A new herpesvirus-caused disease in tortoises

This report describes a highly fatal diphtheroid-necrotizing stomatitis in tortoises of hitherto unknown etiology. The tortoises suffered from dyspnea and anorexia, due to massive diphtheroid membranes in oral and pharyngeal cavities. Histologically, eosinophilic intranuclear inclusion bodies were found in epithelial layers of oral and tracheal mucous membranes. Furthermore, electron microscopy revealed herpesvirus like particles in affected cells. Possible environmental factors influencing the outbreak of the disease are discussed.     

Erythromycin and florfenicol treatment of rainbow trout Oncorhynchus mykiss (Walbaum) experimentally infected with Flavobacterium psychrophilum

Flavobacterium psychrophilum is responsible for significant economic losses in rainbow trout aquaculture. Antimicrobial treatment remains the primary means of control; however, there are limited choices available for use. The objectives of the study were therefore to determine the minimum inhibitory concentrations for erythromycin and florfenicol in selected F. psychrophilum isolates and to evaluate their clinical treatment efficacy in experimentally infected rainbow trout. All isolates tested had moderate susceptibility to florfenicol and erythromycin except one isolate, which had low susceptibility to erythromycin. Two isolates (one with moderate and one with low susceptibility to erythromycin) were used in an experimental infection trial. Rainbow trout juveniles were injected intraperitoneally with 10⁸ cfu/fish and after mortality had begun, fish were given erythromycin‐ and florfenicol‐medicated feed at a rate of 75 mg kg^?1 day^?1 and 10 mg kg^?1 day^?1 fish body weight, respectively, for 10 consecutive days. The splenic F. psychrophilum load was determined using an rpoC quantitative PCR throughout the 30‐day trial. Relative to antibiotic‐free controls, erythromycin treatment significantly (p < 0.05) reduced mortality of rainbow trout juveniles infected with FPG101, even when treatment was initiated after clinical signs developed.     

金乌贼幼体弧菌性坏死病的病原、组织病理特征和药物敏感性

对青岛市黄岛区人工繁育金乌贼(Sepia esculenta)苗种大规模坏死病进行了病原、组织病理、传播途径和防控药物研究。发病金乌贼幼体不摄食,生长缓慢,体色发黑;体表、肌肉、口腕、触腕及内脏团大面积溃烂,死亡率高达90%;组织病理学显示:患病金乌贼皮肤色素细胞崩解,肌丝断裂,鳃丝崩解,肝细胞大量坏死脱落,肠道微绒毛脱落;从患病金乌贼分离出3株优势菌(SE-A、SE-B和SE-C),人工感染实验表明,SE-B、SE-C对金乌贼半数致死量分别为3.98×10~6 CFU/mL和8.91×10~5 CFU/mL,有较强致病力,为该病的致病菌;根据细菌形态、生理生化和16S rDNA序列分析将SE-B和SE-C鉴定为溶藻弧菌(Vibrio alginolyticus)和塔氏弧菌(Vibrio tubiashii)。分析金乌贼幼体、育苗用水和生物饵料糠虾的病原菌来源、分布和优势度,发现致病菌主要由糠虾携带进入育苗系统。分析了2株致病菌的药物敏感性,SE-B对强力霉素和氟苯尼考等敏感,对链霉素、庆大霉素等耐药;SE-C对氟苯尼考和罗美沙星等敏感,对新霉素和克拉霉素等耐药。     

Outbreak of enteric microsporidiosis of hatchery‐bred juvenile groupers,Epinephelus spp., associated with a new intranuclear microporidian in China

A new enteric microsporidian was found to be associated with the mass mortality of hatchery‐bred juvenile groupers, Epinephelus spp., in China. The outbreak usually occurred during the rainy season between May and November when water temperature ranged from 26 to 30 °C and salinity from 28 to 34 ppt, although this microsporidian can be detected year round. External clinical signs included severe emaciation, white faeces syndrome, anorexia, sinking to the bottom of culture ponds and mass mortality (up to 90%). Upon necropsy, severe intestinal oedema and thin and transparent intestinal wall could be observed. The mature spores are tiny, measuring 1.3–1.5 (1.35 ± 0.13) × 1.6–2.4 (2.16 ± 0.31) μm and can be found in the cytoplasm and the nucleoplasm of most enteric epithelial cells of host. Epidemiological investigation showed that this species was distributed throughout most of the culture area of grouper fingerlings in Fujian, Guangdong, Hainan and Guangxi provinces in China, with maximum prevalence of 95%. Molecular analysis based on the partial small subunit rRNA sequence (1045 bp) placed this species within the Enterocytozoonidae, but sequence identities to other species were below 90%. The exact taxonomic position warrants study of the ultrastructural characteristics of the developmental stages.     

Development and regression of soybean meal induced enteritis in Atlantic salmon, Salmo salar L., distal intestine: a comparison with the intestines of fasted fish

The development of a pathological condition in the distal intestine of Atlantic salmon, Salmo salar L., induced by dietary soybean meal, was studied in a 6-week feeding experiment. The fully developed condition, as observed after 3 weeks on the experimental diets, was characterized by: (1) a shortening of heights of the mucosal foldings; (2) a loss of the normal supranuclear vacuolization of the absorptive cells in the intestinal epithelium; (3) a widening of the central stroma within the mucosal foldings, with increased amounts of connective tissue; and (4) a profound infiltration of inflammatory cells in the lamina propria. The first signs of morphological changes were observed after 2 days on a diet containing a solvent extracted soybean meal, and within 7 days, all the above mentioned signs were observed. When the fish were subsequently transferred to a control diet, the mucosal folds were rebuilt from the base, resulting in an apparently functional epithelium after 3 weeks. Starved fish also exhibited characteristic changes of the mucosa, including a finely granular cytoplasm replacing the supranuclear vacuoles seen in the epithelial cells of normal fish. In addition, a pattern of irregularly spaced indentations developed in the epithelium of the simple folds. The condition induced by dietary soybean meal was classified as a no n-infectious subacute enteritis, and a pathogenesis involving immunological mechanisms is suggested.     

The Toxicity of Erythromycin, Minocycline, Malachite Green, and Formalin to Nauplii of the Shrimp Penaeus stylirostris

The toxicities of erythromycin, minocycline, malachite green, and formalin to nauplii of the shrimp Penaeus stylirostris were determined in a static bioassay. Toxicity was assessed on the basis of survival of nauplii after 12 and 24 hours of exposure to the compounds and metamorphosis of the nauplii to protozwa. The results suggested that metamorphosis to protozoea is more susceptible to toxic effects than is naupliar survival. Metamorphosis was either reduced or completely inhibited by lower concentrations of erythromycin, minocycline, and malachite green than was naupliar survival at 12 or 24 hours. Metamorphosis was either reduced or completely inhibited by erythromycin, minocycline, malachite green, and formalin concentrations of 80, 100, 0.08, and 27 mg/ L, respectively. Toxic effects were not observed at erythromycin, minocycline, malachite green, and formalin concentrations of 16, 62.5, 0.016, and 2.7 mg/L, respectively. The results suggest that formalin may be toxic at therapeutic levels frequently recommended for post larvae and older penaeids, but that erythromycin, minocycline and malachite green are not.     

Scanning and transmission electron microscopical observations on Hexamita salmonis (Moore, 1922) related to mortalities in rainbow trout fry Salmo gairdneri Richardson

Abstract. Examination of the digestive tract of rainbow trout fry infected with Hexamita salmonis revealed no pathological changes in the epithelial cells of the pyloric caeca or upper intestine even where the parasites were closely apposed to the brush border of these cells. No invasion of the epithelium by the parasites was observed, although examples of shrinkage necrosis (apoptotic bodies) of epithelial cells were often found. The ultrastructure of the parasite resembled that of the closely related Giardia lamblia . Despite the lack of pathological changes to the intestinal epithelium, treatment of the fish to remove the parasites greatly reduced mortalities.     

Uptake of erythromycin by first-feeding sockeye salmon,Oncorhynchus nerka (Walbaum), fed live or freeze-dried enriched adult Artemia or medicated pellets

The potential to use adult Artemia to deliver erythromycin to first-feeding sockeye salmon, Oncorhynchus nerka (Walbaum), was investigated in three trials. In the first trial, first-feeding sockeye were fed live erythromycin enriched adult Artemia or pellets containing equal amounts of erythromycin for 35 days. At the end of the trial, tissue erythromycin concentration of the fish fed the live Artemia was significantly greater (P < 0.05, 25.52 +/- 1.29 microg mL(-1); mean +/- SEM), than the tissue concentration of the fish fed the pellets (0.72 +/- 0.01 microg mL(-1)). In the second trial, first-feeding sockeye were fed either live or freeze-dried bioencapsulated erythromycin (adult Artemia) or pellets containing erythromycin daily for 21 days. Mean daily erythromycin concentration in fish fed the freeze-dried Artemia, live Artemia, or pellets did not differ significantly. In the third trial, apparent erythromycin digestibility was determined. Significantly more (P < 0.05) erythromycin was retained by juvenile sockeye fed freeze-dried bioencapsulated erythromycin (98.3 +/- 1.0%) compared with medicated pellets (89.2 +/- 1.7%). Uptake of bioencapsulated erythromycin from adult Artemia (live or freeze-dried) appears to be greater than uptake from pellets. Freeze-dried and live Artemia were equally effective at delivery suggesting enriched freeze-dried adult Artemia could be produced into a highly palatable, consistent, off-the-shelf product.     

载铜蒙脱石对嗜水气单胞菌粘附尼罗罗非鱼上皮细胞的影响

采用尼罗罗非鱼上皮细胞培养模型，观察嗜水气单胞菌对鳃上皮、皮肤上皮、肠上皮细胞的粘附率，研究载铜蒙脱石对细菌粘附的阻断作用及其对细菌粘附引起鱼上皮细胞膜生物特性变化的影响。结果显示：嗜水气单胞菌与鱼上皮细胞均有不同程度的粘附作用，粘附率大小顺序为鳃上皮〉皮肤上皮〉肠上皮细胞。载铜蒙脱石均显著降低了嗜水气单胞菌对鳃、皮肤和肠上皮细胞的粘附率（P〈0．05），而且对不同上皮细胞的粘附阻断率无显著差异。嗜水气单胞菌粘附肠上皮细胞后细胞膜生物特性发生了显著变化，与正常对照组相比，嗜水气单胞菌粘附鱼上皮细胞后，胞浆游离钙和细胞膜磷脂酶A2活性显著上升。载铜蒙脱石可显著降低由于嗜水气单胞菌粘附鱼上皮细胞引起的胞浆游离钙和细胞膜磷脂酶A2活性的升高。结果表明，载铜蒙脱石可有效阻断病原菌粘附，从而防治细菌感染和细菌移位。     

Effects of chloramphenicol, erythromycin, and furazolidone on growth of Isochrysis galbana and Chaetoceros gracilis

This study focused on determining the effects of antibiotics on microalgae used as food for scallop larvae. Six different dose levels of chloramphenicol, erythromycin, and furazolidone were added to cultures of Isochrysis galbana and Chaetoceros gracilis. An in vivo experiment was subsequently conducted to determine the effect of chloramphenicol and erythromycin on larval survival of the Pacific calico scallop Argopecten ventricosus in tanks and on the population of its associated bacteria. Results showed that growth of I. galbana was not significantly affected by chloramphenicol or erythromycin at the test doses of 0.5, 1.0, 3.0, 6.0, 9.0, and 12.0 mg/l. C. gracilis was significantly sensitive to erythromycin and chloramphenicol at doses higher than 0.5 and 3.0 mg/l, respectively. Furazolidone inhibited the growth of both I. galbana and C. gracilis at all test doses. Results showed that exposure of scallop larvae to a dose of 6 mg/l chloramphenicol or erythromycin did not significantly affect growth of I. galbana, significantly enhanced survival of the scallop larvae, and inhibited the growth of Vibrio spp. in tanks. This study demonstrated the adverse effect of chloramphenicol, erythromycin and furazolidone on I. galbana and C. gracilis microalgae but the positive effect on survival of the scallop larvae, decreasing associated bacterial population.     

Characterization of the pattern of cytokeratin proteins in the epidermal cells of loach,Misgurnus anguillicaudatus (Cyprinformes)

The pattern of cytokeratin proteins in the epidermal cells of loach was studied by immunotechniques and partial separation of the epidermal cells. Two monoclonal antibodies, namely 8F7 and 1C45, against the cytokeratin proteins of the loach epidermis were prepared. these two monoclonal antibodies exhibit distinctive results in immunohistochemical staining. The 8F7 monoclonal antibody stains mainly with the epithelial cells, while the 1C45 monoclonal antibody stains specifically with the club cells. The pattern of cytokeratin proteins in the club cells and the epithelial cells of various epidermal layers was further determined by partial separation of these cells. Immunoblotting analysis of the cell fractions confirms the cytokeratin proteins to be differentially expressed in the club cells and the epithelial cells. However, the cytokeratin proteins expressed in the epithelial cells of the basal, middle and outer layers are same. The results indicate that differentiation of the epithelial cells seems limited during their translocation from basal to upper layers, but in those cells that do differentiate into club cells, the cytokeratin pattern changes.     

Invasion and survival of Photobacterium damselae subsp. piscicida in non-phagocytic cells of gilthead sea bream, Sparus aurata L.

Fluorescence microscopy and gentamicin protection assays were used to investigate the ability of four Photobacterium damselae subsp. pisicida (Phdp) strains to adhere to and to invade the fish epithelial cell line, SAF-1, derived from Sparus aurata . All strains tested were detected intracellularly using both techniques, although internalization levels varied among strains. Treatment with cytochalasin D and experiments carried out at 4 °C demonstrated that a functional host cell cytoskeleton and active cell metabolism are necessary for bacterial internalization. Intracellular bacteria were detected for up to 7 days with a round morphology and were stained with DAPI, indicating that some bacterial cells may remain viable inside SAF-1 cells. Our in vitro findings indicate that Phdp are capable of adhering, entering and surviving within the non-phagocytic epithelial cell line SAF-1, which may be important for persistence and establishment of a carrier state in S. aurata .     

Acute LD50 and kidney histopathology following injection of erythromycin (Erythro-200) and its carrier in spring chinook salmon, Oncorhynchus tshawytscha (Walbaum)

We evaluated the toxicity of commercial injectable erythromycin (Erythro-200) and the injectable drug carrier alone (PEG-400, ethyl alcohol and ethyl acetate) in sub-adult spring chinook salmon, Oncorhynchus tshawytscha (Walbaum). Both compounds were toxic to salmon when administered in high dosages. The 50% lethal dose (LD₅₀) of the erythromycin within its carrier (Erythro-200) at 12 °C was estimated at 429 mg erythromycin kg^–1 or 2.145 mL kg^–1 when measured by volume. The LD₅₀ of the drug carrier alone was 2.95 mL kg^–1 by volume or equivalent to 3.21 g kg^–1 of the carrier. We evaluated and scored the histopathology in kidney sections removed from fish 96 h after injection with Erythro-200, the drug carrier alone, or sterile phosphate buffered saline (PBS) that served as a reference control. Kidneys from fish injected with saline had lowest average pathology scores; those injected with the carrier had moderate scores; and the pathology score was highest in tissues from erythromycin-injected fish. In tests at 12 °C, vacuoles were more prominent in proximal and distal tubules of fish injected with higher dosages of 403 and 545 mg erythromycin kg^–1 than in tissues from fish injected with 299 mg kg^–1.     

三角帆蚌珍珠囊细胞的分泌活动

本文对三角帆蚌珍珠囊细胞的分泌活动进行了亚显微结构的研究。发现珍珠囊表皮细胞能积极地进行物质合成 ,这些物质主要是蛋白质、硫酸化粘多糖和中性粘多糖 ,并将这些物质以微绒毛分泌、块状物分泌、细胞间隙分泌和大颗粒分泌等多种方式分泌到珍珠囊腔 ;而位于珍珠囊表皮细胞之间的、来源于外套膜结缔组织的腺细胞则含有丰富的中性粘多糖 ,并通过开口式分泌的方式释放到珍珠囊腔 ,同珍珠囊表皮细胞分泌的物质一起 ,共同参与珍珠结晶层的形成 ;珍珠囊细胞分泌的多样性与珍珠组成的复杂有关 ;珍珠囊细胞的分泌活动具有节律性和区段性的特点 ,这种区段性和节律性的分泌与珍珠多层结晶纤层的形成相适应。     

Opportunistic Predation of the Sea Urchin,Lytechinus variegatus,by the Amphipod,Elasmopus levis,in an Intensive Inland Culture System

Invasive and/or opportunistic organisms can negatively influence the success of sea urchin culture operations. To our knowledge, pests associated with sea urchin aquaculture have not been reported. In this study, we report the predatory association of the amphipod, Elasmopus levis (Amphipoda: Melitidae), with the sea urchin, Lytechinus variegatus (Echinodermata: Echinoidea), within an intensive inland culture system. Wild‐caught Ly. variegatus broodstock were placed in an intensive culture system, and within their canopy of spines or within transport seawater, populations of epifauna (including amphipods) and microfauna were most likely transferred into the culture system. A growing population of the amphipod developed over time. Associations of the amphipod with the sea urchin were observed to negatively affect the health of the sea urchin, and in some cases, epithelial tissue from the sea urchin test appeared to be consumed by the amphipod. An infestation of these amphipods in commercial sea urchin cultures could have the potential to be costly, and we recommend quarantine and/or water purification procedures be considered to prevent the introduction of pests to inland culture systems.     

Bioassay Procedure for the Evaluation of Erythromycin Activity in Aquaculture Environments

A new bioassay procedure was developed for the detection of erythromycin in aquaculture samples using a strain of a Stenotrophomonas as an indicator organism. Conventional disk-plate and well-plate radial diffusion assay procedures were developed, as well as a third procedure using the same indicator organism in Luria-Bertani (LB) broth, supplemented with the indicator dye Brilliant Black (40 μg/mL) in a multi-well microtiter plate. For both the disk-plate and well-plate radial diffusion assays, the response reflected in the size (width) of the growth inhibition zone, which was linear over the tested concentration range of 0.05 to 2.0 μg erythromycidml. The limit of quantitation of the bioassay was at 0.05 μg erythro-mycidml. Among the three methods of assay tested with Stenotrophomonas sp., the semi-quantitative dye reduction method is easy to read and is not diffusion dependent. This method allows for processing of more samples and more replication on a single titer plate. This new indicator organism is specific for erythromycin when tested in the presence of other antibacterial agents, i.e., oxytetracycline (Terramycin®) and/or Romet-Ma. This new bioassay procedure is suitable for quantitation of low concentrations of erythromycin in aquaculture water and sediment samples.     

Fine structure and differentiation of the alimentary canal in captive-bred Japanese eel Anguilla japonica preleptocephali

ABSTRACT:   The fine structure of the alimentary canal in preleptocephali produced by artificially matured Japanese eel was examined. At 1 day posthatch (dph), the alimentary canal was found only above the dorsal side of the yolk mass, and the epithelium was composed of a single layer of epithelial cells. By 5 dph, the alimentary canal was divided into three segments based on the structure of the epithelial cells: foregut, midgut and hindgut, corresponding to the future esophagus, intestine and rectum, respectively. After 7 dph, the epithelium in the foregut was surrounded by a circular muscle layer, suggesting a role in the transportation of food materials. The epithelial cells of the midgut exhibited well-developed membranous structures, which are deduced to be invaginations of the cytoplasmic membrane. Pinocytotic invaginations and vacuoles were observed in the epithelial cells of the hindgut; this observation suggests that this region is involved in the uptake of food. Significant changes in morphological features of the epithelial cells in each segment were observed until 7 dph; however, these were not evident between 7 dph and 13 dph. Consequently, the differentiation of the alimentary canal was completed by 7 dph, and preleptocephalus had developed the ability to absorb food by 7 dph.     

A light and electron microscope study of epitheliocystis in juvenile steelhead trout, Salmo gairdneri Richardson

Abstract. Ultrastructural analysis of epitheliocystis organisms from gills of anadromous juvenile steelhead trout, Salmo gairdneri Richardson, revealed the presence of several distinct forms. Two oval infectious organisms resembled previously described agents and had chlamydia-like characteristics. One form had a distinct oval head region from which a tail-like structure projected. These prokaryotic forms have an ultrastructural appearance which has not been described in previous reports of epitheliocystis. Morphological analyses of gill epithelial cells of S. gairdneri suggest that each cyst remains contained within the cytoplasm of a single host cell.