Purification and characterization of hemagglutinating proteins from Poker-chip Venus (Meretrix lusoria) and Corbicula clam (Corbicula fluminea).

Hemagglutinating proteins (HAPs) were purified from Poker-chip Venus (Meretrix lusoria) and Corbicula clam (Corbicula fluminea) using gel-filtration chromatography on a Sephacryl S-300 column. The molecular weights of the HAPs obtained from Poker-chip Venus and Corbicula clam were 358 kDa and 380 kDa, respectively. Purified HAP from Poker-chip Venus yielded two subunits with molecular weights of 26 kDa and 29 kDa. However, only one HAP subunit was purified from Corbicula clam, and its molecular weight was 32 kDa. The two Poker-chip Venus HAPs possessed hemagglutinating ability (HAA) for erythrocytes of some vertebrate animal species, especially tilapia. Moreover, HAA of the HAP purified from Poker-chip Venus was higher than that of the HAP of Corbicula clam. Furthermore, Poker-chip Venus HAPs possessed better HAA at a pH higher than 7.0. When the temperature was at 4°C-10°C or the salinity was less than 0.5‰, the two Poker-chip Venus HAPs possessed better HAA compared with that of Corbicula clam.

Lactococcus lactis subsp. lactis infection in Bester sturgeon, a cultured hybrid of Huso huso × Acipenser ruthenus, in Taiwan.

Approximately 5300 hybrid sturgeons with an average body weight of 600-800 g were farmed in 3 round tankers measuring 3m in diameter each containing 28,000 L of aerated groundwater. According to the owner's description, the diseased fish had anorexia, pale body color, and reddish spots on the abdomen. The morbidity and lethality rates in this outbreak were about 70% (3706/5300) and 100% (3706/3706), respectively. The clinical examination revealed enteritis, enlarged abdomen, and rapid respiration rate. The gross findings revealed a volume of about 4 mL of ascites. The histopathological examination showed multiple massive, hemorrhagic or coagulative necrotic foci in the liver and spleen. Furthermore, there was diffuse infiltration of glycogen in hepatic cells, and a few polymorphonuclear and mononuclear leucocytes were observed surrounding the spleen. Some bacterial clumps were noted around the necrotic foci. We also observed that there was moderate to severe, acute, multifocal, coagulative necrosis in the renal parenchyma, with some necrotic foci present beneath the margin of the kidney. Additionally, multifocal, coagulative necrosis was found in the pancreas. Results of microbiologic examinations, including biochemical characteristics, PCR amplification of 16S rRNA gene, sequencing and comparison, and phylogenetic analysis, revealed the pathogen of this infection was Lactococcus lactis subsp. lactis, and based on the results of an antimicrobial agent sensitivity test the bacterium was only sensitive to ampicillin and florfenicol. Additionally, results of in vivo experimental infections in hybrid tilapia showed that 1×10(8) and 1×10(9) CFU/mL of our isolate caused death in all fish and LD(50) values ranged from 10(2) to 10(5) CFU/mL. To the best of the authors' knowledge, this is the first reported case of Lactococcus lactis subsp. lactis infection in hybrid sturgeon.

Relationship between age, plasma renin activity, and renal resistive index in dogs.

The renal resistive index (RI) value of 0.73 has been proposed as the upper limit in normal adult dogs. In humans, changes in RI with age are associated with plasma renin activity. There are relatively few equivalent reference data for dogs. We obtained reference RI data from 22 clinically healthy dogs <4 months of age and 33 healthy dogs between 4 months and 7 years of age. An association between the RI and plasma renin activity was investigated. The mean RI in the older dogs was 0.65 +/- 0.05 vs. 0.75 +/- 0.05 in dogs <4 months of age. The mean plasma renin activity in the older dogs was 1.18 +/- 1.03 vs. 4.23 +/- 3.09 ng/ml/h in dogs <4 months of age. There was a weak linear relationship between the RI and plasma renin activity (r2 = 0.280, P < 0.01) in dogs <4 months of age. Also in these younger dogs, RI was negatively correlated with age (r2 = 0.682, P < 0.01). The RI was higher in dogs <4 months of age than in older dogs. Therefore, the mean renal RI is slightly higher in young dogs than reported for an older population and interpretation of the RI must include an assessment of patient age.

The effects of different anesthetic agents on short electroretinography protocol in dogs.

The purpose of this article was to investigate the effects of sedatives and general anesthetics, such as tiletamine-zolazepam, medetomidine, and isoflurane on the short ERG protocol. Six healthy mongrel dogs were assessed by a convenient short ERG protocol with the owners' consent. The amplitudes of a-wave and b-wave, as well as the implicit time of ERG under different anesthesia statuses, were recorded and analyzed. The amplitudes of ERG waves were not significantly different between tiletamine-zolazepam and medetomidine groups, except in b-wave after 5 min dark adaptation (140 +/- 42 microV in tiletamine-zolazepam and 101 +/- 32 microV in medetomidine, p<0.01). The amplitude of ERG recorded in isoflurane (5 +/- 3 microV of a-wave and 12 +/- 6 microV of b-wave under light adaptation; 41 +/- 19 microV of b-wave after 1 min dark adaptation; 28 +/- 15 microV of a-wave and 58 +/- 32 microV of b-wave after 5 min dark adaptation) were significantly different from tiletamine-zolazepam (8 +/- 2 microV of a-wave and 24 +/- 9 microV of b-wave under light adaptation; 117 +/- 44 microV of b-wave after 1 min dark adaptation; 59 +/- 18 microV of a-wave and 140 +/- 42 microV of b-wave after 5 min dark adaptation), except in a-wave after 1 min dark adaptation (39 +/- 13 microV in tiletamine-zolazepam and 34 +/- 17 microV in isoflurane). Comment-General anesthesia had significantly lower amplitudes in the dark-adapted group compared with the sedation group. Therefore, tiletamine-zolazepam is a desirable choice for the short ERG protocol in dogs.

Development of an enrofloxacin immunosensor based on label-free electrochemical impedance spectroscopy.

Enrofloxacin is the most widespread antibiotic in the fluoroquinolone family. As such, the development of a rapid and sensitive method for the determination of trace amounts of enrofloxacin is an important issue in the health field. The interaction of the enrofloxacin antigen to a specific antibody (Ab) immobilized on an 11-mercapto-undecanoic acid-coated gold electrode was quantified by electrochemical impedance spectroscopy. Two equivalent circuits were separately used to interpret the obtained impedance spectra. These circuits included one resistor in series with one parallel circuit comprised of a resistor and a capacitor (1R//C), and one resistor in series with two parallel RC circuits (2R//C). The results indicate that the antigen-antibody reaction analyzed using the 1R//C circuit provided a more sensitive resistance increment against the enrofloxacin concentration than that of the 2R//C circuit. However, the 2R//C circuit provided a better fitting for impedance spectra, and therefore supplies more detailed results of the enrofloxacin-antibody interaction, causing the increase of electron transfer resistance selectively to the modified layer, and not the electrical double layer. The antibody-modified electrode allowed for analysis of the dynamic linear range of 1-1000 ng/ml enrofloxacin with a detection limit of 1 ng/ml. The reagentless and label-free impedimetric immunosensors provide a simple and sensitive detection method for the specific determination of enrofloxacin.

Multiple tiny ectopic sebaceous glands discovered throughout entire esophageal tract.

A 45-year-old woman complaining of abdominal fullness was referred for endoscopic examination. She was a non-smoker and non-drinker. An endoscopic examination revealed the presence of more than 100 tiny, rounded, elevated, yellowish lesions <0.5 mm in diameter scattered throughout the upper and lower esophagus. Based on the endoscopic examination results, her stomach manifested symptoms of mildly superficial gastritis. Histopathologic examination of the esophagus biopsy specimen revealed that some of the lobules of the cells displayed typical sebaceous differentiation covered by a squamous epithelium. No evidence of inflammatory reaction, hair follicles, or malignancy was found. The patient's blood and serum findings were unremarkable. Our final diagnosis was multiple tiny ectopic sebaceous glands in the esophagus. This is an interesting and rare case of esophageal sebaceous glands distributed throughout the entire esophagus. Because there were no esophageal symptoms or/and eating problems, the patient did not require endoscopic surgery or other treatment. Follow-up examinations were recommended at intervals between 6 months and 1 year. At the 2-year follow-up, an endoscopic examination revealed no change in the size or the number of the tiny ectopic esophageal sebaceous glands.

Sensitive label-free biosensors by using gap plasmons in gold nanoslits.

The detection sensitivities of gap plasmons in gold nanoslit arrays are studied and compared with surface plasmons on outside surface. The nanoslit arrays were fabricated in a 130 nm-thick gold film with various slit widths. For transverse-magnetic (TM) incident wave, the 600 nm-period nanoslit array shows two distinguishable transmission peaks corresponding to the resonances of gap plasmons and surface plasmons, respectively. The surface sensitivities for both modes were compared by coating thin SiO(2) film and different biomolecules on the nanoslit arrays. Our experimental results verify gap plasmons are more sensitive than conventional surface plasmons. Its detection sensitivity increases with the decrease of slit width. The gap plasmon is one order of magnitude sensitive than the surface plasmon for slit widths smaller than 30 nm. We attribute this high sensitivity to the large overlap between biomolecules and nanometer-sized gap plasmons.

Antibiotic susceptibility and prevalence of erythromycin ribosomal methylase gene, erm(B) in Streptococcus spp.

The aim of this study was to investigate drug resistance and the genetic relatedness of erythromycin-resistant Streptococcus spp. from different animals and humans in Taiwan. Cumulatively, 248 isolates were collected from 15 animal species and human patients and the susceptibilities of the isolates to six antimicrobial agents including azithromycin (AZI), clarithromycin (CLAR), erythromycin (ERY), spiramycin (SPIR), amoxicillin (AMO), and enrofloxacin (ENRO) were determined by the agar dilution method. The results indicated that resistance among the 248 strains was highest for SPIR, followed by ENRO, CLAR, ERY, AZI, and AMO. The most common resistotypes of the isolates from mammals and aquatic animals were AZI-CLAR-ERY-SPIR (27.5%) and SPIR (55.1%), respectively. The presence of ERY-resistant genes was confirmed by PCR. The erm gene was amplified from 28 isolates (20.6%) by PCR for further investigation. The predominant erm gene in the ERY-resistant isolates was the erm(B) gene. The phylogenetic analysis of the erm(B) gene results indicated that there was a close genetic relationship among all the strains but the genotypic clusters did not show clear segregation of the isolates according to the source or region.

Sensitive biosensor array using surface plasmon resonance on metallic nanoslits.

Chip-based biosensor arrays for label-free and high-throughput detection were fabricated and tested. The sensor array was composed of a 150-nm-thick, 50-nm-gap, and 600-nm-period gold nanoslits. Each array size was 100 mumx100 mum. A transverse-magnetic polarized wave in these metallic nanostructures generated resonant surface plasmons at a wavelength of about 800 nm in a water environment. Using the resonant wavelength shift in the nanoslit array, we achieved detection sensitivity up to 668 nm per refractive index unit, about 1.7 times larger than that reported on an array of nanoholes. An antigen-antibody interaction experiment in an aqueous environment verified the sensitivity in a surface binding event.

Simultaneous flow cytometric assessment for cellular types and phagocytic abilities of the haemocytes of the hard clam, Meretrix lusoria.

The aim of this study was to devise a simple protocol for flow cytometric analysis to separate various haemocytic populations of the hard clam Meretrix lusoria based on the mitochondrial membrane potential diversity detected by the fluorescence probe 3,3-dihexyloxacarbocyanine iodide (DiOC6). Compared with the traditional technique for separation of haemocytic populations, continuous Percoll gradient centrifugation, our novel method was more efficient and yielded a higher ratio in separating the clams' haemocytic populations. Based on fluorescence 1 (FL-1) and side scatter (SSC) analysis for haemocytes stained with various fluorescent densities of DiOC6 using flow cytometer, the data showed that there were three obvious cell regions R1, R2, and R3, identified by hyalinocytes, small granulocytes and large granulocytes, respectively. At the same time our results showed that the percentages of haemocytes in R1, R2, and R3 were 49.71+/-0.65%, 19.35+/-00.74%, 30.94+/-0.69%, respectively. After classifying the haemocytic populations, phagocytic activity of the haemocytes was simultaneously analysed with phycoerythrin (PE)-labeled Vibrio vulnificus and detected by flow cytometry. Our results demonstrated that there were higher percentages of large granulocytes compared with hyalinocytes and the percentage of small granulocytes was related to the mitochondrial membrane potential and phagocytic activities.

In vivo effects of adding singular or combined anti-oxidative vitamins and/or minerals to diets on the immune system of tilapia (Oreochromis hybrids) peripheral blood monocyte-derived, anterior kidney-derived, and spleen-derived macrophages.

Macrophage function is an important factor for resistance to infection and anti-oxidative vitamins and minerals can affect how macrophages function in fish. We report the in vivo effect of adding singular or combined vitamins (A, C, and E) and/or minerals (Se, Zn, Cu, Mn, and Fe) in diets on the immune system of tilapia (Oreochromis hybrids) peripheral blood monocyte-derived, anterior kidney-derived, and spleen-derived macrophages. An optimal dose of vitamins and/or minerals in diets increased macrophage proliferation and protective activity, maintained macrophage viability, increased body weight and length, and increased lysozyme activity, however, at improper doses and combinations of vitamins or minerals a decrease was observed. Furthermore, vitamins and/or minerals at any doses and combinations in diets decreased superoxide and nitric oxide production. Therefore, appropriate doses and combinations of vitamins and/or minerals in diets may increase tilapia macrophages immunity.

In vitro effects of singular or combined anti-oxidative vitamins and/or minerals on tilapia (Oreochromis hybrids) peripheral blood monocyte-derived, anterior kidney-derived, and spleen-derived macrophages.

The present study was to determine the in vitro effects of singular or combined anti-oxidative vitamins (A, C, and E) and/or minerals (Se, Zn, Cu, Mn, and Fe) on the immune functions of tilapia, Oreochromis hybrids, peripheral blood monocyte-derived, anterior kidney-derived, and spleen-derived macrophages. An optimal dose of vitamins and minerals increased cell viability and lysozyme activity. On the other hand, the above activities decreased at the high doses of combined vitamins (A+C+E group, each 300 microg mL(-1)) or single mineral (Se, Zn, Cu, Mn, and Fe groups, each 200, 800 or 1000 microg mL(-1)). Combining two of the aforementioned vitamins (A+C, A+E, and C+E groups, each 100 microg mL(-1)) was able to prolong cell viable time up to 72 h compared with singular vitamin addition. Before or after adding vitamins or minerals during infection, addition of vitamins decreased the percentage of dead cells and a greater effect was observed for mineral (each 40 or 80 microg mL(-1)) and vitamin (each 100 microg mL(-1)) combinations. A low dose of vitamins increased nitric oxide production and decreased superoxide production, but high dose of vitamins decreased superoxide and nitric oxide productions. Furthermore, minerals also decreased nitric oxide production at concentrations of 40, 80, 200, 800 or 1000 microg mL(-1). The threshold concentrations for cell death by necrosis and/or apoptosis were >1000 and >800 microg mL(-1) for vitamins and minerals, respectively. In conclusion, appropriate concentration of vitamins or minerals can increase tilapia macrophage immunity; nevertheless, extreme concentrations of vitamins or minerals are lethal to cells.

Purification and localization of nitric oxide synthases from hybrid tilapia (Nile tilapia x Mozambique tilapia).

The aims of this study were to purify and localize the nitric oxide synthases (NOSs) from hybrid tilapia (Nile tilapia Oreochromis niloticus x Mozambique tilapia O. mossambicus). The purification procedures involved affinity chromatography with a 2', 5'-ADP-agarose 4B column and ion exchange with a diethylaminoethanol Bio-Gel A column. The results from gel filtration assays showed that the molecular weights of neuronal NOS (nNOS) and inducible NOS (iNOS) were 178 and 120 kDa, respectively. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis results showed that there were three bands with molecular weights of 89, 47, and 29 kDa from the purified nNOS. However, only one band, with a molecular weight of 120 kDa, appeared on the gel from the purified iNOS. Hybrid tilapia nNOS was a dimer structure, while iNOS appeared to be a monomer structure. Moreover, our results revealed that the activities of nNOS and iNOS were significantly higher after the addition of Ca+2 or Mg+2 ions individually. However, when L-arginine and NADPH were present, the addition of 1 mM of either ion did not further increase the activity. The chemical L-N(G)-methyl-L-arginine could inhibit the activities of the purified NOSs with or without L-arginine. Western blot analyses showed only an 89-kDa immunoreactive band from the extracts of cerebrum; however, we did not find the specific bands in other tissues, such as gill, intestine, liver, spleen, and anterior kidney. We found another 120-kDa immunoreactive protein band with the rabbit antirat iNOS serum against iNOS from the extracts of anterior kidney and spleen. The results of immunohistochemistry with the rabbit antihuman nNOS serum indicated that the nNOS existed in the cerebellum, olfactory bulb, diencephalons, and nerve cell bodies and neuronal fibers of the spinal cord. Interestingly, only macrophages from anterior kidney and spleen showed positive reactions with the rabbit antirat iNOS serum. In the same way, the endothelial NOS (eNOS) located in the heart and epithelial cells of the blood vessels reacted positively with the rabbit antibovine eNOS serum.

The effects of five different glycans on innate immune responses by phagocytes of hybrid tilapia and Japanese eels Anguilla japonica.

The aim of this study was to evaluate the immune responses in hybrid tilapia (Nile tilapia Oreochromis niloticus x Mozambique tilapia O. mossambicus) and Japanese eels Anguilla japonica after treatment with five glycans: barley, krestin, MacroGard, scleroglucan, and zymosan. The effects of the glycans on the innate immune responses of the fish were investigated using the phagocytic index (PI), lysozyme activity, complement opsonization, and activation assay. The results of the lysozyme assay demonstrated that the lysozyme activities increased after treatment with glycans. Moreover, based on the PI, treatment with each of the five glycans resulted in increased phagocytic activities in anterior kidney and peripheral blood phagocytes in both tilapia and Japanese eels. The opsonic effect of complement on phagocytosis in tilapia and Japanese eels were investigated using baker's yeast, which served as the activator in the classical complement pathway (CCP) and in the alternative complement pathway (ACP). Tilapia and Japanese eel sera that were treated with glycans greatly enhanced phagocytosis. The classical pathway--hemolytic complement titer (CH50) of Japanese eels treated with glycans was slightly increased in vitro and in vivo. While glycan treatment enhanced the CCP of both species in vitro and in vivo, the alternative pathway-hemolytic complement titer (ACH50) was only increased in vitro and in vivo in glycan-treated tilapia. Thus, it follows that the ACP must have been activated in tilapia treated with glycans. However, in Japanese eels, the ACH50 of the ACP activation assay was undetected in vitro or in vivo due to possible unknown factors in the Japanese eel serum that caused lysis of the rabbit red blood cells. Our study investigated the effects of glycans used to enhance phagocytosis and activate both of the complement pathways involved in stimulating the innate immune responses of Japanese eels and tilapia.

A miniaturized germanium-doped silicon dioxide-based surface plasmon resonance waveguide sensor for immunoassay detection.

A surface plasmon resonance (SPR) waveguide immunosensor fabricated by germanium-doped silicon dioxide was investigated in this study. The designed waveguide sensor consisted of a 10 microm SiO(2) substrate layer (n=1.469), a 10 microm Ge-SiO(2) channel guide (n=1.492) and a 50 nm gold film layer for immobilization of biomolecules and SPR signal detection. The resultant spectral signal was measured by a portable spectrophotometer, where the sensor was aligned by a custom-designed micro-positioner. The results of the glycerol calibration standards showed that the resonance wavelength shifted from 628 to 758 nm due to changes of refractive index from 1.36 to 1.418. Flow-through immunoassay on waveguide sensors also showed the interactions of protein A, monoclonal antibody (mAb ALV-J) and avian leucosis virus (ALVs) resulted in wavelength shifting of 4.17, 3.03 and 2.18 nm, respectively. The SPR dynamic interaction could also be demonstrated successfully in 4 min as the sensor was integrated with a lateral flow nitrocellulose strip. These results suggest that SPR detection could be carried out on designed waveguide sensor, and the integration of nitrocellulose strip for sample filtering and fluid carrier would facilitate applications in point-of-care portable system.

Inhibition of eosinophil survival by a selective inhibitor of phosphodiesterase 4 via the induction of apoptosis.

Selective inhibitors of phosphodiesterases (PDEs) have been suggested to have anti-inflammatory effects on bronchial asthma through the inhibition of chemotaxis, adhesion, degranulation, the respiratory burst, and survival prolongation of eosinophils. However, the mechanisms by which these agents inhibit eosinophil survival remain unclear. We therefore investigated the possible mechanisms of inhibitory effects of selective inhibitors of PDE 3 (cilostazol) and PDE 4 (rolipram) on granulocyte-macrophage colony-stimulating factor (GM-CSF)-mediated eosinophil survival. Purified blood eosinophils were cultured with medium alone or GM-CSF (0.01 ng/ml) in the presence or absence of the agents for up to 6 d. DNA was extracted from freshly isolated eosinophils and eosinophils cultured for 2 d with medium alone, GM-CSF, or GM-CSF in the presence of the agents, and analyzed using agarose gel electrophoresis. The presence of rolipram (10(-4), 10(-5), 10(-6) M), but not cilostazol, significantly inhibited eosinophil survival at days 2, 4, and 6. A laddering pattern was observed in the DNA of eosinophils cultured with medium alone and with GM-CSF in the presence of rolipram. The results reveal that selective PDE 4 inhibitors inhibit GM-CSF-mediated eosinophil survival through the induction of apoptosis.