Comparative studies of PKX and Sphaerospora spp. from salmonids using lectin and monoclonal antibody staining techniques

The results of comparative immunohisto-chemical staining studies with two lectin probes, GS-I and SBA, and five monoclonal antibodies raised against the PKX myxosporean are presented. In order to investigate the postulated link between PKX and Sphaerospora spp., the lectins and monoclonal antibodies were tested against PKX, Sphaerospora renicola, Sphaerospora oncorhynchi, Sphaerospora elegam, Sphaerospora truttae and Sphaero-spora sp. from Atlantic salmon, Salmo salar L., and Ceratomyxa shasta. SBA appears a useful tool for tbe diagnosis of all Sphaerospora spp. infections and was also positive for C. shasta. GS-I and monoclonal antibodies (12, C5, D4 and A3) appear to be specific for both the extrasporogonic and sporogonic stages of PKX. Although MabB4 was raised to PKX, it appears to stain some of the sporogonic stages of Sphaerospora spp. but not C. shasta. The results of the lectin and monoclonal staining of PKX and Sphaerospora oncorhynchi indicated that further work was required to prove or disprove the hypothesis that PKX and S, oncorhynchi are the same organism.     

The occurrence of proliferative kidney disease (PKD) in cultured and wild fish: further investigations

Abstract. In an investigation of the occurrence of proliferative kidney disease (PKD) in freshwater fish other than rainbow trout, 18 species of wild fish and seven species of fish raised in cultivation wore sampled from waters where the disease occurred annually in rainbow trout, Oncorhynchus mykiss (Richardson). Results revealed that certain wild stocks of brown trout. Salmo trutta L., grayling, Thymallus thymallus L., and pike, Esox lucius L., were infected with PKD, as were cultivated Atlantic salmon, Salmo salar L., parr, brown trout and char, Salvelinus alpinus (L.). Microscopical examination revealed the presence of the PKX cell in these species and also intraluminal protozoa possibly related to the PKX cell, which were not found in the rainbow trout. Other species of freshwater fish had myxosporidan infections but, unlike PKD infection, there was little host/parasite tissue response. The PKX cell as a myxosporidan stage is discussed and the presence of the disease in wild fish is reported.     

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.     

Tracing the route of Sphaerospora truttae from the entry locus to the target organ of the host, Salmo salar L., using an optimized and specific in situ hybridization technique

Sphaerospora truttae is an important pathogen of Atlantic salmon parr in Scottish aquaculture. To trace the early development of S. truttae and to overcome the common problem of detecting low numbers of cryptic, early myxosporean stages, a DNA-based approach was applied in this study. Specific primers were designed for S. truttae from Atlantic salmon, based on 18S rDNA sequences, obtained from isolated myxosporean spores. These were 5' biotin-labelled and used in an optimized and rapid in situ hybridization (ISH) protocol, which provided a strong and specific signal of the parasite within host tissue sections and, at the same time, minimized structural damage to tissues due to processing. This methodology provided a reliable tool enabling the detection of S. truttae stages down to single cell level. Using ISH the epithelium of the gills was identified as the predominant entry locus of the parasite. By 3 days after infection S. truttae had penetrated the vascular epithelia and thereafter proliferated in the blood for at least 10 days before exiting the vascular system through capillary walls. From day 12 post-infection onwards, the kidney, as well as the spleen and the liver, were invaded. Numbers of S. truttae invading the kidney (37.3%) differed little from numbers found in the spleen (35.3%) and the liver (27.4%). The latter organs represented a dead end in the development of S. truttae as all stages in these organs degenerated and sporogony was found to take place exclusively inside the renal tubules. Early sporogonic stages were found from day 25 post-infection but mature spores only developed after at least 15 days of proliferation within the tubules.     

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.     

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.     

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.     

Sphaerospora testicularis sp. nov. (Myxosporea: Sphaerosporidae) in wild and cultured sea bass, Dicentrarchus labrax (L.), from the Spanish Mediterranean area

Abstract. A new myxosporean, Sphaerospora testicularis sp. nov., was found in fresh smears from the organs of wild and cultured sea bass, Dicentrarchus labrax (L.), and seminal fluid from spermiating cultured males. It is distinguished from all previously reported Sphaerospora spp, by the shape and dimensions of the mature spore, its location and the host and its geographical distribution. Only males were parasitized; 2.5% of the wild fish and 6-25% of the cultured fish were infected. In spermiating cultured males, the prevalence of infection increased progressively from 6-9 to 75% during the study period. The parasite causes destruction of the tubular tissue of testes and a reduction or loss of spermatozoa.     

Protozoan parasites of wild and cultured sea bass, Dicentrarchus labrax (L.), from the Mediterranean area

Abstract. In a parasitic survey of wild and cultured sea bass, Dicentrarchus labrax (L.), thirteen protozoan species were found. Ectoparasites included two ciliates ( Trichodina spp.), one dinoflagellate ( Amylodinium sp.) and one zooflagellate ( Cryptobia sp.). Endoparasites were represented by members of Apicomplexa ( Hemogregarina sp., Eimeria spp.). Microsporea (unidentified species) and Myxosporea ( Myxobilatus sp., Ceratomyxa spp., Sphaerospora dicentrarchi, Sphaerospora testicularis). Myxobilatus sp., Eimeria spp. and the microsporean were exclusively found in wild fish, whereas Amyloodinium sp. and Hemogregarina sp. were only detected in cultured fish. Data on prevalence and intensity of infections and on the location in the host are provided. The potential importance of protozoan parasites for sea bass culture is discussed.     

Distribution and prevalence of T. bryosalmonae in Austria: A first survey of trout from rivers with a shrinking population

The first evidence of proliferative kidney disease (PKD) in an Austrian river (the River Kamp) was documented in 2016, and no information on the PKD infection status of trout in other rivers was available. Since then, brown trout (Salmo trutta fario) and rainbow trout (Oncorhynchus mykiss) have been collected from rivers in Upper and Lower Austria for different diagnostic purposes. In this study, we summarize the recent findings of a first survey concerning the distribution of Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease (PKD), from these samples. Between September 2015 and October 2017, a total of 280 brown trout and 39 rainbow trout were collected from 21 rivers in the provinces of Upper and Lower Austria. T. bryosalmonae was detected by PCR of kidney tissue in 17 of 21 sampled rivers and in 138 of 280 brown trout as well as in 11 of 39 rainbow trout. Pathological signs of PKD (e.g., hypertrophy of the kidney) were observed in 33 analysed brown trout and six rainbow trout samples. No correlations between fish infected by T. bryosalmonae and the parameters size and age class, condition factor, geological origin of the streams and distribution within the river course were found, while positively tested fish are significantly increased at sampling sites exceeding water temperatures of 15°C for median periods of 115 days. The prevalence within the affected streams or stream sections is highly variable, and in single rivers, infection rates of up to 90% are confirmed.     

Characterization of glycans in the developmental stages of Myxobolus cerebralis (Myxozoa), the causative agent of whirling disease

Glycans and sugar-binding molecules (lectins) form an interactive recognition system, which may enable parasitic organisms to adhere to host cells and migrate into target tissues. The aim of the present study was to analyse surface-associated glycans in the developmental stages of Myxobolus cerebralis (Hofer), the causative agent of whirling disease. A panel of biotin-labelled plant lectins was used to detect a broad spectrum of glycan motifs with high specificity. Binding sites were detected histochemically in the tissue sections of infected rainbow trout, Oncorhynchus mykiss (Walbaum), and infected Tubifex tubifex (Müller), and were characterized by light, fluorescence and transmission electron microscopy. With mannose-specific lectins [Lens culinaris agglutinin, Pisum sativum agglutinin, Canavalia ensiformis agglutinin (LCA, PSA, CanA)] mannose-containing glycans were detected in all the developmental stages and host tissues. No binding sites for galactose-specific lectins were present in M. cerebralis spores but reactivity with host tissues occurred. Diversity in glycans was detected by N-acetyl-D-galactosamine-specific lectins in sporoplasm cells of M. cerebralis and triactinomyxon spores. In the group of lectins with monosaccharide-specificity for N-acetyl-D-glucosamine (GlcNAc), the reactivity of Datura stramonium agglutinin (DSA), Lycopersicon esculentum agglutinin (LEA) and Solanum tuberosum agglutinin (STA) was restricted to polar capsules whereas Griffonia simplicifolia agglutinin II (GSA II) also bound to sporoplasm cells of stages in the fish host but not in those present in infected T. tubifex. Moreover, Triticum vulgaris (wheat germ) agglutinin (WGA) and succinylated WGA indicated the presence of N-acetyl-D-glucosamine polymers in polar capsules. No specificity for spores was observed concerning 'bisected'N-glycans and no reactivity in parasitic stages was observed with the fucose-binding lectin Ulex europaeus agglutinin (UEA) I, Sambucus nigra agglutinin (SNA) (specific for alpha2,6-sialylated glycans) and Maackia amurensis agglutinin (MAAI) (specific for alpha2,3-sialylated glycans). Arachis hypogaea (peanut) agglutinin (PNA), Erythrina cristagalli agglutinin (ECA), GSA I, Sophora japonica agglutinin (SJA), Dolichos biflorus agglutinin (DBA) and GSA II detected reactive sites solely confined to the developmental stages of M. cerebralis and were not reactive in the fish host. These parasite-specific glycans may play a role in the adhesion process of the parasite to fish epidermis prior to infection, but may provide protection to the host by activating the complement system, or stimulating an adaptive immune response as putative antigens.     

Immunohistochemical characterization of polyclonal antibodies against Enteromyxum leei and Enteromyxum scophthalmi (Myxozoa: Myxosporea), intestinal parasites of fish

The enteric myxozoan parasites Enteromyxum leei (Diamant, Lom et Dyková) and Enteromyxum scophthalmi Palenzuela, Redondo et Álvarez‐Pellitero are responsible for high weight loss in infected fish, which leads to subchronic disease and low mortality rates in gilthead sea bream (GSB), Sparus aurata L., and to high mortality rates in turbot, Psetta maxima (L.). The detection of initial parasite stages in histological sections is particularly difficult, but can be simplified by means of specific antibodies. Rabbit polyclonal antibodies (pAbs) were raised against E. scophthalmi and E. leei, and direct enzyme‐linked immunosorbent assay (ELISA) and immunohistochemistry were used to characterize their sensitivity and specificity. Both pAbs were adsorbed (apAb) with non‐infected intestines to avoid non‐specific labelling of fish tissues and to improve their specificity. The highest titre obtained in ELISA was 1: 32 000 for apAb‐Eleei and 1:16 000 for apAb‐Escoph. Working dilutions in immunohistochemistry were 1:1000 for apAb‐Eleei and 1:8000 for apAb‐Escoph. Both apAbs labelled proliferative and sporogonic stages with high specificity. apAb‐Escoph was very specific, whereas apAb‐Eleei cross‐reacted with Sphaerospora dicentrarchi Sitjà‐Bobadilla et Álvarez‐Pellitero and Sphaerospora testicularis Sitjà‐Bobadilla et Álvarez‐Pellitero, suggesting the presence of shared antigens. These pAbs stand as new tools for antigenic characterization and the diagnosis of both Enteromyxum species.     

Frequency and distribution patterns of Saprolegnia infection in wild and hatchery-reared brown trout Salmo trutta L. and char Salvelinus alpinus (L.)

Abstract. Fungal infection of sexually mature brown trout and char was associated with a particular type of Saprolegnia exhibiting a low degree of homothallic sexuality. Hatchery-reared brown trout were more severely infected (in terms of the % area of the body covered with the fungus) than were wild fish. The fins of hatchery-reared fish were particularly prone to Saprolegnia infection regardless of sex. In wild brown trout, a sexual difference in the pattern of infection was demonstrated. The flanks of the male fish appeared to be more prone to infection when compared with the female and there was a greater susceptibility of the caudal and ventral fin of the female when compared with the male. Evidence is presented which suggests that the incidence of infection in mature male salmonid fish prior to spawning is significantly greater than in the females. This difference may not be apparent in spent fish after spawning. These findings are discussed in relation to the background concentration of fungal spores in the water, the behavioural characteristics of spawning fish and differences in epidermal structure.     

Micro‐scale distribution of brown trout: an opportunity for kin selection?

Abstract – Fourteen microsatellite loci were used to analyse the degree of relatedness between and within cohorts of stream-living brown trout ( Salmo trutta L.). Differences in degree of relatedness were found between cohorts with mature and older having the highest intracohort relatedness. The higher genetic relatedness among the mature and older trout is interpreted as indications of clustering of related mature and older trout prior to spawning in the vicinity of spawning grounds. Young-of-the-year trout showed a lower degree of association with related trout of the same cohort than did mature and older trout. It is argued that the clustering of mature and older trout may facilitate kin selection and that the low degree of association among related young-of-the-year trout could be explained by juvenile fish avoiding competition for territories with related individuals.     

Effect of brown trout body size on post-stocking survival and pike predation

Abstract –  A total of 40 (20 age-3 + 20 age-4) radio-tagged hatchery-reared brown trout ( Salmo trutta L.) and 40 wild radio-tagged northern pike ( Esox lucius L.) were released into a regulated river. Age-3 brown trout were predicted and observed to be highly vulnerable to predation by pike (50% mortality), whereas age-4 brown trout were predicted and observed to enjoy an almost absolute size refuge from predation (5% mortality). Almost half of the fish from both age groups similarly emigrated and survived from the river within 3 days of the release. However, there was a considerable difference in survival between age groups for fish that remained in the river for a longer period. Of these, all except one age-3 brown trout were eaten by pike, whereas all but one age-4 fish survived predation.     

Food consumption rates of piscivorous brown trout (Salmo trutta) foraging on contrasting coregonid prey

Knowledge of predator–prey dynamics is essential to understand ecosystem functioning. Quantification of such interactions is important for fisheries management, in particular in the case of stocking programmes. Here, food consumption rates (FCR) were quantified for wild and stocked piscivorous brown trout, Salmo trutta L., in three subarctic lakes with contrasting coregonid (Coregonus spp.) prey communities, using the Wisconsin and the Elliott–Hurley bioenergetic models. FCR was highest for stocked brown trout in lakes with the lowest predator densities, and lowest for wild brown trout. Although FCR estimate may vary somewhat depending on the specific model used, such tools are imperative for the proper impact assessment of brown trout stocking programmes and for the provision of advice on optimal stocking densities.     

Growth and sexual maturation of six stocks of brown trout Salmo trutta L. in culture

The growth dynamics of six different Swedish stocks (Arjeplog, Arevattnet, Bergnäs, Båthälla, Granbo. Gullspång) of landlocked brown trout were compared. The fish were kept in 1-m² troughs from start of feeding in spring 1988 until September 1989. Thereafter the stocks were reared together in 4-m² troughs until the termination of the experiment in October 1990. After the first summer of growth, Bergnäs trout had the highest mean weight and Granbo and Båthälla trout the lowest. In September 1989 the ranking of stocks with respect to mean weight had not changed. During the rest of the experiment, i.e. when stocks were reared together, Arevattnet trout increased in weight most rapidly and reached the highest final mean weight. Bergnäs trout grew very slowly after the stocks had been merged. Granbo and Båthälla trout ended up with the lowest mean weights. Sexual maturation in 1 + males was very rare. Gullspång trout had the highest proportion (42%) of mature 2+ males, while Granbo trout had the lowest proportion (4%). With the exception of Gullspång trout, mature males generally had a higher mean weight than immature fish. The experiment revealed considerable differences in overall growth, even between stocks with similar life histories in nature. Hatching time, frequency of sexual maturation and hatchery selection were identified as potential mechanisms behind the observed differences. In all, stock differences in important traits for commercial aquaculture are considerable, which makes further stock comparisons necessary before the start of breeding programmes.     

A systemic protozoal disease of cultured salmonids

Abstract. Sporadic cases of a systemic protozoal infection with cells resembling Dermocystidium sp. are described from cultured rainbow trout, Salmo gairdneri Richardson, from France and brown trout, Salmo trutta L., and Atlantic salmon, Salmo salar L., from Ireland. Classical proliferative kidney disease was additionally found in the rainbow trout and PKX-like cells were also seen in the brown trout and salmon.     

Are environmental conditions in Finnish streams limiting to early life-history survival in the nonnative rainbow trout?

The nonnative rainbow trout Oncorhynchus mykiss has been an unsuccessful invader in North European streams, although it has been widely introduced. Here we studied whether early life history stages (egg incubation and hatching, first overwintering) act as filters for the establishment of hatchery rainbow trout. Survival of hatched alevins was approximately 80%, whereas only 47% of the embryos survived. However, the latter value was impacted by the high number of unfertilized eggs. Correlation coefficients with embryo survival rate and environmental variables (pH and temperature) were statistically insignificant. In the overwintering experiments, the survival of rainbow trout was 93%. The growth was generally slowed during the winter, but in the spring the growth of rainbow trout exceeded that of the native brown trout. Our data demonstrated that the survival and growth of rainbow trout during early life-history stages were relatively high and comparable to those of the native brown trout. Based on the variables considered in our study, our results suggest that environmental conditions during early life-history stages are not detrimental for rainbow trout in the study streams.