Light and electron microscopic studies on Epieimeria anguillae (Léger & Hollande, 1922), a coccidium parasitizing the European eel, Anguilla anguilla L.

Abstract. The foregut of eels naturally infected by Epieimeria anguillae (Léger & Hollande, 1922) was studied by light and electron microscopy. It has been established that this parasite, which develops in a characteristic location on the surface of epithelial cells, and was classified on this basis by Dyková & Lom (1981) as a member of the genus Epieimeria , undergoes intracellular merogony and gamogony similarly to other eimerians; however, its sporogony takes place outside the fish or intercellularly. The trophozoites and merogonic and gamogonic stages each develop in a para-sitophorous vacuole which is half embedded in the epithelial cell and protrudes into the intestinal lumen. The parasitophorous vacuote is surrounded by a single membrane; however, towards the intestinal lumen it is covered also by the cell membrane. In its location, Epieimeria anguillae resembles cryptosporidia, but differs from the latter significantly in its relationship with the host cell.     

Corrosion anatomical and scanning electron microscopic studies of the blood vessels in the digital end organ of the hindlimb of cattle

By aid of modern corrosion anatomical methods the afferent and efferent blood vessels as well as the microvasculature of the corium of the pelvic limb digit were investigated. The longest papillae are to be found in the perioptic corium. In the coronary corium the length decreases from proximal to distal. In the caudal section of the digital cushion the papillae show a typical, regular wavy form, whorl formation is rare.     

Morphological characterization of larval stages and first juvenile of the freshwater prawn Cryphiops caementarius (Molina, 1782) (Decapoda: Palaemonidae) under laboratory conditions

Research has been in progress for several years on various aspects of the biology and ecology of the freshwater prawn Cryphiops caementarius, an inhabitant of rivers in northern Chile. The commercial value of this prawn fomented the accomplishment of studies on its reproduction and development with the aim of producing juveniles under controlled conditions, to be followed by growout to commercial size in managed culture systems. The present study describes larval culture of this species from eggs of gravid females obtained in the field, from the first developmental stage (Zoea I) through the first juvenile stage. The larvae were cultured at 25 °C in UV sterilized water at variable salinities based on the requirements of the developmental stages. Larvae were fed with Nannochloris, Isochrysis and Artemia nauplii as required. This report describes in detail the 18 larval stages of this prawn, as well as its first juvenile form.     

Epidermal hyperplasia of walleye, Stizostedion vitreum vitreum (Mitchill), associated with retrovirus-like type-C particles: prevalence, histologic and electron microscopic observations

Epidermal hyperplasia consisting of discrete translucent raised outgrowths of cells were observed on the skin of walleye, Stizostedion vitreum vitreum (Mitchill), during their spawning period in the spring. The cells constituting the hyperplastic growths were limited to the epidermal layer, and were associated with surface budded, 120-nm-diameter, retrovirus-like particles located in the expanded intercellular spaces. These tumour-like growths were distinct from the other virus-associated skin lesions of walleye including dermal sarcoma, lymphocystis disease and herpesvirus-associated hyperplasia. Lesions could be differentiated by careful observation in the field and comparison of portions of each growth by histologic and electron microscopic observations.     

Light and electron microscopic evidence of white spot disease in the giant tiger shrimp, Penaeus monodon (Fabricius), and the kuruma shrimp, Penaeus japonicus (Bate), cultured in Taiwan

Since 1992, mass mortalities among cultured giant tiger shrimp, Penaeus monodon (Fabricius), and kuruma shrimp, Penaeus japonicus (Bate), have been observed in Taiwan. The condition is known as 'white spot disease' (WSD), based on the characteristic white spots on the cuticle of diseased shrimp. With the scanning electron microscope, two sizes of white spots were observed. Each spot represented a protrusion on the inside surface of the carapace. The composition of white spots was similar to that of the cuticule, most calcium, as determined with an energy dispersive spectrometer. Histological studies of moribund, infected specimens revealed degenerated cells, characterized by hypertrophied nuclei, in various meso- and ectodermal tissues. Infected tissues included cuticular epidermis, connective tissue, lymphoid organ, antennal gland, and haematopoietic, gill and nervous tissue. Nuclei were Feulgen-positive and no occlusion body was found in the necrotic tissue. Transmission electron microscopy revealed the presence of rod-shaped and enveloped virions in the hypertrophied nuclei. The virions measured 298 ± 21 × 107 ± 8 nm in the giant tiger shrimp and 248 ± 12 × 104 ± 8 nm in the kuruma shrimp. In an experimental infection trial, cumulative mortality was 40% within 14 days under stress conditions. No mortality was observed in controls or in non-stressed infected shrimp. Experimental infections show that environmental stressors such as ammonia may enhance the severity of WSD virus infections in cultured shrimp.     

Light and electron microscopic observations of sporogenesis in the myxosporida, Ceratomyxa shasta (Noble, 1950)

Abstract. The stages of development leading to sporogenesis of Ceratomyxa shasta (Noble) were investigated by light and electron microscopy. Salmonid fishes were infected by exposing them to water containing the infectious stage and intestinal material was fixed at weekly intervals. Signs of intestinal infection were barely detectable by 7 days following exposure where trophozoites and later developmental forms were present, but by 14 days a large number of pansporoblasts could be detected in varying stages of development. By 21 days the majority of caeca were completely occluded and infection had spread throughout the connective tissues attached to the caeca.
The early developing trophozoites contained two or more nucleated cells within a mother cell. There was some evidence of multiplication of nuclei by fission. The sporoblasts usually contained twelve nucleated cells that gave rise to two groups of six cells (sporonts) and resulted in the formation of two spores in each mother cell. Each spore was formed by two sets of bilaterally arranged cells consisting of the main germinative cell or sporoplasm, the anteriorly placed capsule cells and the outer envelope or spore valve cell that surrounded the others and formed the spore covering.
As the spore matured the two germinative cells interacted with each other by pseudopodial extensions and appeared to fuse to form a diploid cell. The position of the cells laterally and slightly posteriorly to the central suture line formed a bilaterally curved spore. Mature spores when examined with the electron microscope were condensed, dark staining and relatively featureless, with a lateral measurement of 15 μm and an anterior-posterior measurement of 7μm.     

Alterations in the epidermis of the carp,Labeo rohita (Cyprinidae: Cypriniformes), infected by the bacteria,Aeromonas hydrophila: A scanning electron microscopic,histopathological and immunohistochemical investigation

This study was carried out to comprehend the pathogenicity of the bacteria in the epidermis of Labeo rohita inoculated with Aeromonas hydrophila. Alterations in the histopathology of the epidermis were examined using scanning electron microscopy, light microscopy and the localization of iNOS and caspase 3 + ve cells by means of immunohistochemical methods. Skin samples obtained from infected fish at different intervals 2, 4, 6, 8 and 10 days showed significant changes in the cellular components of the epidermis. Epithelial cells often appeared hypertrophied with fragmented and loosely arranged microridges, and in the process of exfoliation. Mucous goblet cells increased significantly in density. Club cells showed degenerative changes, often with simultaneous confluence of adjacent cells and release of their contents. Increase in density of iNOS and caspase 3 + ve cells indicates inflammatory response and apoptosis. This study could provide valuable information on the pathogenesis of the disease, and disease outbreaks in farmed fish. Further, it could provide useful guidelines for fish farmers to take preventive measures for the control of the disease.     

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 microscopic studies on Goussia zarnowskii (Jastrzębski, 1982): an intestinal coccidium parasitizing the three-spined stickleback, Gasterosteus aculeatus (L.)

Abstract. The foregut and midgut of sticklebacks, Gasterosteus aculeatus (L.), naturally infected with Goussia zarnowskii (Jastrzębski, 1982) was studied by light and electron microscopy. Trophozoites, meronts, gamonts and young oocysts developed in the intestinal epithelial cells within a parasitophorous vacuole located in an ectoplasmal position and covered by host cell plasmalemma towards the intestinal lumen. A persistent conoid, together with two polar and preconoidal rings, was present in trophozoites, young meronts and also in young macrogamonts. All developmental forms of the parasite, excluding microgamonts, were surrounded by a trimembranous pellicle consisting of an external continuous unit membrane and double-membranous system of flattened vesicles. The micro-gamont pellicle was reduced to a single surrounding membrane during the development of the microgamont. The manner of merogony remains unknown. The oocyst wall consisted of a double membrane of host cell origin and at least three additional membranes derived from the endoplasmic reticulum of the zygote. Material from inclusions of double electron-density, that had occurred formerly at the periphery of the macrogamont cytoplasm, was disposed outside of the pellicle between the newly-created oocyst wall membranes. The function of this deposit and way it passes across the pellicle membranes remains unknown. The oocyst is surrounded by the membranes of the parasitophorous vacuole and the host cell plasmalemma detached from the surface of the intestinal epithelium facing the intestinal lumen.     

Evaluation of Chuni, a commercially available low-cost cattle fodder, in the diet for rohu, Labeo rohita (Hamilton), fingerlings

An 80-day feeding experiment was conducted to evaluate the suitability of incorporation of Chuni, a commercially available low-cost cattle fodder, comprising cereal grains and leguminous seeds, into the practical diets for rohu, Labeo rohita (Hamilton), an Indian major carp, fingerlings (average weight= 6.23 0.15g). Four experimental diets, incorporating Chuni at levels of 10%, 20%, 30% and 40% into a fish-meal-based control diet (35% protein) were formulated. In terms of fish growth, feed conversion, protein efficiency ratio, apparent nutrient digestibility, and protein and lipid deposition in fish muscle, 10%Chuni incorporated diet showed the best performance of fish and was comparable to those with the control diet. However, the growth of fish was lower with higher levels of Chuni incorporation (20% or more), which resulted in poor nutrient digestibility. The present results indicate that Chuni could be used as a component in the supplementary diet for L. rohita fingerlings, partially substituting the fish-meal-based diet up to the extent of 10%, and it this is the first report on the use of Chuni as a dietary ingredient in diets for carp.     

The glycerinated body wall of the sea cucumber as a suitable preparation for electron microscopic and physiological studies of ‘catch mechanism’

ABSTRACT:   The body wall of the sea cucumber changes its stiffness by ionic environments. The stiff state can be held for a long time, and the mechanism concerned is known as 'catch mechanism'. In the present study, the direct effects of ions on the mechanism using the glycerinated body wall treated with 50% glycerin to clarify how the ions effect changes of stiffness were examined. The glycerinated body walls contained collagen fibers and some broken cells in the connective tissue ultrastructurally. Cell membranes were not clearly present in the broken cells, and cell organelles were dispersed around the cells. The glycerinated body walls went into a limp state during addition of 10 mM ethylenediaminetetraacetic acid (EDTA), and showed height elongation rate in this study's experimental system. In contrast, the elongation rate decreased by the addition of 10 mM CaCl₂, that is, the body wall came to a stiff state. This stiff state could be considered as equivalent to 'catch state' of glycerinated body wall. Collagen fibers in those samples showed more compact arrangements at 10 mM CaCl₂ treatment than the one of 10 mM EDTA ultrastructurally. These features and physiological results suggested that EDTA and/or CaCl₂ from outside affect directly to the main part of the 'catch' mechanism in the glycerinated body wall.     

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.     

Use of the Reflotron system for the determination of creatine kinase (CK) in the blood of swine, sheep, cattle, horses and dogs]

The Reflotron-CK test was evaluated with blood samples of healthy and diseased pigs, sheep, cattle, horses and dogs in relation to the standard CK-NAC test (UV-method). The precision within the series on the day of blood sampling was better than VK = 7.5% (coefficient of variation) with both methods. The day to day precision was estimated by using deep frozen plasma and was in the same order of magnitude with the Reflotron-CK and the UV-method (CV less than 10%). While fresh whole blood of sheep, cattle, horses and dogs respectively should be applied directly onto the dry reagent carrier with the Reflotron-pipette (32 microliters), the blood samples of pigs must be diluted (200 microliters 0.9% NaCl-solution and 32 microliters blood, factor 7.25) before determination with Reflotron-CK. In the five species mentioned the correlation between Reflotron-CK and CK values obtained with the standard UV-method was satisfactory (r greater than 0.96). Systemic deviations of the Reflotron-CK values should be corrected by calculation formulae, given in the paper.     

Pathology of experimental infections of the sablefish, Anoplopoma fimbria (Pallas), with Renibacterium salmoninarum, the agent of bacterial kidney disease in salmonids

Abstract. The susceptibility of sablefish, Anoplopoma fimbria (Pallas), a potential candidate for commercial mariculture, to the devastating salmonid pathogen Renibacterium salmoninarum (Rs) and the pathology of the infection were investigated. Sablefish became moribund or died of apparently uncomplicated infections of Rs within 50-71 days following intrapcritoncal inoculation of the pathogen and pure cultures of Rs were re-isolated from some affected individuals. At least one sablefish carried the pathogen up to 165 days post-inoculation when the experiment was terminated. The pathology of the Rs infection in this strictly marine fish is described and the implication of the findings to the operation of salmon marinefarms is discussed.     

Growth of common carp Cyprinus carpio in Anatolia (Turkey), with a comparison to native and invasive areas worldwide

Common carp Cyprinus carpio occurs in several non‐native areas worldwide, where it is generally regarded as either naturalised or invasive. Anatolia (Turkey) represents a unique region for evaluating common carp growth, due both to its location at the southernmost range of expansion of the species' wild form and to most of its water bodies having been stocked with domesticated strains. Based on a review of length‐at‐age data for common carp stocks from 45 water bodies sampled between 1953 and 2007, regional patterns in growth across climates, water body types, scalation variants and sexes, along with altitudinal gradients in growth performance and mortality, were investigated. Growth rates were lower in cold and arid relative to temperate climates, and also under hot or dry summers; this was true also of the mirror relative to the scale variant, males to females, but not of water body types (i.e., man‐made reservoirs, natural lakes, water courses). Growth performance and mortality decreased with increasing altitude and decreasing temperature, likely due to optimisation of resource allocation between growth and reproduction. Growth rate of common carp from Anatolia was consistently lower compared to its native (Eurasian) and, especially, invasive (North American) counterparts, which reflected an opportunistic life‐history strategy. Lower growth rates in Anatolia were ascribed to lower resilience of the widespread mirror variant together with limited habitat for spawning in man‐made reservoirs. Better knowledge of common carp growth in Anatolia will improve stock management and conservation efforts. Further studies will help clarify the mechanisms responsible for evolutionary genotype–phenotype inter‐relationships.     

Cytogenetic and histological studies of the brook trout, Salvelinus fontinalis (Mitchill), and the Arctic char, S. alpinus (L.) hybrids

Although brook trout and the Arctic char hybrids are able to reproduce, individuals with decreased fertility or even fish that are unable to produce any gametes have been also described. Abnormal gonadal development and disturbances in the gamete production in the char hybrid offspring may be triggered by the odd chromosome number and disturbances in their pairing during meiosis. To verify this hypothesis, cytogenetic examination and the gonadal histology analysis of the brook trout x Arctic char hybrids were carried out. Diploid chromosome number in the studied char (F₁) hybrids varied from 82 to 84 (FN = 99–102). Among 28 hybrids, 12 males, three females, nine intersex individuals and two sterile specimens were described. In the case of two individuals, gonads were not found. Diploid chromosome numbers in the males and intersex individuals varied from 82 to 84. Chromosome numbers in the females were 82 and 83 chromosomes. Two sterile fish exhibited karyotypes composed of 82 and 84 chromosomes. Predominance of the ovarian component in the intersex gonads and gonadal sex ratio distortion towards the males suggested hybrid females had problems with gonadal differentiation. However, the lack of the clear relationship between chromosome number and gonadal development in the studied hybrids did not support our hypothesis that odd chromosome number may be responsible for such reproductive disturbances in the hybrid individuals. We have presumed that sterility and intersexual development of the gonads may be caused by interactions between brook trout and Arctic char genes on the sex chromosomes and autosomes rather than unpairing of the parental chromosomes.     

Effect of feed supplementation with kynurenic acid on the morphology of the liver,kidney and gills in rainbow trout (Oncorhynchus mykiss Walbaum, 1792), healthy and experimentally infected with Yersinia ruckeri

Kynurenic acid (KYNA) is an endogenous substance produced on the kynurenine pathway which is primarily known for its neuroactive properties. Recently, it has been proven that KYNA is a selective ligand for G protein‐coupled receptor (GPR 35), presented on immunocompetent cells such as T lymphocytes. This opens up new possibilities of its application as an immunostimulating substance in aquaculture. Thus far, no histopathological investigations in fish have been completed to evaluate influence of KYNA supplementation in feed. This study has been undertaken to determine the effect of feed supplementation with KYNA (2.5, 25, 250 mg kg^?1 of feed) for 28 days on the liver, gills and kidney in healthy fish and experimentally infected with Yersinia ruckeri. In a control group were observed a fatty liver, which is natural for this fish species in the autumn and winter season. As the dose of the supplement was increased, the fat liver changed, it decreased or completely disappeared. Additionally, inflammatory changes occurred in all the analysed organs, and their intensification was dose dependent. In the fish experimentally infected, KYNA caused aggravation of the signs in the liver, kidneys and gills, and the effect was dose dependent. The results implicate that KYNA may be a stressor for fish.     

Establishment and characterization of a new cell line derived from kidney of grouper, Epinephelus akaara (Temminck & Schlegel), susceptible to Singapore grouper iridovirus (SGIV)

A marine fish cell line derived from the kidney of red-spotted grouper, Epinephelus akaara, designated as EAGK was established and characterized. The EAGK cells multiplied well in Leibovitz's L-15 medium containing 10% foetal bovine serum at 25 °C and have been subcultured for more than 90 passages. Karyotyping, chromosomal typing and ribosomal RNA (rRNA) genotyping analysis revealed that EAGK had a modal diploid chromosome number of 82 and was a fibroblast cell line originated from grouper. A severe cytopathic effect was observed in EAGK cells incubated with Singapore grouper iridovirus (SGIV), but not with soft-shelled turtle iridovirus, viral nervous necrosis virus or spring viraemia of carp virus. SGIV replication was further confirmed by immunofluorescence, electron microscopy and virus titre determination. Bright fluorescence was observed after transfection with fluorescent protein reporter plasmids, indicating that EAGK cells can be used to identify gene functions in vitro. In addition, the cell organelles including mitochondria and endoplasm reticulum changed and aggregated around virus factories after SGIV infection, suggested that the EAGK cell line could be an important tool for investigation of iridovirus-host interactions.