands of mitochondria and a reticulate hypertrophic nucleus. Vegetative developmental stages, meronts, are plasmodial and divided by plasmotomy. These stages are enclosed by host membranes. Sporogonic stages are free in the cytoplasm and divide by multiple exogenous budding. Uninucleate spores (2·4μm × 4·2μm) possess 7–9 coils of the polar filament and a lamellar polaroplast. Xenomas are associated with liver connective tissue, and cause necrosis of adjacent liver cells in certain circumstances. Host response to infection involves leucocyte infiltration and granuloma formation, with spores being destroyed by repeated macrophage phagocytosis and necrosis and resulting in gradual resolution of the lesion. Although juveniles apparently tolerate large parasite burdens there is some evidence of a contribution by the parasite to stress-related mortality. The transmission of the disease and its potentially high pathogenicity to larval fishes is discussed.     

Tissue reaction of the three-spined stickleback Gasterosteus aculeatus L. to infection with Glugea anomala (Moniez, 1887)

Abstract. Studies on an experimental infection of the stickleback (Gasterosteus aculeatus L.) with the microsporidian Glugea anomala conducted until day 126 post infection confirmed that xenoparasitic formations typical of the intracellular development of this microsporidian were responsible for a marked tissue reaction in the host. After a single oral infection, xenoparasitic formations developed in the subepithelial connective tissue of the glandular part of the stomach. The course of xenoma development was asynchronous. Up to the 'cyst' stage (xenomas filled with mature spores), their growth and development caused a pressure atrophy in the surrounding tissue. Later, the response to the presence of the xenoparasitic formation was the development of a granulomatous inflammatory reaction. Locally, the host responded to the infection by phagocytosis of mature spores by locally derived macrophages. In massive infections, changes occurred in the subepithelial connective tissue of the glandular part of the stomach (often the complete disappearance of tubular glands), which may result in a permanent influence on the function of this organ.     

Macrophages and giant cells associated with a microsporidian parasite causing liquefaction of the skeletal muscle of the Norway pout, Trisopterus esmarkii (Nilsson)

Abstract. A mature microsporidian infection of the skeletal muscles of Norway pout, Trisopterus esmarkii Nilsson, is described. Infected fish had noticeable liquefaction of the muscle. The white foci of infection contained packed microsporidian spores, and the surrounding infected muscle fibres were degenerate. The host response involved invasion by phagocytic cells and encapsulation by fibroblasts, forming large granulomas. Macrophages, 17–20 μm in diameter, were actively ingesting the spores and typically had a foamy cytoplasm. Some had formed Langhans-type giant cells, 35 μm in diameter, with peripherally arranged nuclei. Sporulation was complete and appeared to have occurred within sporo- phorous vacuoles in which numerous spores were formed. Individual spores measured 2·8 × 1·5 μm and were characterized by irregular electron dense areas, a short polar filament with four coils and a large posterior vacuole with three inclusions.     

Ultrastructure and development of Pleistophora senegalensis sp. nov. (Protozoa, Microspora) from the gilt-head sea bream, Sparus aurata L. (Teleost, Sparidae) from the coast of Senegal

Abstract. The microsporidian Pleistophora senegalensis sp. nov. parasitizes the gilt-head sea bream, Sparus aurata L, The parasite is found in the intestinal wall where it forms small xenomas in the muscularis. The development cycle of this species is described by light and electron microscopy. Meronts are rounded plasmodia dividing by plasmotomy and bounded by an amorphous and very regular wall. At the onset of sporogony, sporophorous vesicles are formed by separation of the plasma membrane from the external wall which then becomes a characteristic mesh. Mature spores (4.45 × 2.37μm) are ovoid and slightly pyriform with a large posterior vacuole.     

Loma salmonae-associated xenoma onset and clearance in rainbow trout, Oncorhynchus mykiss (Walbaum): comparisons of per os and cohabitation exposure using survival analysis

Survival analysis techniques were used to compare experimental exposure methods of Loma salmonae (Microspora) in rainbow trout (RBT) by measuring xenoma onset and clearance time. Twenty‐eight naive RBT were exposed per os (fed L. salmonae spores) and 28 RBT were exposed by cohabitation with 28 L. salmonae‐infected RBT. Exposed fish were examined once every week (7 days) post exposure (PE). For xenoma onset, the median survival time, the time to first appearance of branchial xenomas, was 6 weeks PE for both per os and cohabitation exposure. For xenoma clearance, the median survival time, the time to total clearance of branchial xenomas, was 10 weeks for per os exposure and 12 weeks for cohabitation exposure. There was a significant difference between the survival curves of per os fish compared with cohabited fish for both xenoma onset and xenoma clearance. The incidence rate of xenoma development was greater for per os exposure (0.1745 cases per fish‐week) compared with cohabitation exposure (0.1342 cases per fish‐week). Similarly, the rate of xenoma clearance was greater for per os exposure (0.1028 cases per fish‐week) compared with cohabitation exposure (0.0885 cases per fish‐week). Differences between exposure methods were attributed to differences in the frequency and duration of exposure to spores. Survival analysis was useful for examining the onset and clearance of L. salmonae and may have applications for understanding disease dynamics in aquaculture.     

Loma salmonae-associated growth rate suppression in rainbow trout, Oncorhynchus mykiss (Walbaum), occurs during early onset xenoma dissolution as determined by in situ hybridization and immunohistochemistry

Experimental infection of rainbow trout, Oncorhynchus mykiss (Walbaum), juveniles with Loma salmonae at a water temperature of 15 °C yielded detectable parasite DNA within the gills by week 2 post-exposure (PE) and detectable spore-wall antigen within developing xenomas by week 3 PE, as determined by in situ hybridization and monoclonal antibody (Mab) based immunohistochemistry, respectively. The microsporidian was most commonly located within endothelial cells of lamellar basal channels. Whereas the onset of xenoma formation appeared to be relatively synchronous, as expected from previous studies, xenoma dissolution followed an unexpected biphasic pattern with peaks at weeks 4 and 9 PE. The onset of significant growth rate suppression, at week 4 PE in exposed fish, was temporally associated with the appearance of gill lesions which, in turn, were centred about sites of premature xenoma dissolution. The latter was determined by the detection of spore-wall antigen within lesions. Co-habitant control fish began developing xenomas by week 10, indicating the infective potential of those spores released from the principal fish during early xenoma dissolution. Although infection with L. salmonae significantly affects fish growth rates, the time-course of this suppression is limited, and as an unexpected finding, growth rate recovery commences prior to the infection’s resolution.     

Evaluating protection against Loma salmonae generated from primary exposure of rainbow trout, Oncorhynchus mykiss (Walbaum), outside of the xenoma-expression temperature boundaries

A series of challenge and re-challenge studies was conducted in which juvenile rainbow trout were exposed to the pathogen Loma salmonae , a microsporidian which typically causes xenoma formation during sporogony and inflammation in the gills as the xenomas undergo dissolution. The specific goal was to determine if a primary exposure, conducted at a water temperature outside of the range which permits the parasite to undergo sporogony and form branchial xenomas, would stimulate a protective response in the fish to a later challenge conducted under temperature conditions optimal for the parasite. Primary challenge of fish to L. salmonae at 7 °C or 21 °C blocked or limited xenoma formation, as discussed in a previous study. However, these fish had a relative percentage protection (RPP) against a second optimized exposure which matched, or was not significantly less than, the degree of protection (100%) that developed in other groups of fish that received a primary exposure throughout the range of water temperatures which permits xenoma formation. When the primary exposure was conducted at 5 °C, the RPP against the second exposure was adversely affected and declined to 61%. These findings have application to the control of L. salmonae within aquaculture, in that it may be possible to expose hatchery stocks of susceptible salmonid species to spores of L. salmonae when hatchery water temperature is at 7 °C. At this temperature, the risks of disease stemming from this primary exposure appear minimal, since xenomas fail to form. However, the degree of protection appears promising, and may be sufficient to protect fish from spore exposure occurring at netpen marine sites where the parasite may be endemic.     

Whole body net ion fluxes, plasma electrolyte concentrations and haematology during a Loma salmonae infection in juvenile rainbow trout, Oncorhynchus mykiss (Walbaum)

Loma salmonae infections of salmonids culminate in the development of branchial xenomas and subsequent focal hyperplasia of the lamellar or filament epithelium following xenoma rupture and spore release. The effects of this acute branchial disruption upon net ionic flux rates and plasma electrolyte concentrations were determined in juvenile rainbow trout given an experimental oral exposure to L. salmonae. Mean numbers of branchial xenomas peaked at week 5 post-exposure (PE), which coincided with a reduction in the specific growth rate, although there were no significant differences in mass, length or condition of Loma-exposed fish compared with unexposed controls. Following exposure, negative net whole body Na(+) and K(+) fluxes decreased, whereas net Cl(-) fluxes remained unchanged compared with non-exposed control fish. At week 3 PE during the initial branchial xenoma formation stage, there was a significant negative whole body net K(+) flux in Loma-exposed trout compared with other points during the exposure and subsequent infection. Additionally, Loma-exposed fish had marginally elevated plasma Na(+) and Cl(-) concentrations, whilst K(+) levels remained unchanged, compared with control fish. Although there was a progressive decrease in leucocrit, haematocrit remained unchanged over the course of the Loma exposure and subsequent infection. These results suggest that ionic compensation can occur at the gills during the development of xenomas during exposure to L. salmonae and the resultant infection, therefore allowing defence of plasma electrolyte concentrations, unlike the acute ionic disturbances seen with some other parasitic diseases.     

Spraguea (Microsporida: Spraguidae) infections in the nervous system of the Japanese anglerfish, Lophius litulon (Jordan), with comments on transmission routes and host pathology

Anglerfish from the genus Lophius are a globally important commercial fishery. The microsporidian Spraguea infects the nervous system of these fish resulting in the formation of large, visible parasitic xenomas. Lophius litulon from Japan were investigated to evaluate the intensity and distribution of Spraguea xenomas throughout the nervous system and to assess pathogenicity to the host and possible transmission routes of the parasite. Spraguea infections in L. litulon had a high prevalence; all fish over 403 mm in standard length being infected, with larger fish usually more heavily infected than smaller fish. Seventy percent of all fish examined had some gross visible sign of infection. The initial site of development is the supramedullary cells on the dorsal surface of the medulla oblongata, where all infected fish have parasitic xenomas. As the disease progresses, a number of secondary sites typically become infected such as the spinal, trigeminal and vagus nerves. Fish with infection in the vagus nerve bundles often have simultaneous sites of infection, in particular the spinal nerves and along the ventral nerve towards the urinary bladder. Advanced vagus nerve infections sometimes form xenomas adjacent to kidney tissue. Spraguea DNA was amplified from the contents of the urinary bladders of two fish, suggesting that microsporidian spores may be excreted in the urine. We conclude that supramedullary cells on the hindbrain are the primary site of infection, which is probably initiated at the cutaneous mucous glands where supramedullary cells are known to extend their peripheral axons. The prevalence of Spraguea infections in L. litulon was very high, and infections often extremely heavy; however, no associated pathogenicity was observed, and heavily infected fish were otherwise normal.     

Morphological characterization and notes on the life cycle of a newly discovered variant of Loma salmonae (Putz, Hoffman & Dunbar) from a natural infection of chinook salmon, Oncorhynchus tshawytscha (Walbaum)

Two variants of Loma salmonae occur in net-pen reared chinook salmon, Oncorhynchus tshawytscha. The typical variant (OA) has a host specificity for salmonids of the genus Oncorhynchus whereas the atypical variant (SV) has a host specificity for brook trout, Salvelinus fontinalis, and in this study, the ultrastructure of the two are compared. In fish at 8 weeks post-exposure xenomas of the SV variant have a very high proportion of mature spores compared with other developmental stages, while in xenomas of the OA variant there are fewer spores and many other developmental stages. Spores of the SV variant had up to 20 turns of their polar tube whereas those of the OA variant only had 17. Furthermore, the spores of the SV variant were significantly larger than those of the OA variant. The sporophorous vesicle for both variants appears to form around a sporogonial plasmodia, which results in many spores developing within the vesicle.     

Loma salmonae (Protozoa: Microspora) infections in seawater reared coho salmon Oncorhynchus kisutch

Loma salmonae (Putz et al., 1965) infections were observed in five groups of coho salmon, Oncorhynchus kisutch, reared in seawater net-pens in Washington State, U.S.A. in 1984–1986. Ultrastructural characteristics, size of spores, tissues and host infected, and geographical location identified the microsporidium as Loma salmonae. Preserved spores measured 4.4×2.3 (4–5.6×2–2.4) μm and exhibited 14–17 turns of the polar filament. Infections were evident in the gills of some fish before seawater entry, but few parasites were observed and they caused little tissue damage. Infections observed in fish after transfer to seawater were associated with significant pathological changes in the gills. A mixed inflammatory infiltrate was associated with ruptured microsporidian xenomas within the vessels and interstitium of the primary lamellae. Microsporidian spores were dispersed throughout the lesions and were often seen inside phagocytes. The parasite was also observed in the heart, spleen, kidney and pseudobranchs; however, the inflammatory lesions were common only in the heart.

Monthly examination of fish after transfer to seawater showed peak prevalences (33–65%) of gill infections during the summer. Although moribund fish were often infected with other pathogens, the high prevalence of L. salmonae infections and the severity of the lesions it caused, suggested that this parasite significantly contributed to the recurrent summer mortalities observed at this net-pen site.     

An ultrastructural study of Myxobolus exlguus Thelohan, 1895 (Myxosporea) from grey mullet, Crenimugil labrosus (Risso)

Abstract. An ultrastructural study of Myxobolus exiguus Thelohan, 1895 using interlamellar and subdermal plasmodia from thick-lipped grey mullet, Crenimugil labrosus , revealed differences in the structure of the plasmodia and host tissue reaction. Both plasmodia showed structural evidence of pinocytotic activity, but the surface area of subdermal plasmodia was considerably increased by digitiform extensions of the ectoplasm. A filamentous glycocalyx was a feature of the surface membrane of plasmodia, being more distinct on the interlamellar form. Sporo-genesis involved two morphologically distinct types of generative cells, producing a disporous pansporoblast. Mature spores were oval, measuring 9-0 μ m×7.5 μ m. Sporogenesis and capsulogenesis were essentially similar to other myxosporea. In the juvenile fish examined the host tissue reaction was slight, involving hyperplasia of epithelial cells associated with interlamellar plasmodia and some collagen deposition and localized melanization associated with subdermal plasmodia.     

Induced resistance in rainbow trout, Oncorhynchus mykiss (Walbaum), to gill disease associated with the microsporidian gill parasite Loma salmonae

Branchial xenomas were detected by week 5 and disappeared by week 10 after naive juvenile rainbow trout, held at 14.5 °C, were fed or intubated with Loma salmonae ‐infected gill tissue preparations. Upon re‐challenge with L. salmonae , these fish were protected from disease and branchial xenomas did not develop. Branchial xenomas were never detected in naive fish held at 10 °C and exposed to L. salmonae . When these fish were re‐challenged with L. salmonae at 14.5 °C, they were also protected from the disease. Branchial xenomas also developed after naive fish, held at 14.5 °C, were injected intraperitoneally with a semipurified preparation of fresh spores, but generally did not develop after intraperitoneal injection with a preparation of spores subjected to freezing and thawing before use. Fish that had received fresh spores intraperitoneally were completely resistant to disease when re‐challenged via oral delivery of spores, whereas those that had received frozen spores were incompletely, but significantly, protected from disease compared with naive fish. We conclude that infection with L. salmonae induces strong protection towards the disease upon re‐exposure to spores, and that the protection does not depend on the completion of the parasite's life cycle, thus establishing the basis for further research on vaccine development for this disease.     

Interaction of water temperature and challenge model on xenoma development rates for Loma salmonae (Microspora) in rainbow trout, Oncorhynchus mykiss (Walbaum)

This study evaluated the regulatory effects of water temperature on the development of branchial xenomas caused by Loma salmonae using a high-dose per os-challenge model compared with a low-dose cohabitation-challenge model. Approximately 275 juvenile rainbow trout (RBT), Oncorhynchus mykiss, were randomly distributed to six tanks with two tanks each maintained at 11, 15 and 19 degrees C. Fish in one tank from each temperature setting were exposed per os to macerated L. salmonae-infected gill material and fish in the other tank from each temperature setting were exposed to L. salmonae using the cohabitation-challenge model. Fish were monitored for the development of branchial xenomas beginning at day 21 post-exposure. Survival analyses were used to evaluate the effect of water temperature and challenge model on the number of days until the first visible branchial xenoma was detected. The survivor curves for the per os-challenge model revealed that there was at least one significant difference, whereas the cohabitation challenge did not reveal any significant differences amongst the temperature settings. The proportional hazards model revealed a significant interaction between the challenge model used and water temperature. This indicated that the effect of water temperature was different depending on challenge model. Additionally, from the mean xenoma intensities, on average, the per os-challenged fish showed higher xenoma intensity compared with the cohabitation-challenged fish. Overall, the impact of water temperature on disease pathogenesis was greater when the RBT were per os challenged compared with using the cohabitation model.     

Effect of water temperature manipulation on a thermal unit predictive model for Loma salmonae

Loma salmonae is an important microsporidian pathogen affecting the mariculture of Pacific salmon, Oncorhynchus tshawytscha (Walbaum). Clinical signs associated with infection arise when the parasite enters sporogony and forms xenoparasitic complexes (xenomas) within the gill. The present study tested a thermal unit (TU) model, which was devised to predict the timing of xenoma formation, under conditions in which water temperature changed during the course of infection. In vivo trials with juvenile trout showed that the model TU for xenoma onset= (days post exposure) (C above 7C) accurately predicted the onset time for xenomas when fish were moved from either 11 or 5C to 15C at various intervals after exposure. These findings suggest that the TU model may allow aquaculturists to predict disease onset. However, the model failed when fish were moved from 15 to 5C at intervals after exposure. This finding suggests that the temperature-constrained phase of the life-cycle of L. salmonae occurs early on after the spore is ingested by the fish.     

A comparative study of the tissue response to invasion and encystment by Stephanochasmus baccatus (Nicoll, 1907) (Digenea: Acanthocolpidae) in four species of flatfish

Abstract. Four species of flatfish [plaice, Pleuronectes platessa L.; turbot, Scophthalmus maximus (L.); Dover sole, Solea solea (L.) and the common dab, Limanda limanda (L.)] were experimentally infected with cercariae of Stephanochasmus baccatus under similar conditions and examined at intervals up to 7 weeks post-infection- The tissue response was found to be qualitatively the same in all four hosts and was consistent with a chronic granulomatous inflammatory reaction induced by resistant particulate foreign bodies. The intensity and rate of the response varied between host species but the capsule formation was essentially similar in all. The mature capsule consisted of an inner layer composed of leucocytes which underwent epithelioid transformation. The cellular structure of this tissue gradually degenerated from the oldest layer to form a dense eosinophilic ground substance in which was embedded nuclear debris. The outer layer of the capsule consisted of fibroblasts and reticulin collagen fibres. Muscle and fin cysts differed, with muscle cysts tending to be less discrete and more fibrous. The rate and intensity of the response to the parasite varied between different host species. Plaice and turbot showed the most intensive response and in turbot the reaction was considerably accelerated. Mortality of encysted metacercariae was only seen in turbot. The overall effect on larger 1-group fish was minimal, but the smaller 0-group plaice were more severely affected by the relatively large cvsts that interfered with organ function.     

Tissue reactions to microsporidian infections in fish

Abstract. Histopathological studies on natural and experimental infections of nineteen microsporidian species from fishes distinguished two types of tissue reactions.
The first type is characteristic of infections with xenoma-inducing microsporidian species and comprises three successive stages: a weakly reactive stage, a productive stage with the formation of granulomas and a stage of granuloma involution. Following the first stage, tissue reactions are directed towards the isolation of the parasite and result in its complete elimination and host tissue repair. The extent of pathological changes probably depends on the number of parasite cells which initiated the infection.
The second type is represented by Pleistophora species infecting muscles or oocytes. Host tissue reaction is surprisingly slight during the schizogony and sporogony and does not tend to isolate the invaded muscle fibres. A slight lymphocytic infiltration of myosepta indicates the first stage of tissue reaction. The tissue reaction only reaches the productive stage when mature spores completely fill the contents of the infected muscle fibre. A thick wall of fibroblasts may be formed to demarcate the parasite mass as soon as it undergoes necrotic changes. The extent of pathological changes probably depends on the ability of early developmental stages of the parasite to spread the infection within the host.
In both types of tissue reaction, the spores are destroyed by complete digestion within host phagocytic cells.     

Undulatory fish swimming: from muscles to flow

Undulatory swimming is employed by many fish for routine swimming and extended sprints. In this biomechanical review, we address two questions: (i) how the fish's axial muscles power swimming; and (ii) how the fish's body and fins generate thrust. Fish have adapted the morphology of their axial musculature for high power output and efficiency. All but the superficial muscle fibres are arranged along curved trajectories, and the myomeres form nested cones. Two conflicting performance goals shape the fibre trajectories of the axial muscles. Maximum power output requires that all fibres contract uniformly. In a bending fish, uniform contraction in a single myomere can be ensured by curved fibre trajectories. However, uniform strain is only desirable if all muscle fibres have the same contractile properties. The fish needs several muscle‐fibre types that generate maximum power at different contraction speeds to ensure effective muscle power generation across a range of swimming speeds. Consequently, these different muscle‐fibre types are better served by non‐uniform contractions. High power output at a range of swimming speeds requires that muscle fibres with the same contractile properties contract uniformly. The ensuing helical fibre trajectories require cone‐shaped myomeres to reduce wasteful internal deformation of the entire muscle when it contracts. It can be shown that the cone‐shaped myomeres of fish can be explained by two design criteria: uniform contraction (uniform strain hypothesis) and minimal internal deformation (mechanical stability hypothesis). So far, only the latter hypothesis has found strong support. The contracting muscle causes the fish body to undulate. These body undulations interact with the surrounding water to generate thrust. The resulting flow behind the swimming fish forms vortex rings, whose arrangement reflects the fish's swimming performance. Anguilliform swimmers shed individual vortex rings during steady swimming. Carangiform swimmers shed a connected chain of vortex rings. The currently available sections through the total flow fields are often not an honest representation of the total momentum in the water – the wake of carangiform swimmers shows a net backward momentum without the fish accelerating – suggesting that our current picture of the generated flow is incomplete. To accelerate, undulatory swimmers decrease the angle of the vortex rings with the mean path of motion, which is consistent with an increased rate of backward momentum transfer. Carangiform swimmers also enlarge their vortex rings to accelerate and to swim at a higher speed, while eel, which are anguilliform swimmers, shed stronger vortex rings.     

Myxosporean infection causing intestinal disease in farmed turbot, Scophthalmus maximus (L.), (Teleostei: Scophthalmidae)

The present report describes an intestinal disease which causes important losses in farmed turbot. Mortality rates were higher in summer and reached 100% in all tanks where the disease was confirmed. Affected fish showed external signs consisting of anorexia, sunken eyes and a typical prominent bony ridge on the skull. These signs can be considered the pathognomonic signs of the disease, together with the gut lesions seen in the histological study. Pallor of the internal organs, intestinal haemorrhages and the presence of liquid in the intestine were also observed, with ascites in heavily infected fish. Histopathological damage was evident in the gut, with severe enteritis, detachment of epithelium, haemorrhages and inflammation of the subepithelial connective tissue. The myxosporean aetiology was demonstrated in all the fish showing the characteristic signs of disease. Myxosporean stages, including scarce spores, were found in the affected epithelium or free in the intestinal lumen together with epithelial debris. The present study demonstrates the importance of this myxosporean disease which represents a serious threat for turbot culture. This is also the first record of a member of the Myxosporea in turbot.