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.     

An indirect fluorescent antibody test for the rapid detection of infectious pancreatic necrosis virus in tissues

Abstract. An indirect fluorescent antibody (IFA) test was developed to detect viral antigen in tissue sections prepared from rainbow trout experimentally inoculated with infectious pancreatic necrosis virus (IPNV). Specific fluorescence was present in the pancreatic acinar tissue and occurred as brilliant fluorescence throughout the mesentery surrounding the pyloric caeca and intestines. In addition, multiple foci of fluorescent cells were seen occasionally in the kidneys and liver of infected fry. Fluorescence was not observed in tissues other than the pancreas, kidneys, and liver. The IFA test was found to be quite specific and offers a rapid means of diagnosing IPN during acute outbreaks.     

Salmonid macrophages: separation, in vitro culture and characterization

Abstract. A method is described for the separation of fish leucocytes and the establishment of pure monolayers of fish macrophages in vitro. The method makes it possible to study the important role of cellular immunity in fish. Fish leucocytes were obtained from the pronephros of rainbow trout, Salmo gairdneri Richardson, and compared to those obtained from the pronephros of Atlantic salmon, Salmo salar L. Single cell suspensions were separated by density gradient centrifugation and seeded on glass cover slips for maintenance in culture. After 20 h in culture a subpopulation of the cells had adhered and spread out on the cover slips and were macrophage-like by morphological criteria. About 90–99% of these cells had the ability to phagocytose a variety of particles, including fixed sheep erythrocytes, latex, carbon particles, yeast and Vibrio anguillarum. Opsonization of particles with mammalian immunoglobulins and mammalian complement did not enhance the phagocytic activity.     

A case history of whirling disease in a drainage system: Battle Creek drainage of the upper Sacramento River basin, California, USA

Abstract. Whirling disease was diagnosed in steelhead trout, Salmo gairdneri Richardson, during 1985 at Coleman National Fish Hatchery in the Battle Creek drainage of the upper Sacramento River basin, California, USA. Early confirmation of the aetiologic agent, Myxobolus cerebralis Hofer, 1903, was difficult. However, later investigation confirmed the identity of the agent and led to a management decision to destroy the 1985 brood year of steelhead trout at the hatchery. Sentinel fish and collections of feral trout were used to survey Battle Creek watershed to determine the source of infectivity. Feral rainbow trout, Salmo gairdneri Richardson, from South Fork Battle Creek were found to be infected with M. cerebralis and other infected trout were later found at two commercial rainbow trout hatcheries. No reinfection with M. cerebralis has been detected at the Coleman hatchery since ozonation was begun in early 1986.     

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.     

An updated geographic distribution of Myxobolus cerebralis (Hofer, 1903) (Bivalvulida: Myxobolidae) and the first diagnosed case of whirling disease in wild-caught trout in the south-eastern United States

Myxobolus cerebralis (Bivalvulida: Myxobolidae), the aetiological agent of salmonid whirling disease, was detected in 2 river basins of North Carolina during 2015, which initiated the largest spatial–temporal monitoring project for the disease ever conducted within the south-eastern United States (focused mainly in eastern Tennessee and western North Carolina). A total of 2072 rainbow trout Oncorhynchus mykiss, 1,004 brown trout Salmo trutta and 468 brook trout Salvelinus fontinalis were screened from 113 localities within 7 river basins during June 2017 through October 2019. Infections were detected by pepsin–trypsin digest, microscopy and the species-specific nested polymerase chain reaction (PCR) in 19 localities across 6 river basins. Myxospore morphology was indistinguishable from the published literature. In 2019, five rainbow trout that symptomatic for whirling disease (sloping neurocranium and lordosis) were captured and processed for histopathology. Myxospores were detected in the calvarial cartilage of two deformed trout with associated erosion of the cartilage consistent with reported whirling disease lesions. This is the first report of M. cerebralis in Tennessee and the first histologically confirmed cases of whirling disease in southern Appalachian (south-eastern United States) rivers and streams and expands the distribution of M. cerebralis throughout western North Carolina and eastern Tennessee.     

Salmonid alphavirus (SAV) and pancreas disease (PD) in Atlantic salmon,Salmo salar L., in freshwater and seawater sites in Norway from 2006 to 2008

A cohort study was initiated in the spring of 2006 to investigate epidemiological aspects and pathogenesis of salmonid alphavirus (SAV) subtype 3 infections and pancreas disease (PD). The aims were to assess involvement of the freshwater production phase, the extent and frequency of subclinical infections and to follow PD‐affected populations throughout the entire seawater production cycle, as well as investigate possible risk factors for PD outbreaks. Fish groups from 46 different Atlantic salmon freshwater sites in six counties were sampled once prior to seawater transfer and followed onto their seawater sites. A total of 51 Atlantic salmon seawater sites were included, and fish groups were sampled three times during the seawater production phase. SAV subtype 3 was not identified by real‐time RT‐PCR from samples collected in the freshwater phase, nor were any SAV‐neutralizing antibodies or histopathological changes consistent with PD. In the seawater phase, SAV was detected in samples from 23 of 36 (63.9%) studied sites located within the endemic region. No SAV subtype 3 was detected in samples from seawater sites located outside the endemic region. The cumulative incidence of PD during the production cycle amongst sites with SAV detected was 87% (20 of 23 sites). Average fish weight at time of PD diagnosis ranged from 461 to 5978 g, because of a wide variation in the timing of disease occurrence throughout the production cycle. Mortality levels following a PD diagnosis varied greatly between populations. The mean percentage mortality was 6.9% (±7.06) (range 0.7–26.9), while the mean duration of increased mortality following PD diagnosis was 2.8 months (±1.11) (range 1–6).     

Carbohydrates in fish nutrition: digestion and absorption in postlarval stages

This review summarizes information regarding digestion and absorption of carbohydrates in cultivated fish. Relevant results of studies of digestive enzymes, e.g. amylase, chitinase, cellulase and brush border disaccharidases are presented. Fish amylases appear to be molecularly closely related and to have characteristics comparable to mammalian amylases. Whether chitinases and cellulases are endogenous enzymes of some fish species is still a matter of speculation, although recent molecular evidence, at least for chitinase seems to settle the issue in favour of endogenous sources. Feed and intestinal microbes may be the source of polysaccharidases in fish feeding on nutrients‐containing non‐starch polysaccharides. Knowledge regarding monosaccharide transport in fish intestine as interpreted from studies of brush border membrane vesicles, everted sleeves of fish intestinal sections and molecular biology is discussed. Glucose transporters of the intestinal brush border show characteristics similar to those found in mammals. A tabulatory presentation of experimental details and results reported in the literature regarding starch digestibility is included as a basis for discussion. Although numerous investigations on digestion of starch and other carbohydrates in fish have been published, the existing information is highly fragmentary. As yet, it is impossible to derive a cohesive picture on the integrated process of carbohydrate hydrolysis and absorption and interaction with diet composition for any of the fish species under cultivation. The physiological mechanisms behind the species differences are not known.     

Spring viraemia of carp virus (SVCV): comparison of immunoperoxidase, fluorescent antibody and cell culture isolation techniques for detection of antigen

Abstract. The sensitivity of the immunoperoxidase (IP) and fluorescent antibody (FA) techniques applied to frozen sections of organs of carp infected with spring viraemia virus (SVCV) was similar, both in respect of the intensity of the reaction and in the detection rate of the antigen. Seven days after a waterborne infection both methods detected the antigen in the kidneys and to a lesser extent in the liver and spleen. Quantification of virus gave a titre of 10^2.8 to 10^5.5 TCID₅₀/g of kidney, whereas the agent could not be isolated from liver and spleen tissue. In contrast, at 24¹/₂ days post–infection the viral antigen could be readily detected in liver and spleen but only occasionally in the kidneys using the IP and FA techniques. At this time, liver and spleen showed infectivity titres of 10^3.8 to 10^7.2 TCID₅₀/g tissue, whereas the kidneys were found to be free of infective virus. It is concluded that using the IP and FA techniques SVCV can be detected most frequently in the kidneys from 7 to 14 days post–infection and in the liver and spleen from 14 to 24 days post-infection.     

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.     

Spinal curvatures and an encephalotropic myxosporean, Triangula percae sp. nov. (Myxozoa: Ortholineidae), enzootic in redfin perch, Perca fluviatilis L., in Australia

Abstract. A new myxosporean, Triangula percae sp. nov., (Myxozoa: Ortholineidae) from the brain of redfin perch, Perca fluviatilis L., is described. Spinal curvatures are prevalent in many perch populations in Victoria, Australia, Triangula percae infection was detected in all perch displaying spinal curvatures and in 67% of normal perch from Lake Nillahcootie in north east Victoria. Perch populations exhibiting spinal curvatures in several other waterways were also infected with Triangula percae but the infection was absent in one population which was apparently normal. Triangula percae infection is, thus, considered to be the cause of a proportion of the spinal curvatures. Certain fish surviving outbreaks of epizootic haematopoietic necrosis virus (EHNV) also developed spinal curvatures, which may have been due to viral infection of the central nervous system.     

陕西省重大动物疫病秋防免疫抗体的检测与分析

猪瘟、口蹄疫、禽流感和新城疫四种重大动物疫病对畜禽业的危害非常严重,目前疫苗免疫是预防和控制动物疫病最有效的途径,免疫抗体检测能很好的反映出免疫效果的好坏。为了及时了解陕西省重大动物疫病免疫状况,2010年11月份在全省11个市(区)的21个县随机采集牛、羊、猪、鸡秋防免疫血清共2359份,按照农业部规定的检测方法,对其进行了重大动物疫病免疫抗体水平的检测,检测结果均达到或超过农业部规定的合格标准,说明陕西省今年秋防动物免疫效果总体较好。     

罗非鱼温和气单胞菌病的病原研究和药敏试验

从患病濒死尼罗罗非鱼肝脏分离到99-5-A和99-7-28菌株.经细菌学鉴定均为温和气单胞菌(Aeromonas sobria).用这2菌株对尼罗罗非鱼、 奥利亚罗非鱼及尼奥罗非鱼(尼罗罗非鱼♀×奥利亚罗非鱼♂)进行人工感染,均可使其致病.发病鱼症状与自然病鱼症状一致,呈出血性败血症.再分离菌株的各种特性与原分离菌株相同,确认这2菌株皆为温和气单胞菌.采用纸片扩散法,用35 种药物对上述2菌株进行药物抑菌试验,其中,丁胺卡那霉素、庆大霉素、多粘菌素、 妥布霉素、氟哌酸、氯霉素、氨曲南抑菌效果最佳;99-5-A菌株对呋喃妥因敏感,但 99-7-28菌株对其耐药;99-7-28对复方新诺明敏感,而99-5-A菌株对其则无反应.     

Wetland creation: early stages in colonization of phytoplankton and submerged macrophytes in hypereutrophic freshwater lagoons

1. Colonization by macrophyte and phytoplankton communities was recorded in the newly created freshwater wetland complex at the Wildfowl and Wetlands Trust (WWT), The Wetlands Centre, London. To attract particular bird species and to increase overall invertebrate and vertebrate biodiversity it was originally planned to establish a number of different submerged aquatic macrophyte communities in the water bodies. Early planting schemes were abandoned due to extensive growths of filamentous macroalgae. 2. During 1997 and 1998 hypereutrophic conditions prevailed in the lagoons with peak concentrations of total phosphorus (TP) in excess of 1000 mg m⁻³. Turbidity was high and dense algal crops and cyanobacterial blooms common, with chlorophyll a concentrations in excess of 40 mg m⁻³. 3. In 1999, a ‘switch’ in state occurred in the Sheltered Lagoon from a turbid lake dominated by cyanobacteria and filamentous macroalgae in 1998, to a clear lake, dominated by the macrophyte Myriophyllum spicatum L. with a mean biomass of 245 g dry weight m⁻²; concentrations of TP remained high (>200 mg m⁻³). 4. Possible causes for the switch in the Sheltered Lagoon are considered. Reduction in TP was not considered to be sufficient to trigger a switch. The smaller size (1.8 ha), with smaller fetch and greater protection from wind mixing may have reduced sediment suspension in the Sheltered Lagoon and, coupled with water level changes, may have created favourable light conditions for the establishment of M. spicatum. Implications for future design and management of the water bodies are considered. Copyright © 2000 John Wiley & Sons, Ltd.     

Microsatellite loci in tench: isolation and variability in a test population

Because of their high variability and rapid evolution, microsatellites became increasingly important in genetic research, e.g. population structure and differentiation studies, gene mapping and parentage analysis. However, such loci have not been isolated in tench so far. Applying a PCR based method of generating microsatellite enriched DNA fragment libraries we were able to identify nine loci (MTT-1 to MTT-9). The variability of these microsatellite loci was determined in 50 tench individuals originating from a wild population of Lake Döllnsee, Germany. Three loci were found to be monomorphic. The remaining six loci segregated for two to nine alleles. The observed heterozygosities at polymorphic loci were high (0.500–0.959) with only one exception: locus MTT-8 (0.167). These polymorphic microsatellite loci showed a much higher level of genetic variability than the allozyme loci previously studied in the same individuals. Thus, they seem to be more suitable for genetic studies of tench. On the other hand, it remains to be checked in other populations if the three loci that did not show any variation in this population are generally monomorphic in this species.     

Distribution of X-cell disease in common dab, Limanda limanda L., in the North Sea, and ultrastructural observations of previously undescribed developmental stages

Abstract. The distribution and prevalence of common dab, Limanda limanda L., affected with X-cell lesions was investigated at various localities around the British Isles. Extreme variability in the prevalence of affected gills was recorded in the Moray Firth (NE Scotland), where samples obtained from hauls taken in close proximity ranged from nil to 60% of the collected dab. The condition was not detected in dab from the Irish Sea, and was found only sporadically in samples in the southern North Sea and the northwest coast of Scotland, Areas of high prevalence are typically coastal or relatively shallow habitats. The patchy distribution of the disease throughout dab populations suggests that an infectious agent may be involved in its aetiology. A method is described for rapid, accurate screening of fish samples for X-cell disease, which utilizes the autofluorescence of the X-cell. Ultrastructural studies of lesions from external and internal organs in dab disclosed some previously undescribed features of X-cells. Stages of the X-cell development which occurred in'nests', and possibly preceded the commonly found X-cells, were found in gill lesions. Electron dense inclusions found in X-cells in the spleen and kidney were rod- or dumbbell-shaped structures and possibly represented secretory granules.