Sterile Phosphate Buffer Solution Research Articles

Impaired Testicular Function without Altering Testosterone Concentration Using an Anti-Follicular-Stimulating Hormone Receptor (Anti-FSHr) Single-Chain Variable Fragment (scFv) in Long-Tailed Macaques (Macaca fascicularis).

FSHr antibodies have been shown to inhibit the differentiation of spermatogonia to primary spermatocytes, resulting in infertility without a pathological effect on reproductive organs. The aim of this study was to develop single-chain variable fragments (scFvs) against the follicular-stimulating hormone receptor (anti-FSHr) using phage-display technology and to evaluate the effects of intratesticular administration of the anti-FSHr scFv on testicular function and testosterone production. A phage clone against the extracellular domain of FSHr selected from a scFv phagemid library was analyzed for binding kinetics by surface plasmon resonance. Using ultrasound guidance, three adult macaques (M. fascicularis) were administered with 1 mL of 0.4 mg/mL anti-FSHr scFv (treatment) and 1 mL sterile phosphate buffer solution (control) into the left and right rete testis, respectively. Testicular appearance and volume, ejaculate quality, and serum testosterone levels were recorded on day 0 (before injection) and on days 7, 28, and 56 (after injection). Testicular tissue biopsies were performed on day 7 and day 56 to quantify the mRNA expressions of androgen binding protein (ABP), inhibin subunit beta B (IHBB), and vascular endothelial growth factor A (VEGFA). The results demonstrated that the anti-FSHr scFv molecule was calculated as 27 kDa with a dissociation constant (KD) of 1.03 µM. The volume of the anti-FSHr scFv-injected testicle was reduced on days 28 and 56 compared with day 0 (p < 0.05). Total sperm number was reduced from day 0 (36.4 × 106 cells) to day 56 (1.6 × 106 cells) (p < 0.05). The percentage of sperm motility decreased from day 0 (81.7 ± 1.0%) to day 7 (23.3 ± 1.9%), day 28 (41.7 ± 53.4%), and day 56 (8.3 ± 1.9%) (p < 0.05). Sperm viability on day 0 was 86.8 ± 0.5%, which reduced to 64.2 ± 1.5%, 67.1 ± 2.2%, and 9.3 ± 1.1% on days 7, 28, and 56, respectively (p < 0.05). The expression of ABP and VEGFA on days 7 (14.2- and 3.2-fold) and 56 (5.6- and 5.5-fold) was less in the scFv-treated testicle compared with the controls (p < 0.05). On day 56, the expression of IHBB was less (p < 0.05) in the treated testis (1.3-fold) compared with the controls. Serum testosterone levels were unchanged throughout the study period (p > 0.05). This study characterized the anti-FSHr scFv and demonstrated that treatment with anti-FSHr ameliorates testicular function without altering testosterone levels, offering a potential alternative contraceptive for the long-tailed macaques.

The 16S rRNA Gene Sequencing of Gut Microbiota in Chickens Infected with Different Virulent Newcastle Disease Virus Strains.

Simple SummaryNewcastle disease (ND), which is caused by virulent Newcastle disease virus (NDV), is one of the most important viral diseases for chickens and birds. However, the intestinal pathogenesis of NDV is still poorly understood. To preliminarily investigate its intestinal pathogenesis mechanisms from the aspect of gut microbiota, the 16S rRNA gene sequencing technology was used to evaluate the gut microbiota composition changes post different virulent NDV infection. Results showed that different virulent NDV infection resulted in a different alteration of the gut microbiota in chickens, including a loss of probiotic bacteria and an expansion of some pathogenic bacteria. The above results suggest that NDV strains with different virulence have different impacts on chicken gut microbiota.Newcastle disease virus (NDV) which is pathogenic to chickens is characterized by dyspnea, diarrhea, nervous disorder and hemorrhages. However, the influence of different virulent NDV strain infection on the host gut microbiota composition is still poorly understood. In this study, twenty 21-day-old specific pathogen free (SFP) chickens were inoculated with either the velogenic Herts33 NDV strain, lentogenic La Sota NDV strain or sterile phosphate buffer solution (PBS). Subsequently, the fecal samples of each group were collected for 16S rRNA sequencing. The results showed that the gut microbiota were mainly dominated by Firmicutes, Bacteroidetes and Proteobacteria in both healthy and NDV infected chickens. NDV infection altered the structure and composition of gut microbiota. As compared to the PBS group, phylum Firmicutes were remarkably reduced, whereas Proteobacteria was significantly increased in the velogenic NDV infected group; the gut community structure had no significant differences between the lentogenic NDV infected group and the PBS group at phylum level. At genus level, Escherichia-Shigella was significantly increased in both the velogenic and lentogenic NDV infected groups, but the lactobacillus was only remarkably decreased in the velogenic NDV infected group. Collectively, different virulent strain NDV infection resulted in a different alteration of the gut microbiota in chickens, including a loss of probiotic bacteria and an expansion of some pathogenic bacteria. These results indicated that NDV strains with different virulence have different impacts on chicken gut microbiota and may provide new insights into the intestinal pathogenesis of NDV.

Assessment of Histopathological Changes in the Liver and Spleen of Mice Infected with Salmonella typhi Following Treatment with Ciprofloxacin

This study evaluated the toxicity of ciprofloxacin to spleen and liver when used for the treatment of mice infected with S. typhi for seven days. The dose concentration used in these experiments was 100mg/kg. Mice were divided into two groups . The first group (negative control) was not given ciprofloxacin, but rather a sterile phosphate buffer solution (PBS) as an alternative. Ciprofloxacin was administered to the second group. After seven days , the animals were sacrificed and organs (liver and spleen) were collected . The histopathological examination showed normal hepatocytes in the liver and normal structure of spleen cells in animals of control group . However, the treated group showed dilated and congested blood vessels with perivascular inflammatory cell cuffing and acute cell swelling in the liver, as well as white pulp activation with an increased number of megakaryocyte cells in the spleen. Therefore, the current study suggests that the concentration of 100 mg/kg of ciprofloxacin is considered to be toxic to hepatocytes and splenocytes of mice during the treatment period.

Mechanism study of dendritic epidermal T lymphocytes in promoting healing of full-thickness skin defects wound on mice by regulating the proliferation and differentiation of epidermal stem cells in mice

Mechanism study of dendritic epidermal T lymphocytes in promoting healing of full-thickness skin defects wound on mice by regulating the proliferation and differentiation of epidermal stem cells in mice

Effects of sodium butyrate on intestinal barrier of severe scald mice and the related mechanism

Objective: To investigate the effects of sodium butyrate on intestinal barrier of the severe scald mice and the related mechanism. Methods: Eighteen C57BL/6 female mice, aged eight to twelve weeks, were divided into sham scald group, pure scald group, and scald+ sodium butyrate group according to random number table, with 6 mice in each group. Back of each mouse in pure scald group and scald+ sodium butyrate group were immersed into 90 ℃ water for 9 s, causing full-thickness scald of 30% total body surface area, while back of each mouse in sham scald group were immersed into 37 ℃ water for 9 s, causing sham injury. All of the mice in 3 groups were intraperitoneally injected with 1 mL sterile lactated Ringer's solution immediately after injury. Besides, mice in scald+ sodium butyrate group were intraperitoneally injected with 300 mg/kg sodium butyrate at 30 min before injury and immediately after injury, while mice in sham scald group and pure scald group were intraperitoneally injected with the same volume of sterile phosphate buffer solution. At post injury hour (PIH) 24, portal vein of mice in 3 groups was harvested, intestinal permeability was measured by fluorescin isothiocyanate-dextran fluorescence probe tracing method, then lileal tissue of mice in 3 groups was harvested, protein expressions of zonula occludens l (ZO-1), occludin, claudin-1, claudin-2, nucleotide-binding oligomerization domain-containing protein-like receptor family pyrin domain containing 3 (NLRP3), interleukin-1β (IL-1β), and IL-18 were detected by Western blotting, and distribution of ZO-1 in intestinal mucosa was observed by indirect immunofluorescence. Data were processed with one-way analysis of variance, least-significant difference test, and Bonferroni correction. Results: (1) At PIH 24, the intestinal permeability of mice in sham scald group, pure scald group, and scald+ sodium butyrate group was 0.88±0.19, 2.62±0.48, 1.23±0.16, respectively. Compared with that in sham scald group, the intestinal permeability of mice in pure scald group was significantly elevated (P<0.01), while the intestinal permeability of mice in scald+ sodium butyrate group showed no obvious change (P>0.05). Compared with that in pure scald group, the intestinal permeability of mice in scald+ sodium butyrate group was significantly decreased (P<0.01). (2) At PIH 24, compared with those in sham scald group, the protein expressions of ZO-1, occludin, and claudin-1 of mice in pure scald group and scald+ sodium butyrate group were significantly decreased (P<0.05), while the protein expression of claudin-2 was significantly increased (P<0.05). At PIH 24, compared with those of pure scald group, the protein expressions of ZO-1 and occludin of mice in scald+ sodium butyrate group were significantly elevated (P<0.05), while the protein expression of claudin-2 was significantly decreased (P<0.05), the protein expression of claudin-1 showed no significant difference (P>0.05). (3) At PIH 24, compared with those in sham scald group, the protein expressions of NLRP3, IL-1β, and IL-18 of mice in pure scald group and scald+ sodium butyrate group were significantly increased (P<0.05). Compared with those of pure scald group, the protein expressions of NLRP3, IL-1β, and IL-18 of mice in scald+ sodium butyrate group were significantly decreased (P<0.05). (4) At PIH 24, ZO-1 in intestinal mucosa of mice in sham scald group was distributed smoothly, continuously and homogeneously along the membrane. ZO-1 in intestinal mucosa of mice in pure scald group was distributed unsmoothly with breaks. The distribution of ZO-1 in intestinal mucosa of mice in scald+ sodium butyrate group was ameliorated compared with that in pure scald group. Conclusions: Sodium butyrate can inhibit the activation of NLRP3 inflammasome and decrease the production of IL-1β and IL-18 in intestinal mucosa of severe scald mice, which protects the intestinal barrier function by alleviating the alteration of tight junction protein expression and localization.

Effect of intratracheal injection of JNK siRNA on ischemia-reperfusion injury in a rat model of lung transplantation

Objective To investigate the effect of intratracheal injection of c-Jun N-terminal kinase (JNK) siRNA plasmid on ischemia-reperfusion (I/R) injury in a rat model of lung transplantation. Methods ExperimentⅠ Thirty-two male Wistar rats, weighing 250-280 g, were divided into 2 groups (n=16 each) using a random number table method: control group (group C) and JNK siRNA group.JNK siRNA plasmid 2 mg/kg (diluted to 0.2 ml in sterile phosphate buffer solution) was intratracheally injected in JNK siRNA group.Scrambled siRNA plasmid 2 mg/kg (diluted to 0.2 ml in sterile phosphate buffer solution) was intratracheally injected in group C. Six rats in each group were sacrificed at 48 h of administration, and left lung tissues were removed for determination of the expression of JNK and JNK mRNA (by Western blot and real-time polymerase chain reaction, respectively). The other 10 rats left in each group were used for left lung transplantation.Experiment Ⅱ Thirty male Wistar rats, weighing 250-280 g, were divided into 3 groups (n=10 each) using a random number table method: sham operation group (group S), transplanted lung I/R group (group I/R) and transplanted lung I/R+ JNK siRNA group (group I/R+ JNK siRNA). In group I/R and group I/R+ JNK siRNA, the left lung transplantation was performed, and the donor lungs were obtained from the living donors in group C and group JNK siRNA, respectively.At 15 min of mechanical ventilation and 30, 60, 90 and 120 min of reperfusion, arterial blood samples were obtained for blood gas analysis, PaO2 was recorded, and the oxygenation index (PaO2÷FiO2) was calculated.Arterial blood samples were obtained at 120 min of reperfusion in transplanted lungs for determination of concentrations of interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-α) and interferon-γ (IFN-γ) in serum (using enzyme-linked immunosorbent assay), and the rats were sacrificed and left lung tissues were removed for microscopic examination of the pathological changes which were scored and for determination of wet/dry lung weight ratio (W/D ratio), and nuclear factor kappa B (NF-κB) and activator protein-1 (AP-1) activities (using enzyme-linked immunosorbent assay). Results ExperimentⅠ Compared with group C, the expression of JNK and JNK mRNA was significantly down-regulated in group JNK siRNA (P<0.05). ExperimentⅡ Compared with group S, the oxygenation index was significantly decreased, and the concentrations of serum IL-8, TNF-α and IFN-γ, W/D ratio, lung injury score and activities of NF-κB and AP-1 were increased in I/R and I/R+ JNK siRNA groups (P<0.05). Compared with group I/R, the oxygenation index of receptors were significantly increased, and the concentrations of serum IL-8, TNF-α and IFN-γ, W/D ratio, lung injury score and activities of NF-κB and AP-1 were decreased in group I/R+ JNK siRNA (P<0.05). Conclusion Intratracheal injection of JNK siRNA can reduce transplanted lung I/R injury, and the mechanism may be related to inhibiting inflammatory responses of rats. Key words: JNK mitogen-activated protein kinases; Lung transplantation; Reperfusion injury; Injection, intratracheal

Fungi isolated from food samples for an efficient stereoselective production of phenylethanols

Abstract Twenty strains of fungi isolated from food samples were screened for the asymmetric reduction of acetophenone. A dimorphic fungus denominated initially as GZ1 and isolated from a carrot sour rot showed very high reduction activities, and based on phenotypic and genetic characteristics was identified as Galactomyces candidus. The only product of the reduction was confirmed to be (R)-1-phenylethanol. The bioreduction conditions mediated by Gal. candidus GZ1 were investigated, and the optimal conditions in terms of acetophenone concentration, temperature, pH media, co-solvent, the charge of inoculum and shaking speed were established. Under the optimal conditions of 1.875 g/L of acetophenone dissolved in DMSO (1.25% v/v), 112.5 g/L of microorganism cells in 100 mM sterile phosphate buffer solution at pH 7.0, 25 °C and 150 rpm, the conversion and enantiomeric excess values after 48 h reaction were 99% and > 99.9%, respectively. A preparative scale reaction of (R)-1-phenylethanol was made and the isolated yield was 82% with > 99.9 e.e.%. Additionally, a study using several substituted acetophenones was carried out, and the advantages and scope of GZ1 in the production of chiral 1-phenylethanols were established. In this sense, good results were obtained with 3′-hydroxyacetophenone and a preparative scale reaction was performed which give (R)-1-(3′-hydroxyphenyl)ethanol (key intermediate of Rivastigmine) with 53% of isolated yield and > 99.9 e.e.%.

IL-12 Expressing oncolytic herpes simplex virus promotes anti-tumor activity and immunologic control of metastatic ovarian cancer in mice.

BackgroundDespite advances in surgical aggressiveness and conventional chemotherapy, ovarian cancer remains the most lethal cause of gynecologic cancer mortality; consequently there is a need for new therapeutic agents and innovative treatment paradigms for the treatment of ovarian cancer. Several studies have demonstrated that ovarian cancer is an immunogenic disease and immunotherapy represents a promising and novel approach that has not been completely evaluated in ovarian cancer. Our objective was to evaluate the anti-tumor activity of an oncolytic herpes simplex virus “armed” with murine interleukin-12 and its ability to elicit tumor-specific immune responses. We evaluated the ability of interleukin−12-expressing and control oncolytic herpes simplex virus to kill murine and human ovarian cancer cell lines in vitro. We also administered interleukin−12-expressing oncolytic herpes simplex virus to the peritoneal cavity of mice that had developed spontaneous, metastatic ovarian cancer and determined overall survival and tumor burden at 95 days. We used flow cytometry to quantify the tumor antigen-specific CD8+ T cell response in the omentum and peritoneal cavity.ResultsAll ovarian cancer cell lines demonstrated susceptibility to oncolytic herpes simplex virus in vitro. Compared to controls, mice treated with interleukin−12-expressing oncolytic herpes simplex virus demonstrated a more robust tumor antigen-specific CD8+ T-cell immune response in the omentum (471.6 cells vs 33.1 cells; p = 0.02) and peritoneal cavity (962.3 cells vs 179.5 cells; p = 0.05). Compared to controls, mice treated with interleukin−12-expressing oncolytic herpes simplex virus were more likely to control ovarian cancer metastases (81.2 % vs 18.2 %; p = 0.008) and had a significantly longer overall survival (p = 0.02). Finally, five of 6 mice treated with interleukin−12-expressing oHSV had no evidence of metastatic tumor when euthanized at 6 months, compared to two of 4 mice treated with sterile phosphate buffer solution.ConclusionOur pilot study demonstrates that an interleukin−12-expressing oncolytic herpes simplex virus effectively kills both murine and human ovarian cancer cell lines and promotes tumor antigen-specific CD8+ T-cell responses in the peritoneal cavity and omentum, leading to reduced peritoneal metastasis and improved survival in a mouse model.

Removal of MS2, Qβ and GA bacteriophages during drinking water treatment at pilot scale

The removal of MS2, Qβ and GA, F-specific RNA bacteriophages, potential surrogates for pathogenic waterborne viruses, was investigated during a conventional drinking water treatment at pilot scale by using river water, artificially and independently spiked with these bacteriophages. The objective of this work is to develop a standard system for assessing the effectiveness of drinking water plants with respect to the removal of MS2, Qβ and GA bacteriophages by a conventional pre-treatment process (coagulation–flocculation–settling-sand filtration) followed or not by an ultrafiltration (UF) membrane (complete treatment process). The specific performances of three UF membranes alone were assessed by using (i) pre-treated water and (ii) 0.1 mM sterile phosphate buffer solution (PBS), spiked with bacteriophages. These UF membranes tested in this work were designed for drinking water treatment market and were also selected for research purpose.The hypothesis serving as base for this study was that the interfacial properties for these three bacteriophages, in terms of electrostatic charge and the degree of hydrophobicity, could induce variations in the removal performances achieved by drinking water treatments.The comparison of the results showed a similar behaviour for both MS2 and Qβ surrogates whereas it was particularly atypical for the GA surrogate. The infectious character of MS2 and Qβ bacteriophages was mostly removed after clarification followed by sand filtration processes (more than a 4.8-log reduction) while genomic copies were removed at more than a 4.0-log after the complete treatment process. On the contrary, GA bacteriophage was only slightly removed by clarification followed by sand filtration, with less than1.7-log and 1.2-log reduction, respectively. After the complete treatment process achieved, GA bacteriophage was removed with less than 2.2-log and 1.6-log reduction, respectively.The effectiveness of the three UF membranes tested in terms of bacteriophages removal showed significant differences, especially for GA bacteriophage. These results could provide recommendations for drinking water suppliers in terms of selection criteria for membranes.MS2 bacteriophage is widely used as a surrogate for pathogenic waterborne viruses in Europe and the United States. In this study, the choice of MS2 bacteriophage as the best surrogate to be used for assessment of the effectiveness of drinking water treatment in removal of pathogenic waterborne viruses in worst conditions is clearly challenged. It was shown that GA bacteriophage is potentially a better surrogate as a worst case than MS2. Considering GA bacteriophage as the best surrogate in this study, a chlorine disinfection step could guaranteed a complete removal of this model and ensure the safety character of drinking water plants.

Sex hormone profiles and cellular changes of reproductive organs of mice experimentally infected with C. pseudotuberculosis and its exotoxin phospholipase D (PLD)

Caseous lymphadenitis has been globally a prevalent disease among farmed small ruminants for over a century. As its chronic disease, sex hormone profiles and cellular changes of reproductive organs in infections associated with Corynebacterium pseudotuberculosisis and its exotoxin phospholipase D have been very few or absent. This study was conducted to acquire a better way of understanding the reproductive pathophysiology response of Corynebacterium pseudotuberculosisis and its exotoxin in mouse model. In this study, 64 healthy mice, 2-3 weeks of old, were divided equally into 3 groups, where the first group of mice were interperitoneally inoculated with 1.0 ml of sterile phosphate buffer solution (PBS), pH 7, the second group of mice were interperitoneally inoculated with 1.0 ml of 10 colony forming unit (CFU) of live C. pseudotuberculosis and the third group of mice were interperitoneally inoculated with 1.0 ml of single dose of exotoxin (PLD) extracted from C. pseudotuberculosis. Following infection, clinical signs were observed and blood samples were collected by cardiac puncture for sex hormone analysis. For microscopic examinations, the mice were euthanized using cervical dislocation approach and the reproductive organs were collected. The results revealed that there was no significant differences (p<0.05) in the concentration of estrogen and progesterone levels between whole cell and exotoxin groups. Concentration of progesterone in PLD treated group (15.37 ± 2.32 pg/ml) was lower than control group (17.61 ± 2.91pg/ml). The concentration of testosterone in C. pseudotuberculosis (2.98 ± 3.70 pg/ml) was significantly (p<0.05) different from both control group (16.58 ± 3.67 pg/ml) and PLD (11.84 ± 3.19 pg/ml). Microscopically, cellular changes were observed in organs included ovaries, uterus, testicles and epididymis. In C. pseudotuberculosis infected group, infiltration of polymorph nuclear leukocytes with congestion, degeneration and necrosis were documented in almost all investigated parameters. For exotoxin PLD infected group, the observed cellular changes included severe hemorrhage, thrombus formation, degeneration, vacuolation and necrosis. Therefore, the results of this study indicated that there were significant differences in sex hormones and cellular changes of reproductive organs associated with infection Corynebacterium pseudotuberculosisis and its exotoxin phospholipase D (PLD).

A new model of rectal cancer with regional lymph node metastasis allowing in vivo evaluation by imaging biomarkers

Object The work is aimed to develop a murine model of rectal cancer, which could be used to monitor lymph node metastasis development by magnetic resonance imaging (MRI) and optical imaging (OI) techniques. Subjects and methods Ht-29 cancer cells were directly injected into the submucosal layer of the rectum of athymic nude mice using trans-anal rectal cancer cell injection (TARCI). Thirty-six mice were inoculated with 10 × 10 5 cells and five mice were treated with sterile phosphate buffer solution. One to 4 weeks after cell injection, tumor growth was evaluated in vivo using T2-weighted MRI at 4.7T. A further group of animal ( n = 6) treated with ht-29_luc cells, with the same protocol, was monitored by optical imaging. In both groups, the presence of the primary tumor and of lymph nodes metastasis was confirmed by histology. Results In all animals, primary tumors were detectable by MRI, 1 week from TARCI. After 4 weeks primary tumors showed a mean longitudinal diameter of about 2 cm. All animals developed regional lymph node metastases. Others organs (e.g. lung or liver) were not affected. In fat-suppressed, T2-weighted MRI, lymph nodes appeared as small areas characterized by hyper-intense signal compared to muscle. OI permitted evaluation of the primary tumor growth in perineal region. Conclusions TARCI of ht-29 cells into the rectum of nude mice is a feasible way to obtain a easily reproducible model of regional lymph node metastases could be monitored by magnetic resonance and optical imaging techniques.

Revisiting therapeutic strategies in radiation casualties

Nuclear/radiological threats have evolved and scenarios for terrorist attacks involving radioactive material have been identified as complex situations. Mass casualty scenarios may happen, and individuals may be exposed to intentionally hidden sources of high activity, resulting in delayed diagnosis and treatment of acute radiation syndrome (ARS). Moreover, ARS must be considered as an emergency in order to better anticipate delayed radiation toxicity. In this context, therapeutic strategies in radiation casualties have to be revisited and new pharmacological approaches developed. B6D2F1 mice were total-body irradiated (TBI) with a 9 Gy gamma dose and then received intraperitoneal doses of either early (stem cell factor + FLT-3 ligand + thrombopoietin + interleukin-3 [SFT3] +/- keratinocyte growth factor (KGF); stem cell factor + erythropoietin + Peg-filgrastim [SEG]) or delayed treatments (SFT3 +/- KGF, erythropoietin, or hyaluronic acid). Survival was monitored and bone marrow hematopoiesis evaluated at 300 days following early treatments. SFT3 anti-apoptotic cytokine combination administered early (2 hours and 24 hours) after lethal TBI induced 60% survival versus 5% in controls. Early SEG treatment may be an alternative to SFT3 in terms of survival (55%), but SEG benefit might be obtained at the expense of long-term hematopoiesis. SFT3 + KGF induced 75% survival. No effectiveness was observed, over antimicrobial supportive care, when administration of SFT3 or its tested combinations was delayed at 48 hours. As a potentially multi-organ failure, ARS requires global therapy, beyond the hematopoietic syndrome, which may include pleiotropic cytokines such as KGF.

Retinal toxicity of intravitreally injected plain and liposome formulation of fluconazole in rabbit eye

Candidal endophthalmitis is a sight-threatening ocular infection that most frequently occurs as a complication of candidemia. Fluconazole has been effective against Candida albicans in various animal models. Our objective was to evaluate retinal toxicity of plain and liposome formulation of fluconazole at various dose levels after intravitreal injection. Twelve New Zealand albino rabbits weighing 2-2.5 kg were used. Two rabbits were used for every dose level. Liposome formulation containing 100 and 200 microg of fluconazole in sterile phosphate buffer solution and plain fluconazole at concentrations of 100, 200, 400 and 800 microg in 0.1 ml of sterile normal saline were injected intravitreally into the right eyes. The left eyes received 0.1 ml normal saline or 0.1 ml of liposome formulation without fluconazole. One week later, the animals were sacrificed, their eyes enucleated and processed for light microscopy and scanning electron microscopy. It showed that plain fluconazole at a concentration of 100 microg and above caused retinal changes, with disorganization of the photoreceptor outer segments. However, liposome formulation of fluconazole (200 microg/0.1 ml) did not show any significant microscopic changes of the retina. The liposome formulation decreased the retinal toxicity of fluconazole up to the studied concentration of 200 microg/0.1 ml.

Indigenous microflora and opportunistic pathogens of the freshwater zebra mussel, Dreissena polymorpha

Freshwater fouling invertebrate zebra mussels (Dreissena polymorpha) harbor a diverse population of microorganisms in the Great Lakes of North America. Among the indigenous microorganisms, selective species are opportunistic pathogens to zebra mussels. Pathogenicity to zebra mussels by opportunistic bacteria isolated from the mussels was investigated in this study. Among the more than 30 bacteria isolated from temperature-stressed mussels, Aeromonas media, A. veronii, A. salmonicida subsp. salmonicida, and Shewanella putrefaciens are virulent pathogens to juvenile zebra mussels. Inoculation of a bacterial concentration of A. media, A. salmonicida subsp. salmonicida and S. putrefaciens at 107 cells per zebra mussel resulted in 100% mortality within 5 days, and only 64.9% for A. veronii. In contrast, mortality was less than 12.3% following inoculation of a sterile phosphate buffer solution as a control. In addition, mortality was dependent on the size of the pathogen population used in inoculation and the incubation temperature, indicating the close relationship between the bacterial population and subsequent death. On the mussel tissue, a dense microbial population was evident from the moribund mussels viewed with Scanning Electron Microscope (SEM). Opportunistic bacteria invaded and destroyed the D. polymorpha tissue after 7 days of incubation when the bacterial inoculation was larger than 105 per zebra mussel. Our results suggest that mussels are reservoirs of opportunistic pathogenic microorganisms to aquatic organisms and humans and a better understanding of the microbial ecology of the mussels will provide insights to the possible health hazards from these microorganisms.

7038 Is automated cleaning as good as manual cleaning in the reprocessing of flexible gi endoscopes?

Cleaning is an essential step in the reprocessing of GI endoscopes. Because manual cleaning is tedious and technician dependent, automated cleaning may have some advantages over the manual method. In simulated use testing, the efficacy of automated cleaning (System 83-2+DT, Custom Ultrasonics) was compared with a standardized manual cleaning method previously reported by the investigators (Amer J Gastroenterol 1999;94:1546). The automated reprocessor simulates the mechanical effects of a brush or sponge by pulsing enzymatic solution through most channels at a frequency of 60 Hz, and against the exterior surface of the insertion tube at 40,000 Hz. METHODS: One Olympus CF100TL videocolonoscope was contaminated with a simulated soil consisting of 2 parts sterile milk powder, 10 parts fetal calf serum, 10 parts Mycobacteria chelonae, for a final mycobacterial concentration of 10 9 CFU/mL. The suction housing, air-water housing, valves, insertion tube, instrument channel and auxillary channel were independently inoculated, and then air-dried for 60 min prior to cleaning. The size of inoculum (total CFU) was established for each site through control experiments. Each inoculated site was flushed with sterile phosphate buffer solution; the effluent was poured through a 0.45μm membrane filter, cultured in Middlebrook 7H11 agar, and incubated for 10 days at 37° C. Three to five experiments were conducted for controls and for manual and automated cleaning methods using the same scope. RESULTS: See table. CONCLUSIONS: (1) In simulated use testing under worst-case scenario, automated cleaning achieved a 3 to 5 log reduction in mycobacterial bioburden, which was at least equivalent to standardized manual cleaning. (2) Manual cleaning of the auxillary channel is potentially problematic because its small channel caliber precludes the use of a cleaning brush. (3) Used in conjunction with rigorous manual cleaning, automated cleaning may add an extra margin of safety for reprocessing of flexible GI endoscopes.

Effect of animal and vegetal protein diets on feed intake and apparent digestibility of nutrients in rainbow trout (Oncorhynchus mykiss) infected by Aeromonas salmonicida, with and without chronic hypoxia stress

This study was undertaken to investigate the effects of bacterial infection (Aeromonas salmonicida) and chronic hypoxia on mortality, feed intake, and apparent digestibility of each of two diets in rainbow trout (Oncorhynchus mykiss). The diets were isonitrogenous (gross basis); one was based mainly on animal protein and the other on vegetal protein. In each of two separate and successive experiments, where the first served as a control for the second, fish were immersed on day 0 (d0) in either a sterile phosphate buffer solution (first experiment) or a suspension of A. salmonicida (second experiment). Thereafter, the fish were either exposed on d2 for 12 days to hypoxic conditions (60% of water oxygen saturation) or maintained under normal conditions. Feed intake was measured daily, while apparent digestibility coefficients for gross energy, dry matter, and crude protein were measured on d0, d2, d5, d9, d11, and d14. Among all infected fish, feed intakes were reduced (P &lt; 0.05), regardless of the source of dietary protein or the degree of water oxygenation. Also, regardless of infection, the feed intakes of hypoxic fish that were fed the vegetal protein based diet were significantly less than those of fish that were fed the animal protein based diet. This response, however, was transitory, since normal levels of feed intake in the former fish were found by d3. The apparent digestibility coefficients for all parameters were unaltered by hypoxia (P &gt; 0.05), but they were significantly reduced (P &lt; 0.05) by A. salmonicida infection, especially during the critical period of infection (d5 and d9).