Fluorescence microscopy detection of dermatophytes with Blankophor

It is the first time that a fluorescent microscopic rapid-stain process is compared with the alkali method and with mycological culture examination. 810 skin scrapings primarily from horse, cat, and dog were available for analysis. The preparation of the samples for the fluorescent microscope test is easy and quick. The fluorescent stain solution keeps sufficiently long. When the slide preparations are looked at under the fluorescent microscope, the mycological elements light up and can easily be distinguished under survey enlargement. This leads to a considerable reduction of evaluation time. The microscopic proof of dermatophytes is considerably improved by the introduction of the fluorescent microscopic technique into mycological diagnosis, as this process is clearly superior to the alkali method. Dermatophytes are identified and finally evaluated after finishing the cultural analysis. Furthermore, the fluorescent microscopic proof of yeasts of the kind of Pityrosporum yeasts and of Demodex mites is possible. The not inconsiderable costs of the technical equipment may stand in the way of general and routine use of the fluorescent microscope for diagnosing dermatophytes. However, for laboratories with a large number of submitted skin scrapings, this process can be rated as a useful enrichment of their diagnostic potential.     

Parapox in a pigmy chimpanzee]

Parapoxvirus was found using electron microscopy in skin scrapings from a pygmy chimpanzee with a pustular skin disease. Clinical findings are described and possible ways of transmission are discussed. It seems remarkable that this is the first time parapoxvirus has been diagnosed in a nonhuman primate.     

'No-mucus skin disease' of rainbow trout, Oncorhynchus mykiss (Walbaum): a case report

Abstract A skin disease of intensively reared salmonids in Ontario hatcheries, known to the farmers as'no-mucus skin disease', is reported for the first time. It was characterized by erosive and ulcerative lesions found mainly on the flanks of fingerlings, which resulted in exposure of the tips of the scales. Associated with these lesions were colonies of bacteria seen in the SEM to be clustered round the mucous cell pores and under-running the margins of epithelial cells. The cause of this condition is unknown, although the response of fish to formalin treatment, and the presence of bacilli seen in skin scrapings and in the SEM, suggest that bacteria are responsible.     

Cutaneous antibodies in excised skin from channel catfish, Ictalurus punctatus Rafinesque, immune to Ichthyophthirius multifiliis

This study determined whether cutaneous antibodies were present in the excised skin of channel catfish, Ictalurus punctatus Rafinesque, immune to Ichthyophthirius multifiliis Fouquet (Ich). Theronts were immobilized on or near the excised skin from immune fish. The survival of immobilized theronts was significantly reduced after exposure for 8 h to the culture of excised skin from immune fish. Culture fluids from excised skin of immune fish immobilized theronts with a peak in the immobilization titre at 24 h post-exposure. Immobility of theronts in the culture fluid from immune skin was removed after immunoabsorption with theronts. Indirect immunofluorescent staining of theronts treated with culture fluid from excised skin of immune fish revealed strong and uniform fluorescence on the cilia and cell surface of theronts. Western blot analysis of the culture fluid from immune fish revealed a 70-kDa band which corresponded to the molecular weight of catfish immunoglobulin heavy chain. The results of this study show that cutaneous antibodies to Ich theronts were present in and released from the excised skin from fish immune to Ich. Immobilization and killing of the theronts are two characteristics of the antibody response that appear to prevent the successful invasion of theronts into excised skin.     

Quantitative PCR demonstrates a positive correlation between a Rickettsia-like organism and severity of strawberry disease lesions in rainbow trout, Oncorhynchus mykiss (Walbaum)

Strawberry disease (SD) is an inflammatory skin disorder in rainbow trout, Oncorhynchus mykiss (Walbaum). The aetiology of SD is unknown although the 16S rDNA sequence of a Rickettsia-like organism (RLO) has been associated with SD lesions using a nested PCR assay. In this study, we developed a Taqman quantitative PCR assay (qPCR) that targeted the RLO 16S rDNA sequence to examine the distribution of RLO relative to lesion status. We compared 18 lesion samples from 13 fish representing high or low lesion severity as judged by gross examination. QPCR results showed that there was a higher number of RLO sequences in high severity lesions (mean of 12,068 copies) compared with fewer copies of RLO sequence in low severity lesions (mean of 3287 copies, P = 0.012). Grossly normal skin samples (n = 13) from SD-affected fish were all negative by qPCR except two samples (121 and 139 copies). The qPCR assay described herein is a useful tool to investigate the role of RLO in SD in the absence of a culture system for RLO. Our results demonstrate a positive correlation between copy number and lesion severity consistent with the hypothesis that the RLO is the aetiologic agent of SD.     

Antibody mediated immune response against Ichthyophthirius multifiliis using excised skin from channel catfish, Ictalurus punctatus (Rafinesque), immune to Ichthyophthirius

This study investigated antibody mediated immune response against Ichthyophthirius multifiliis (Ich) by determining whether theronts would retain the potential for reinfection, both in vitro and in vivo, after treatment with the culture fluid of excised skin from channel catfish, Ictalurus punctatus , immune to Ich. The invasion was reduced significantly ( P  < 0.05) for theronts treated with the immune culture fluid compared with those treated with the culture fluid from naive fish. The treatment of theronts with the immune culture fluid greatly reduced the size and survival of trophonts compared with those treated with the culture fluid from naive fish. Fewer fish were infected and the infection density was less for fish exposed to theronts treated with immune culture fluid. The infection was severe for fish invaded by theronts treated with the culture fluid from naive fish, with a high number of infected fish and heavy density of trophonts per fish. All fish were infected by Ich when exposed to the theronts treated with the immunoadsorbed culture fluid. In summary, results of this study show that cutaneous antibodies in the culture fluid of excised skin from immune fish significantly reduces theront infectivity by immobilizing or weakening theronts.     

Health Status of Ornamental Freshwater Fishes Imported to South Africa: A Pilot Study

Keeping fish is a popular pastime in South Africa and the majority of ornamental fish are imported. A pilot study was initiated to examine the health status of ornamental freshwater fishes imported to South Africa. Four groups of thirty fish each were examined for the presence of external parasites, and processed for virus isolation and for bacterial and mycobacterial culture. The groups consisted of goldfish (Carassius auratus), koi (Cyprinus carpio), guppies (Poecilia reticulata) and cardinal tetras (Cheirodon axelrodi). Mycobacterium fortuitum was isolated from the goldfish and koi. No other significant bacteria were isolated and virus culture was negative for all groups. Skin scrapings and wet gill preparations were made to detect external parasites. Parasites were identified from fixed material. External parasites included Trichodina mutabilis, Ichthyophthirius multifiliis, ciliophorans of the genus Tetrahymena, and monogeneans belonging to the genera Dactylogyrus and Gyrodactylus. This is the first report of Trichodina mutabilis in South Africa. Diseases imported with ornamental fish pose a risk to both indigenous fish populations and the aquaculture industry. This revised version was published online in August 2006 with corrections to the Cover Date.     

Cutaneous antibodies from channel catfish, Ictalurus punctatus (Rafinesque), immune to Ichthyophthirius multifiliis (Ich) may induce apoptosis of Ich theronts

This study explored the existence of apoptosis (programmed cell death) in Ichthyophthirius multifiliis Fouquet (Ich) theronts and determined the effect of cutaneous antibodies in skin culture fluid from fish immune to Ich on theront apoptosis. Apoptosis was detected in theronts and was clearly distinguished by fluorescent microscopy after staining with acridine orange and propidium iodide. The apoptotic theronts showed characteristic chromatin condensation and nuclear fragments containing chromatin pieces. The externalization of phosphatidylserine on the plasma membrane of apoptotic theronts was detected with fluorescein isothiocyanate-conjugated annexin using flow cytometry. Theront apoptosis was induced using the skin culture fluid from fish immune to Ich, which contained cutaneous antibodies against Ich. The highest apoptosis appeared in theronts exposed to immune skin culture fluid at a 1:10 dilution, compared with those at 1:20 and 1:40 dilutions. A direct correlation was noted between the percentage of apoptotic theronts and exposure duration to immune skin culture fluid. The study indicated that antibody reaction with theronts (immobilization) played an important role in theront apoptosis, but it could not be excluded that other components released from the excised skin had effects on theronts.     

A molecular tool for parentage analysis in the Mediterranean mussel (Mytilus galloprovincialis)

Estimation of selection response and genealogical tracing in family mixtures require an appropriate tool for parentage analysis. In this study, we tested 19 marker loci for parentage analysis allocation in Mediterranean mussel (Mytilus galloprovincialis). To this aim, we reared families in tanks isolated from wild mussel seed, analysed them using the 19 marker loci and characterized their performances based on Mendelian rules. Probabilities of exclusion of a false parent were estimated for different groups of loci and contrasted to the real paternity assignment. Based on this, we chose nine microsatellites with the highest exclusion probabilities and a real paternity assignment of 99.6%. Next, we analysed 600 individuals reared as in the usual production process, where contamination from wild seed is likely. We obtained a real assignment of 94.7% and were able to identify individuals from the wild as the most likely hypothesis to explain the observed incompatibilities with candidate parents. This information was used to evaluate parental contribution in offspring obtained from gamete mixtures of several parents, which bestowed results of interest for future breeding programs of Mediterranean mussel.     

Measurements of oxygen consumption and ammonia excretion of Atlantic salmon (Salmo salar L.) in commercial-scale, single-pass freshwater and seawater landbased culture systems

The oxygen consumption of Atlantic salmon was measured in large culture tanks for a period of 20 months from the parr to the adult stage. In addition, diurnal sampling was conducted for estimation of both oxygen consumption and ammonia excretion. The oxygen consumption was affected especially by temperature, season and smoltification. For parr the oxygen consumption rate was 1–6 mg O₂/kg min and the ammonia excretion rate was 0·037–0·13 mg N/kg min from autumn to spring. The corresponding rates for adult salmon during the period October to July were 1·5–4·5 mg O₂/kg min and 0·075–0·13 mg N/kg min.     

Detection and identification of aquatic mycobacteria in formalin-fixed, paraffin-embedded fish tissues

The isolation of mycobacteria from field samples is problematic, and isolation of the bacterium is sometimes not even attempted. The detection of mycobacteria through traditional histology using formalin‐fixed, paraffin‐embedded (FFPE) tissues is neither sensitive nor specific. However, detection of mycobacterial DNA from FFPE specimens, suspected of being infected with mammalian mycobacteriosis, is a routine clinical procedure. In the present study, a polymerase chain reaction (PCR)‐based method was used to detect and identify mycobacteria in FFPE specimens sampled from fish suspected of being infected with fish mycobacteriosis. A total of 45 fish tissue samples, comprising of 12 tissue samples obtained from experimentally infected fish and the remainder from fish naturally infected with mycobacteria, were analysed using a PCR protocol which amplifies a fragment of the mycobacterial 65 kDa heat‐shock protein (hsp65) gene. PCR‐restriction enzyme analysis and/or sequencing were employed to further analyse the PCR amplicons. The PCR results were compared with those obtained by histology and culture. Mycobacterial DNA was detected in 34 of the 45 samples examined, of which 16 samples (47%) showed granulomatous reactions on histological examination. Using histology as the gold standard, no false‐negative PCR results were obtained. Also, considering the presence or absence of granulomas as a diagnostic criterion, the sensitivity and specificity of PCR in 42 of the FFPE tissues were 16/16 (100%) and 8/26 (～30.8%), respectively. Corresponding microbiological cultures were available for 15 cases, of which 13 were pure Mycobacterium cultures. Of these, 13 were PCR positive (100% sensitivity and 50% specificity). The PCR‐based methods used here proved sensitive, specific and rapid for the detection of mycobacteria in routinely processed paraffin wax‐embedded and formalin‐fixed histological samples, and the results of the study suggest that this method has potential use in retrospective epidemiological studies.     

中华鳖5种组织细胞的离体培养实验

采用组织块移植培养技术,对来源于中华鳖(Trionyx sinesis)肝脏、脾脏、肾脏、心脏、皮肤及裙边组织细胞进行了原代培养,细胞均可从组织块中迁出并生长成单层细胞。对原代培养的单层细胞用胰酶-EDTA消化后传代培养,初步建立了可连续传代的中华鳖肝脏、脾脏、肾脏、心脏及皮肤组织细胞系;还对细胞的培养条件、冷冻保藏与复苏、染色体数目等进行了研究,初步确定中华鳖细胞培养的条件为32℃,MEME或R1640培养基,血清浓度为20%(V/V)。二甲基亚砜(DMSO)冻存保藏后,细胞复苏生长良好,中华鳖细胞染色体数目为2n=66。     

越冬期体色变异红罗非鱼差异表达miRNA的鉴定

为了探究红罗非鱼越冬期体色变异的分子机制，实验构建了其2种体色(正常粉红体色和变黑体色)皮肤组织的小RNA文库，每组4个重复，组装后平均每组分别获得12 190 544和11 891 890条过滤后的读长(clean reads)，获得miRNA成熟序列669个，其中337个是已知的miRNAs。鉴定出越冬期变黑体色相对于正常粉红体色鱼的差异表达miRNAs 26个，其中上调11个，下调15个，且12个为已知的miRNAs，可能在红罗非鱼越冬期体色变异过程中发挥重要作用。富集分析发现了大量代谢和体色调控相关的通路。研究表明，红罗非鱼越冬期的体色变异可能是色素细胞的增殖和迁移形成的，与代谢相关的调控通路在其中发挥重要作用，为解析红罗非鱼越冬期体色变异的机制奠定了基础。     

Fishing gear-induced skin ulcerations in Baltic cod, Gadus morhua L

In 1982 a high prevalence of skin ulcerations was observed in Baltic cod in the vicinity of the Danish island of Bornholm. In March the prevalence varied from 6 to 13%, and in May it had increased to between 26 and 48%. The ulcerations had a sequential development. The initial stage appeared as severe skin abrasions in the area from the pectoral fin posterior to the level of the anus. The skin abrasions developed into large spots of necrotic dermal tissue with a diameter of 2-5 cm. The central part of the necrotic area sloughed off exposing a large haemorrhagic ulcer. Characteristically, most of the ulcerations on the trunk occurred bilaterally. A common histopathological finding was coagulation necrosis of the muscular tissue. The ulcerated fish were mainly 24-28 cm in length. A correlation between high concentrations of cod around Bornholm, high fishing activity in the area, length of fish escaping from the nets, combined with bilateral occurrence of the ulcers, strongly indicates that the skin ulcers were induced by the fishing gear. Features of the pathology could be linked to the temporary retention of cod in trawl meshes.     

Pharmacokinetics of Dietary 13C-Labeled Docosahexaenoic Acid and Docosapentaenoic Acid in Red Sea Bream Chrysophrys major

The objectives of this study were to investigate: 1) the pharmacokinetics of dietary docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA) using ¹³C-labeled fatty acids; 2) the interorgan transport of DHA in the red sea bream by monitoring the DHA level of several organs; and 3) the relationship between the plasma DHA level and optimum dietary DHA level in the plasma of the red sea bream Chrysophrys major . For this purpose, a mixture of 38.5% of [¹³C]DHA, 8.5% of [¹³C]DPA, and 4.2% of [¹³C]palmitic acid were given to the red sea bream at dose level of 8.0, 16.0, and 47.9 mg/kg by a single oral administration. For [¹³C]DHA, the maximum plasma concentration (t_max) occurred at 2.00–3.00 h after the oral administration. The peak plasma concentration (C_max) and the area under the plasma concentration-time curve to 24 h (AUC_0-24 for [¹³C]DHA level linearly increased with respect to dosage. [¹³C]DHA appeared in each organ (plasma, erythrocyte and the fat body of the orbit, liver, intestine, skin, brain, heart and muscle) at 0.5 h and was observed until 24 h. From the values determined for the pharmacokinetic parameters, the range of the effective plasma DHA level for normal growth of the red sea bream was suggested to be between 21.0 and 40.3 μg/mL. For [¹³C]DPA, the AUC_0-24 and C_max values also linearly increased with the dosage, but t_max did not depend on it.     

Primary and secondary structures of grammistins, peptide toxins isolated from the skin secretion of the soapfish Pogonoperca punctata

SUMMARY: Six peptides (grammistins Pp 1, 2a, 2b, 3, 4a and 4b) with both hemolytic and ichthyotoxic activities were isolated from the skin secretion of the soapfish Pogonoperca punctata by gel filtration and reverse-phase high performance liquid chromatography. They were completely sequenced and found to be simple peptides differing from the previous suggestion that they are peculiar peptides with tertiary amine or quaternary ammonium base moieties. Grammistins Pp 1, 2a and 2b were composed of 13 residues, Pp 3 of 25 residues and Pp 4a and 4b of 24 residues. While grammistins Pp 4a and 4b were identical and highly homologous with grammistin Gs 2 previously isolated from another soapfish Grammistes sexlineatus , the other grammistins did not have homologies with any proteinic or peptidic toxins known to date. Judging from circular dichroism data, helical wheel projections and hydrophobic moment plots, all the isolated grammistins are surface-seeking peptides with an abundance in amphiphilic α-helicity, similar to pardaxins from the skin secretion of two species of soles and melittin from bee venom as well as the G. sexlineatus grammistins.     

Natural abundance of 15N and 13C in fish tissues and the use of stable isotopes as dietary protein tracers in rainbow trout and gilthead sea bream

For developing efficient diets, two sets of experiments examined whether the use and allocation of dietary protein can be traced by labelling with stable isotopes (¹⁵N and ¹³C) in two culture fish ( Oncorhynchus mykiss and Sparus aurata) . In the first experiment, natural abundance and tissue distribution of these isotopes were determined, by measuring the δ¹³C and δ¹⁵N values by isotopic ratio mass spectrometry, in fingerlings (14–17 g) adapted to diets differing in the percentage of fish meal replacement by plant protein sources. For both species, δ¹⁵N and δ¹³C were greater in tissues with higher protein and lower lipid content. Delta ¹⁵N of diets and tissues decreased as replacement increased, suggesting δ¹⁵N can be used as a marker for dietary protein origin. The ¹⁵N fractionation (δ¹⁵N fish − δ¹⁵N diet) differed between groups, and could thus be used to indicate protein catabolism. In the second experiment, fish (75–90 g) of each species ingested a diet enriched with ¹⁵N-protein (10 g kg⁻¹ diet) and ¹³C-protein (30 g kg⁻¹ diet). These proportions were suitable for determining that the delta values of tissue components were high enough above natural levels to allow protein allocation to be traced at 11 and 24 h after feeding, and revealed clear metabolic differences between species.     

Mechanism of Pseudoalbinism in Flatfish: An Association Between Pigment Cell and Skin Differentiation

Two groups of flounder P. olivaceus larvae were reared under different conditions to provide either normally pigmented or pseudoalbinic metamorphosed juveniles. The process of the differentiation of skin and pigment cells during postembryonic development was analyzed by means of histological, histochemical and immunofluorescent assay methods. In parallel with these assays, the differentiation of pigment cells was examined with the use of organ and cell culture in vitro . The results obtained strongly suggested that pseudoalbinism was evoked as a result of disruption of the mechanisms that controlled the establishment of asymmetric skin structures during metamorphosis.