Light and electron microscope observations on extrasporogonic and sporogonic stages of a myxosporean parasite of the genus Sphaerospora Thelohan, 1892 from Atlantic salmon, Salmo solar L., in Scotland

Abstract. Juvenile Atlantic salmon from a number of freshwater hatcheries in Scotland were found to be infected by a myxosporean parasite of the genus Sphaerospora. Fish first became infected in June by extrasporogonic stages which could be found in the blood and kidney interstitium. These consisted of a primary cell containing one to over one hundred secondary cells. Some secondary cells contained one or two tertiary cells. Sporogony was disporous and occurred later in the kidney tubules. The development of both extrasporogonic and sporogonic stages was studied by light and transmission electron microscopy.     

Infectivity of internal tissues of Atlantic salmon, Salmo salar L., experimentally infected with the aetiological agent of infectious salmon anaemia (ISA)

Abstract. Infectivity of internal organs and cells of Atlantic salmon, Salmo salar L., was studied at various times after infection with the aetiological agent of infectious salmon anaemia (ISA). Experimentally infected salmon smolts developed anaemia just prior to the onset of ISA-mortality. ISA-infectivity of preparations made from liver, kidney, spleen, plasma, red blood cells (RBC) and head kidney leucocytes was determined by inoculating Atlantic salmon parr with the respective preparations. ISA-mortality was observed after inoculation of salmon parr with preparations of kidney and liver, and to a minor degree, with spleen and a fraction of head kidney leucocytes (WBC1) collected 7 days post-infection. At 11 days post-infection, the infectivity of these preparations increased and ISA-infectivity was also observed with a second fraction of head kidney leucocytes (WBC2), red blood cells (RBC) and blood plasma. At this time, the ISA-infectivity of kidney was not significantly higher (P > 0.05) than that of liver but was significantly higher than all other preparations as judged by Cox-regression analysis. At days 14 and 18 post-infection, the infectivity of kidney was significantly higher (P < 0.05) than that of liver, but not at 21 and 25 days post-infection. Generally, the ISA-infectivity of kidney was higher than spleen, head kidney leucocytes (WBC1 and WBC2), RBC and plasma, although the difference was not significant at all time points. For example, at day 25 post-infection, the infectivity of kidney was only significantly higher than that of spleen and plasma. On a per gram basis, head kidney leucocytes proved to contain higher amounts of infectious matter than RBC. ISA-infective leucocytes present in the kidney tissue may have contributed to a major part of the infectivity recognized in the kidney preparations. Thus, head kidney leucocytes and other kidney leucocytes also may be considered to be among the most important target cells of the aetiological agent of ISA.     

Mass mortality associated with a Sphaerospora-like myxosporidean infestation in juvenile cobia, Rachycentron canadum (L.), marine cage cultured in Taiwan

Cultured cobia, Rachycentron canadum , of 45–80 g exhibited anaemia and ascites, and a mottled red and grey, extremely enlarged kidney with cream-coloured patches or spherical nodules. Cumulative mortality was about 90% within 1 month. Extrasporogonic or sporogonic stages of a myxosporean appeared in the blood, glomerulus, renal tubules and renal interstitium. The renal tubules were the main target tissue of the parasite and were completely occluded by sporogonic pseudoplasmodia at various degrees of maturity. Many sporogonic stages were attached to the brush border of the epithelium of the renal tubules. Mature spores were seen in the lumen of the tubules. They were elongated or spherical with numerous refractile granules in the cytoplasm. The polar filament formed 3–5 coils. No bacteria or viruses were isolated from the diseased fish. Based on the results of microbiological, histopathological and electron microscopical examinations, the cobia disease was believed to be caused by a Sphaerospora -like myxosporean. This is the first report of a myxosporean in cobia in aquaculture.     

Detection of the intranuclear microsporidian Enterospora nucleophila in gilthead sea bream by in situ hybridization

Enterospora nucleophila is an intranuclear microsporidian responsible for emaciative microsporidiosis of gilthead sea bream (GSB). Its minute size and cryptic nature make it easily misdiagnosed. An in situ hybridization (ISH) technique based on antisense oligonucleotide probes specific for the parasite was developed and used in clinically infected GSB in combination with calcofluor white stain (CW) and other histopathological techniques. The ISH method was found to label very conspicuously the cells containing parasite stages, with the signal concentrating in merogonial and sporogonial plasmodia within the infected cell nuclei. Comparison with CW demonstrated limited ISH signal in cells containing mature spores, which was attributed mostly to the scarcity of probe targets present in these stages. Although spores were detected in other organs of the digestive system as well as in the peripheral blood, proliferative stages or parasite reservoirs were not found in this work outside the intestines. The study demonstrated a frequent disassociation between the presence of abundant spores and the intensity of the infections as determined by the parasite activity. The ISH allows confirmatory diagnosis of GSB microsporidiosis and estimation of infection intensity and will be a valuable tool for a more precise determination of parasite dissemination pathways and pathogeny mechanisms.     

Molecular diagnosis of Myxobolus spirosulcatus associated with encephalomyelitis of cultured yellowtail, Seriola quinqueradiata Temminck & Schlegel

Mass mortality of cultured yellowtail, Seriola quinqueradiata, has recently been reported from fish farms in western Japan. Previous studies revealed that diseased fish were characterized by encephalomyelitis and presporogonic stages of a myxosporean‐like parasite in the spinal cord. However, the parasite has remained unidentified because of the lack of mature stages being present. Thus, in the present study, analysis of the small subunit ribosomal DNA (18S rDNA) of the parasite as well as in situ hybridization (ISH) studies using histological sections of the infected tissue was conducted. The 18S rDNA of the myxosporean had higher sequence similarities with those of bile‐duct‐infecting myxosporeans rather than those infecting nervous tissues and was identified as Myxobolus spirosulcatus. The ISH using specific probes demonstrated that the DNA amplified was derived from the multinuclear organisms found in histological sections. A highly sensitive and specific PCR‐based assay for M. spirosulcatus was developed, which revealed a high prevalence of infection in cultured yellowtail that exhibited the clinical signs of encephalomyelitis.     

Experimental identification of the actinosporean stage of Sphaerospora renicola Dykova & Lom 1982 (Myxosporea: Sphaerosporidae) in oligochaete alternate hosts

The extrapiscine development of Sphaerospora renicola, a myxosporean parasite of the kidney of common carp, Cyprinus carpio L., was studied in the experimentally infected oligochaetes Tubifex tubifex (Müller) and Branchiura sowerbyi (Beddard). After the infection of these tubificids with homogenized common carp kidneys containing myxospores of S. renicola, the development of actinosporean stages was first observed under light microscopy 8 days after infection in pathogen-free T. tubifex. Infection of B. sowerbyi with mature actinosporean stages was first observed 91 days after infection. At that stage of development, pansporocysts containing neoactinospores filled the intestinal epithelium of the worm. Ninety-five days after infection, pansporocysts containing actinospores and free actinospores were found in the gut lumen of B. sowerbyi. Actinospores of S. renicola emerged from B. sowerbyi after 98 days of intraoligochaete development. These were floating in the water and showed the typical form of neoactinospores. The shape of the spores was triangular in apical view and elliptical in lateral view. The prevalence of infection reached 37%. Control specimens of B. sowerbyi proved to be free of neoactinospores. Except for a single specimen of B. sowerbyi, the only early developmental stages (pansporocysts) were found in T. tubifex.     

Duration and method of fixation affects the sensitivity of a digoxygenin-labelled DNA probe in detecting Kudoa thyrsites in Atlantic salmon skeletal muscle

The effect of fixative and duration of fixation on the sensitivity of a non-radioactive in situ hybridisation (ISH) protocol to detect Kudoa thyrsites small subunit ribosomal deoxyribonucleic acid (DNA) was investigated. Strong ISH reactions were detected in 5-μm sections of paraffin-embedded Atlantic salmon muscle after fixation for 1 day in Davidson's solution (DS). Reactions were weak following 3 or 5 days fixation and absent after 17 or 28 days fixation. Strong ISH reactions were observed after 1, 3 or 5 days fixation in neutral buffered formalin (NBF). The reactions were weak after 17 days and weak to nonexistent after 28 days of fixation. Reactions were consistently strong after fixation in 95% ethanol for up to 28 days. Some mature spores reacted weakly or not at all by ISH. Parasite DNA was weakly amplified by polymerase chain reaction (PCR) from paraffin-embedded muscle after 1 day of fixation in DS but not after fixation for 3, 5, 17 or 28 days. Amplified DNA was detected after fixation in NBF for 1, 3 and 5 days, but not after 17 or 28 days. In contrast, PCR consistently amplified DNA from paraffin-embedded, ethanol-fixed muscle. Caution should be used in the choice of fixative and duration of fixation when preserving Atlantic salmon tissues for molecular diagnosis of K. thyrsites.     

Salmonid whirling disease: myxosporean and actinosporean stages cross-react in direct fluorescent antibody test

Abstract. Serologic relatedness of the two life stages of the salmonid whirling disease parasite Myxosoma cerebralis Hofer, 1903 — myxosporean spores from fish cartilage and actinosporean triactinomyxon spores from aquatic tubificids — were investigated. When the direct fluorescent antibody technique was used, anti-triactinomyxon and anti- M. cerebralis rabbit sera conjugated with fluorescein isothiocyanate cross-reacted with the respective heterologous life stage. Both stages showed similar locations of specific fluorescence with conjugates of either homologous or heterologous serum. Thus, serology supports the relatedness of the myxosporean M. cerebralis and the actinosporean triactinomyxon stages.     

Experimental mycobacteriosis in Atlantic cod, Gadus morhua L

Piscine mycobacteriosis causes losses in a number of fish species both in the wild and in aquaculture worldwide. Mycobacterium salmoniphilum infections have on several occasions been reported in farmed Atlantic salmon, Salmo salar L. The present study tested and confirmed the susceptibility of Atlantic cod, Gadus morhua L., an important yet relatively novel aquaculture species, to infection with M. salmoniphilum. Atlantic cod injected intraperitoneally with a suspension of this bacterium were maintained together with cohabitant (COH) fish in a flow-through marine water system at 10-11 °C. The fish were supervised daily and samples taken at 2, 7, 14, 23, 34 and 53 weeks post-infection and examined pathologically, bacteriologically and using molecular biology. Injected mycobacteria were re-isolated in high concentrations from both injected and COH fish groups. Death attributable to mycobacterial infection was observed in both injected (47%) and COH (28%) fish groups. Extensive development of granuloma in visceral organs, mainly the mesenteries, spleen, kidney and liver (lesser extent) and at later stages of the infection in heart tissues and gills, was observed in both injected and COH fish. Granulomas underwent a temporal progression of distinct morphological stages, culminating in well-circumscribed lesions surrounded by normal or healing tissue. Acid-fast bacilli were detected in both granulomas and non-granulomatous tissues. This study confirms that Atlantic cod is highly susceptible to M. salmoniphilum infection and that this bacterial species may be a threat to cod both in the wild and in the aquaculture.     

Lectin histochemical studies on Sphaerospora sp. (Myxosporea) from Italian brown trout, Salmo trutta L.

A Sphaerospora sp. (Myxosporea) infection (presumably S. truttae ) was identified on a trout farm in northeastern Italy. Parasites were detected in kidneys from infected brown trout, Salmo trutta L., over a 2-year period. Extrasporogonic, sporogonic stages and mature spores were simultaneously detected in the same fish. Traditional diagnostic methods for Sphaerospora spp. rely on the detection of the myxosporean developmental stages in Giemsa-stained kidney smears or haematoxylin-eosin stained tissue sections. A histochemical method was employed where 10 biotinylated lectins (Con-A, DBA, SBA, GS-I, PHA-P, LEA, PWM, RCA₁, WGA and UEA-I) and the avidin-biotin-peroxidase complex (ABC) were used on Sphaerospora -infected brown trout renal tissues and kidney imprints. Five monoclonal antibodies against PKX (Mab12, MabA3, MabC5, MabD4 and MabB4) were also tested. A lectin glycoconjugate binding pattern for Sphaerospora spp. is presented. This staining method shows that SBA lectin ( Glycine max agglutinin) is a useful tool for the detection of the Sphaerospora spp. Only MabB4 bound some of the most mature sporogonic stages. In contrast Mabs12, A3, C5 and D4, and GS-I lectin ( Griffonia simplicifolia agglutinin) did not stain any of the Sphaerospora spp. stages, but did bind very specifically to the sporogonic and extrasporogonic stages of PKX, the causative agent of proliferative kidney disease (PKD).     

Immunohistochemical and PCR studies of wild fish for Tetracapsula bryosalmonae (PKX), the causative organism of proliferative kidney disease

Tetracapsula bryosalmonae, previously referred to as PKX, causes proliferative kidney disease (PKD) in salmonids and is an economically important myxozoan pathogen in salmonid culture. A variety of molecular and immunological tools have been developed to detect the parasite. To determine the specificity of four monoclonal antibodies (MAbs) raised against T. bryosalmonae, archive material of fish infected with various myxosporean species was obtained and immunostained. Wild fish were also collected from enzootic waters and examined for T. bryosalmonae infection using immunohistochemistry and the polymerase chain reaction (PCR). Three of the MAb probes appear to be specific for T. bryosalmonae while only two of the five sets of primers tested appeared to specifically amplify T. bryosalmonae DNA. The results of the immunostaining and the PCR demonstrate that T. bryosalmonae occurs in the tubules of grayling Thymallus thymallus L., brown trout, Salmo trutta L. and Atlantic salmon, Salmo salar L. outside of the PKD season (June‐September) in the UK. This confirms the results of previous studies that these species are the preferred fish hosts for the parasite in the UK.     

Notes on kidney-infecting species of the genus Sphaerospora Thélohan (Myxosporea), including a new species S. gobionis sp.nov., and on myxosporean life cycle stages in the blood of some freshwater fish

Abstract Freshwater fish in Czechoslovakia were examined for species of the genus Sphaerospora Thélohan, 1892 and for myxosporean life cycle stages in the blood. In addition to perch infected with S. pectinacea Bocharova & Donets, 1974, renal tubules of seven host species harboured thus far unidentified Sphaerospora species. A new species, S. gobionis sp.nov. is described from renal tubules of Gobio gobio. In populations of Gobio gobio, Tinea tinea and Rutilus rutilus harbouring infections with different Sphaerospora species, organisms identified as mobile myxosporean life cycle stages were detected in the blood, where they undergo a proliferative cycle. These organisms were reminiscent of stages in the blood of common carp fingerlings, supposedly identical with Sphaerospora renicola Dyková & Lorn. It is possible that the blood stages found in the three cyprinid hosts represent stages of the life cycle of their respective Sphaerospora species. If this is correct, further studies may show if the presence of proliferative stages in the bloodstream is a character distinctive of the genus Sphaerospora.     

Application of in situ detection techniques to determine the systemic condition of lymphocystis disease virus infection in cultured gilt-head seabream, Sparus aurata L

Immunohistochemistry (IHC) and in situ hybridization (ISH) techniques have been used for the detection of lymphocystis disease virus (LCDV) in formalin-fixed, paraffin-embedded tissues from gilt-head seabream, Sparus aurata L. Diseased and recovered fish from the same population were analysed. IHC was performed with a polyclonal antibody against a 60-kDa viral protein. A specific digoxigenin-labelled probe, obtained by PCR amplification of a 270-bp fragment of the gene coding the LCDV major capsid protein, was used for ISH. LCDV was detected in skin dermis and gill lamellae, as well as in several internal organs such as the intestine, liver, spleen and kidney using both techniques. Fibroblasts, hepatocytes and macrophages seem to be target cells for virus replication. The presence of lymphocystis cells in the dermis of the skin and caudal fin, and necrotic changes in the epithelium of proximal renal tubules were the only histological alterations observed in fish showing signs of the disease.     

Epizootiology of Parvicapsulaminibicornis in Fraser River sockeye salmon, Oncorhynchusnerka (Walbaum)

Late-spawning Fraser River sockeye salmon, Oncorhynchus nerka , stocks have suffered significant prespawn mortality associated with an unusually early freshwater migration pattern and the myxosporean parasite Parvicapsula minibicornis . Surveys of migrating adult salmon from several spawning populations were conducted in 1999 and 2000 to determine the extent of infection with P. minibicornis , when and where the parasite first becomes detectable during migration, and whether early migrating stocks might be used as sentinels to assess risk of infection in late-spawning stocks. Posterior kidney, preserved in 95% ethanol, was examined for P. minibicornis in stained histological sections and using a polymerase chain reaction (PCR) test. The prevalence of this parasite in all Fraser River sockeye salmon stocks examined was high (range 47–100% infected). In contrast, P. minibicornis was not detected in the fish tested from the two sockeye salmon stocks outside the Fraser River drainage in either 1999 or 2000. The parasite was also not detected histologically or by PCR in the kidney tissue of the fish from the Fraser River that were sampled in salt water or early during their freshwater migration up the river. These findings and the progression in the prevalence and intensity of infection as the fish from three stocks (early Stuart, Weaver Creek and Cultus Lake) were monitored over time, suggest salmon acquired the parasite either in the lower Strait of Georgia or in the lower Fraser River before the confluence of the Harrison River. In both 1999 and 2000 the parasite was present in all Fraser River sockeye salmon stocks sampled, which suggests that early Stuart salmon may be valuable as a sentinel stock for the presence of the parasite in later-spawning stocks.     

The life cycle of Henneguya nuesslini Schuberg & Schroder, 1905 (Myxozoa) involves a triactinomyxon-type actinospore

The life cycle of the histozoic myxozoan parasite Henneguya nuesslini was investigated in two salmonid host species. Naive brown trout, Salmo trutta, and brook trout, Salvelinus fontinalis, were experimentally infected in two trials by triactinomyxon type actinospores from naturally infected Tubifex tubifex. In exposed common carp, Cyprinus carpio, no myxospore production was detected. The parasite formed cysts with mature myxospores in the connective tissue of the fish 102 days post-exposure. The morphology of both actinosporean and myxosporean stages was described by light microscopy and a 1417-bp fragment of the 18S rDNA gene was sequenced. Sequence analysis confirmed the absolute congruence of the two developmental stages and assisted in determining species identity. Host range, tissue specificity and myxospore measurements provided sufficiently distinctive features to confirm species validity and were thus crucial for identification. The triactinomyxon spores had 16 secondary germ cells, unique dimensions, a very opaque sporoplasm matrix and three conspicuously protruding, pyriform polar capsules. This is the first record of a Henneguya sp. life cycle with a triactinomyxon-type actinospore, which suggests a close relationship with the Myxobolus group and a polyphyletic origin of the genus Henneguya.     

细鳞鲑早期发育过程中免疫器官发生

应用连续切片技术和显微观察法,对胚胎末期至仔鱼期的细鳞鲑(Brachymystax lenok)免疫器官(头肾、胸腺及脾组织)早期发育进行了系统研究。研究表明,在实验水温为10.0～16.2℃时,细鳞鲑的免疫器官原基发生及其淋巴化的先后顺序均为胸腺、头肾和脾。仔鱼出膜前5 d胸腺原基形成,出膜前1 d胸腺即出现少量淋巴细胞,仔鱼19日龄后,结缔组织和毛细血管渗入胸腺实质而形成小梁,仔鱼35日龄左右,胸腺己经分化出模糊的内区和外区;仔鱼出膜前2 d,出现头肾原基,由未分化的造血干细胞组成,仔鱼3日龄后,头肾由肾小管构成,10日龄后,组织开始淋巴化,仔鱼35～45日龄,肾小管上皮细胞开始退化,头肾内出现血窦,并进一步淋巴化,50日龄后,肾小管退化完毕,头肾主要由淋巴组织和大量血窦构成;5日龄细鳞鲑仔鱼,脾原基出现,16日龄后,脾开始淋巴化,35～50日龄,脾内建立起发达的微血管系统以及血窦结构。细鳞鲑的免疫器官分化较早,其发育速度较其他淡水硬骨鱼类相对滞后,但组织淋巴化速度较快。本研究旨在揭示细鳞鲑早期死亡率高的根本原因,为实现细鳞鲑的规模化养殖提供科学基础。     

Accumulation of immunomodulatory laminaran [β(1,3)-D-glucan] in the spleen and kidney of Atlantic salmon, Salmo solar L.

Abstract. The tissue distribution of ³H- and FITC-laminaran in Atlantic salmon, Salmo salar L., was investigated after intravenous administration. Liquid scintillation counting and whole-body autoradiography revealed that the concentration of ³H-labelled laminaran in the spleen and anterior kidney was higher than in blood throughout the experimental period (1 h to 12 days). Renal excretion and intestinal exsorption were the main elimination pathways for laminaran. Microscopic examination revealed accumulation of FITC-labelled laminaran in macrophages in kidney and spleen. In addition, endothelial cells in the kidney, spleen, liver and intestine contained the immunomodulator. Thus, these experiments show that laminaran accumulates and is retained in immunologically relevant cells in the kidney and spleen.     

In vitro hydroxylation of vitamin D3 and 25-hydroxy vitamin D3 in tissues of Atlantic salmon Salmo salar, Atlantic mackerel Scomber scombrus, Atlantic halibut Hippoglossus hippoglossus and Atlantic cod Gadus morhua

The in vitro metabolism of ¹⁴CD₃ and ³H25OHD₃ was investigated in different tissues from Atlantic salmon Salmo salar , Atlantic mackerel Scomber scombrus , Atlantic halibut Hippoglossus hippoglossus and Atlantic cod Gadus morhua . The tissues analysed were liver, kidney, head kidney, gills, spleen and intestine. The metabolites were extracted in methanol–chloroform and separated by normal-phase high-pressure liquid chromatography (HPLC) followed by scintillation counting. Identification of the metabolites was by comigration with standards on normal and reversed-phase HPLC systems and by protein-binding assays. All tissues from all species analysed produced hydroxylated derivatives identified as 25OHD₃, 24,25(OH)₂D₃ and 1,25(OH)₂D₃. In addition, some unidentified derivatives were recorded, one probably being 25,26(OH)₂D₃. Organs producing great amounts of one metabolite also produced considerable amounts of the other possible derivatives, suggesting a lower degree of specificity in fish organs than in human organs. The predominating metabolite was 24,25(OH)₂D₃ in all organs from salmon and mackerel during incubation with ¹⁴CD₃ and within most organs from all species during ³H25OHD₃ incubation. The latter observation probably results from the need for decreasing rather than increasing the calcium absorption in these species, which live at least some periods of life in a marine environment.