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 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.     

Outbreak of mass mortality of yearling groupers of Epinephelus (Perciformes,Serranidae) associated with the infection of a suspected new enteric Sphaerospora (Myxozoa: Myxosporea) species in South China Sea

A suspected new enteric Sphaerospora species was believed to be directly associated with the mass mortality of yearling groupers of Epinephelus spp. in South China. The epizootic generally emerged from late September to late April of the following year. The infection prevalence and mortality rate were significantly negatively correlated with fish size. Clinical signs included anorexia, cachexia and extrusion of white pulp‐like substance from anus after gentle pressure on the abdomen. Upon necropsy, severe intestinal oedema, thin and transparent intestinal wall, swollen spleen, kidney and gall bladder could be observed. Wet preparation of the infected samples showed large amount of typical disporous plasmodia of the genus Sphaerospora, but no mature spores were observed. Epidemiological investigation showed that this parasite exclusively infected Epinephelus groupers. Histopathologically, this species mainly infected the epithelium of intestine and kidney tubules and caused severe epithelia sloughing and the collapse of intestinal villus. Interestingly, this enteric myxosporidiosis did not cause severe emaciation of infected fish for mass mortality usually emerged within 2–3 days after appearance of clinical signs. The species was most genetically related to Sphaerospora fugu (89% sequence identity) and phylogenetically positioned within marine Sphaerospora lineage. This is the first report of enteric sphaerosporosis of groupers.     

Characterization of a host response to the myxosporean parasite, Ceratomyxa shasta (Noble), by histology, scanning electron microscopy and immunological techniques

Abstract. The tissue response of Salmo gairdneri Richardson, against the myxosporean parasite. Ceratomyxa shasta (Noble), was investigated using histological techniques, scanning electron microscopy and immunological methods. The progress of infection in C. shasta -susceptible and resistant steelhead and rainbow trout was examined by standard histological techniques and by indirect fluorescent antibody methods using monoclonal antibodies directed against C. shasta antigens. Trophozoite stages were first observed in the posterior intestine and there was indication that resistance was due to the inability of the parasite to penetrate this tissue rather than to an inflammatory response. Examination of a severely infected intestine by scanning electron microscopy showed extensive destruction of the mucosal folds of the posterior intestine. Western blotting and indirect fluorescent antibody techniques were used to investigate the immunological component of the host response. No antibodies specific for C. shasta were detected by either method.     

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.     

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.     

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.     

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.     

Association of red‐mark syndrome with a Rickettsia‐like organism and its connection with strawberry disease in the USA

Red‐mark syndrome (RMS), a disease seen mostly in rainbow trout, Oncorhynchus mykiss, is of unknown aetiology. The research presented here indicates the presence of an intracellular bacterium in RMS‐affected fish. A positive reaction was observed using immunohistochemistry (IHC) with skin lesions, liver, kidney and spleen of affected fish sampled from several locations within the United Kingdom using two different polyclonal antisera raised against Piscirickettsia salmonis. The same reaction was also seen with a number of different anti‐P. salmonis monoclonal antibodies (MAbs). A disease with similar clinical signs to RMS, referred to as strawberry disease (SD), has been reported in the USA. A Rickettsia‐like organism (RLO) has recently been associated with SD based on analysis of 16S rDNA sequences. Using the same panel of anti‐P. salmonis antibodies used to screen the RMS samples, similar staining was obtained in tissue of SD‐affected fish by IHC. A polymerase chain reaction (PCR) using RLO‐specific primers was also performed on RMS‐affected fish from the United Kingdom, and the samples were positive for the RLO 16S rRNA sequence. These findings suggest that the same aetiological agent may be responsible for RMS in the United Kingdom and SD in the USA.     

Studies into the possible protozoan aetiology of swimbladder inflammation in carp fry

Abstract. Laboratory examinations of pond-reared common carp, Cyprinus carpio L., revealed a close correlation between the prevalence of swimbladder inflammation (SBI), renal sphaerosporosis and infection by C-blood-protozoan among the carp fry. In impression smears as well as light and electron microscopic preparations we detected developmental stages of intercellular protozoa, mainly in the loose fibrous tissue of the swimbladder. The parasites multiplied by internal budding so that 20–46 secondary cells were formed in each primary cell and two tertiary cells were formed in each secondary cell. The final stage of development was a unit consisting of a secondary cell enclosing two tertiary cells, i.e. a so-called triple formation, which bore a close resemblance to the early sporogonic stages of the renal sphaerosporan Sphaerospora angulata Fujita, 1912. Certain morphological similarities and the frequent simultaneous presence of the swimbladder protozoan, C-bloodprotozoan, and S. angulata in hosts with clinical SBI have led us to postulate that the former two parasites could represent the hitherto unknown presporogonic stages of S. angulata. In view of the pathological changes caused by the parasites in the hosts with clinical SBI, and negative bacteriological and virological findings we have postulated that the swimbladder protozoan is the primary cause of SBI in carp fry.     

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.     

A synthesis of large-scale patterns in the planktonic prey of larval and juvenile cod (Gadus morhua)

Data from 40 published studies of the diet composition of larval and juvenile cod (Gadus morhua) from around the northern North Atlantic were summarized to assess generic patterns in ontogenetic and regional variability in the key prey. The results showed that larvae at the northern edge of the latitudinal range of cod depend primarily on development stages of the copepod Calanus finmarchicus, whilst those at the southern edge depend on Para‐ and Pseudocalanus species. Juvenile cod preyed on a wider range of taxa than larvae, but euphausiids were the main target prey. Analysis of regional variations in the relative abundances of C. finmarchicus and Para/Pseudocalanus spp. in the plankton, as estimated by the continuous plankton recorder (CPR) surveys, showed a similar geographical pattern to the larval cod stomach contents. Comparison of CPR data from the 1960s and 70s with data from the 1990s showed that the boundary between C. finmarchicus and Para/Pseudocalanus spp. dominance has shifted northwards on both sides of the Atlantic, whilst the abundance of euphausiids in the southern cod stock regions has declined. The results are discussed in relation to regional differences in the response of cod stocks to climate variability.     

In Search of the Cause of Proliferative Gill Disease in Channel Catfish,Ictalurus punctatus:

A two-year study was conducted on three ponds at a commercial channel catfish, Ictalurus punctatus, farm that experienced severe losses of channel catfish to proliferative gill disease (PGD) during the winter of 1988/1989. Low levels of mild PGD persisted throughout the year. Outbreaks of PGD with resulting mortalities occurred in both hot and cool months. Mortalities associated with PGD occurred in Pond 37 in its first vear of production and in Pond 42 in both its fist and second years of production; Pond 42 had been drained and dried following a PGD outbreak in the spring of its first year. PGD-related mortalities did not occur in Pond 21 in either its third or fourth years of continual production, despite the presence of histologically detectable PGD in the third year. Hennegiya exilis and Sphaerospora were only occasionally observed in PGD-affected channel catfish. Seasonal changes in PGD prevalence were correlated with changes in myxozoan infection of small mud-dwelling worms, Dero digitata (Oligochaeta: Naididae). The myxozoan is an undescribed species of Aurantiactinomyxon (Actinomyxea: Triactinomyxidae). D. digitata and isolated spores of Aurantiactinomyxon sp. were the only pond organisms that produced PGD in laboratory experiments. Comparison of the oligochaete populations of the study ponds suggested that differences in PGD prevalence in channel catfish raised in "old" and "new" ponds may be related to oligochaete population diversity. Channel catfish naturally infected with PGD recovered completely when held in tanks supplied with well water.     

Use of monoclonal antibodies in the indirect fluorescent antibody technique (IFA) for the diagnosis of Edwardsiella ictaluri*

Abstract. Forty-five channel catfish, Ictalurus pitnctaius (Rafinesque), fingerlings were inoculated with Edwardsiella ictaluri to determine the usefulness of monoclonal antibodies for indirect fluorescent antibody techniques for confirming clinical diagnosis of enteric septicaemia of catfish. Bacteriological and indirect fluorescent antibody results were compared statistically and found to correlate in 90·3% of the cases. A method for incorporating monoclonals for indirect fluorescent antibody into the speciation of Edwardsiella ictaluri and the use of monoclonal antibodies in diagnosis is described.     

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.     

Isolation and use of beneficial microbiota from the digestive tract of lions‐paw scallop Nodipecten subnodosus and winged pearl oyster Pteria sterna in oyster aquaculture

We isolated microbiota from the digestive tract of Nodipecten subnodosus and Pteria sterna and determined in vitro their haemolytic activity, antagonism against Vibrio spp., bacterial hydrophobicity, production of extracellular enzymes and molecular identification. Five bacterial strains were selected: RL5 and C3 (Lactobacillus spp.) and PB1‐1, PB1‐5 and PB1‐6 (Bacillus spp.). The RL5 and C3 isolates showed antimicrobial activity against Vibrio spp. and the PB1‐1, PB1‐5 and PB1‐6 isolates showed enzymatic activity for amylase, protease, lipase and cellulose; the C3 and PB1‐5 isolates were highly hydrophobic. The selected strains of bacteria were tested in vivo as probiotics, together with a treatment of ampicillin and a control without bacteria on juvenile Kumamoto oysters Crassostrea sikamea. The strains were provided individually and as mixes of isolates. Survival, growth and biochemical composition of the juveniles were determined as in vivo indicators. Juveniles grew significantly larger and faster when treated with a specific mix of isolates (MIX‐B), compared with the control. The protein, lipid and carbohydrate concentrations were also significantly higher in oysters exposed to probiotic treatments, compared with the control and the antibiotic treatment. The selected microbiota showed probiotic proprieties for cultivating C. sikamea juveniles.     

Characterization of a monoclonal antibody against the 47- kDa antigen of Cryptobia salmositica Katz and its use in an antigen-capture enzyme-linked immunosorbent assay for detection of parasite antigen in infected rainbow trout, Oncorhynchus mykiss (Walbaum)

A monoclonal antibody (designated MAb-007) was produced against the pathogenic haemoflagellate Cryptobia salmositica Katz. This IgG3 antibody recognized the 47-kDa antigenic polypeptide of C. salmositica (SDS-PAGE and Western immuno-blotting). The antibody did not agglutinate live parasites, and there was no change in the staining intensity of the 47-kDa band on Western immunoblots after immunoabsorption of MAb-007 with live intact parasites. The 47-kDa antigen recognized by MAb-007 was localized in the cytoplasm of the parasite (immunogold labelling and electron microscopy). The monoclonal antibody cross- reacted with the 47-kDa polypeptides of C. bullocki Srrout and C. catostomi Bower & Woo. It was used in an antigen-capture ELISA for the detection of parasite antigen in the plasma of rainbow trout inoculated with the parasite, or with an attenuated vaccine strain of C. salmositica. All pre-infection plasma were negative while all infected fish with detectable parasitaemias were positive for antigen at 1–9 weeks after infection. Parasite antigen was even detected in vaccinated fish that were negative for parasites using the wet mount microscopic technique. The antigen-capture ELISA detected C, salmositica antigen in whole cell lysate preparations at concentrations as low as 0.5 μg ml^-1. Fifty microlitres of fish plasma was required in the antigen-capture ELISA, and the use of a plate reader and 96-well plates facilitated rapid analysis of a large number of plasma samples. The sensitivity of the assay makes it a potentially useful tool for detection of Cryptobia infections.     

Histochemical and immunohistochemical characterization of rodlet cells in the intestine of two teleosts,Anguilla anguilla and Cyprinus carpio

Rodlet cells (RC) are characterized by a distinctive cell cortex and conspicuous inclusions named “rodlets.” These cells are particularly abundant and large in size in intestine of eels. Histochemical, immunohistochemical and ultrastructural investigations were carried out on European eel Anguilla anguilla and Common carp Cyprinus carpio from Northern Italy. Eight biotinylated lectins were used to probe for specific carbohydrate residues in deparaffinized, hydrated intestinal sections of eel and carp. Five antibodies were tested on intestinal sections of both fish species: inducible nitric oxide synthase (i‐NOS), leu‐enkephalin, lysozyme, serotonin and tumour necrosis factor‐α. Lectin histochemistry revealed rodlet cells (RCs) of the eel intestine to react with two of the eight lectins tested, specifically Concanavalin A (ConA) and Sambucus Nigra Agglutinin (SNA). This contrasted to lectin staining of RCs in the intestine of common carp, where four of the eight lectins showed a positive reaction; Dolichos Biflorus Agglutinin (DBA), Wheat Germ Agglutinin (WGA), SNA and ConA. RCs in eel and carp intestine were immunoreactive with antibodies to lysozyme and i‐NOS. The occurrence of the inflammatory peptides lysozyme and i‐NOS in RCs of the eel and common carp poses in favour that these cells are involved in the mechanism of defence against pathogens.     

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.