Sterile Phosphate-buffered Saline Research Articles

The impact of hydrogen sulfide on the heme oxygenase-1/carbon monoxide system in Coxsackie virus B3-induced myocarditis in mice

Objective: To explore the impact of hydrogen sulfide (H(2)S) on the heme oxygenase-1/carbon monoxide pathway in Coxsackie virus B3 (CVB3)-induced murine myocarditis (VMC) model. Method: A total of 70 inbred male Balb/c mouse (4-6 weeks old) were randomized into the following four groups: Normal, VMC, PAG and NaHS (n=10 for Normal, n=20 for VMC, PAG and NaHS groups). Mice in Normal group were non-infected mice treated with intraperitoneal injection of sterile phosphate-buffered saline daily for 10 days.Mice in VMC group received intraperitoneal CVB3 injection (0.1 ml 10(-5.69)TCID(50)m·ml(-1)·d(-1) and PBS for 10 days), and mice in PAG group received additional intraperitoneal DL-proparglygylcine injection (40 mg·kg(-1)·d(-1) for 10 days), mice in NaHS group received additional intraperitoneal NaHS injection (50 μmol·kg(-1)·d(-1) for 10 days). All mice were sacrificed on day 10th, and body weight and heart weight, the ratio of heart weight to body weight were compared among groups.Pathological changes of heart tissues were observed microscopically by HE and the histopathologic scores were valued.The content of COHb was tested after the gathering of blood specimens while reverse transcription-polymerase chain reaction was used to detect myocardial HO-1 mRNA expression. Results: (1) Pathological findings in myocardium: hearts sections in Normal group were normal and no inflammatory cells and necrosis were found.A notable cellular infiltration, interstitial edema, vascular hyperemia and necrosis were observed in heart section of VMC, PAG and NaHS group.Extensive inflammations and larger area of myocardial cells necrosis were evidenced in PAG group and above changes were significantly reduced in NaHS group.(2) Comparison of the ratio of heart weight to body weight and histological scores of myocardium: the ratio was significantly higher in the VMC, PAG, NaHS groups than in Normal group (P<0.05), which was higher in PAG group and lower in NaHS group as compared with VMC group (both P<0.05). The histopathologic scores of all CVB3 innoculation groups were higher than in the Normal group, which was higher in PAG group and lower in NaHS group as compared to VMC group (both P<0.05). (3) The content of blood COHb in VMC, PAG or NaHS group was significantly higher than that in Normal group (P<0.05), which was significantly lower in PAG group, and higher in NaHS group as compared to VMC group (both P<0.05). (4) The mRNA expression of myocardial HO-1 detected by RT-PCR: weak expression was observed in Normal group, which was significantly upregulated in VMC, PAG and NaHS groups (P<0.05), which was downregulated in PAG group and upregulated in NaHS group as compared to VMC group (both P<0.05). (5) Correlation analysis: blood COHb concentration was positively correlated with myocardial HO-1 mRNA expression(r=0.927, P=0.000), negatively correlated with histopathologic scores(r=-0.753, P=0.000)and the histopathologic scores were negatively correlated with the myocardial HO-1 mRNA expression (r=-0.754, P=0.000). Conclusions: H(2)S could play a protective role in murine CVB3 myocarditis model through inducing HO-1 expression and upregulating HO-1/CO pathway.

Effect of nano-selenium and nano-zinc particles during in vitro maturation on the developmental competence of bovine oocytes

The objectives of the current study were to evaluate the effects of supplemental nano-selenium (NSe) and nano-zinc oxide (NZn-O) particles during in vitro maturation (IVM) on DNA damage of cumulus cells, glutathione (GSH) concentration in bovine oocytes, subsequent embryo development and re-expansion rate of vitrified warmed blastocysts. The current study was conducted on bovine ovaries obtained from a local abattoir and transported to the laboratory in sterile phosphate buffer saline with antibiotics at 37°C, within 1 h after slaughter. Ovaries were pooled, regardless of stage of the oestrous cycle of the donor. Only cumulus-intact complexes with evenly granulated cytoplasm were selected for IVM. Experimental design included the following: Experiment 1 studied the effect of addition of 1.0 µg/mL NSe or NZn-O to IVM medium on DNA damage of cumulus cells; Experiment 2 evaluated the effects of NSe or NZn-O on intracellular glutathione in oocytes and cumulus cells; in Experiment 3, the development of oocytes matured in IVM medium supplemented with 1.0 µg/mL NSe or NZn-O was investigated; and in Experiment 4, the effects of adding 1.0 µg/mL NSe and NZn-O to in vitro fertilisation media on vitrified oocytes and embryos were investigated. The DNA damage in cumulus cells decreased with supplemental NSe and NZn-O at concentration of 1 µg/mL in the IVM medium (180.2 ± 21.4, 55.8 ± 4.3 and 56.6 ± 3.9 for the control and NSe and NZn-O groups respectively). Total GSH concentrations increased following supplementation with 1 µg/mL NSe and 1 µg/mL NZn-O, compared with the control group. Re-expansion rate of vitrified warmed blastocysts in experimental media containing NSe and NZn-O with ethylene glycol was higher than that of the control. In conclusion, providing NSe and NZn-O during oocyte maturation significantly increased both intracellular GSH concentration and DNA integrity of cumulus cells. Optimal embryo development was partially dependent on the presence of NSe and NZn-O during IVM. NSe and NZn-O during oocyte maturation act as a good cryoprotective agents of vitrified, warmed blastocysts.

Immunization of sheep against Echinococcus granulosus with protoscolex tegumental surface antigens.

Aim:Cystic echinococcosis (CE) has potential economic effects to both animal products and human health. A vaccine to protect livestock against CE can be effective in reducing economic costs and increasing the livestock products. Protoscolex tegumental surface antigens (PSTSA) used to induce the production of specific antibodies against Echinococcus granulosus in sheep. The tegumental antigens were extracted from viable protoscolices by solubilization in sterile phosphate-buffered saline containing decanoyl-N-methylglucamine.Materials and Methods:Ten lambs which were infected with CE (positive control), 10 negative control, and 10 test groups of sheep were included in the study. 300 µg emulsion of purified-PSTSA was injected intramuscularly in a two-step immunization on the first and 30 days. Sera were collected immediately before immunization and 6 times with 10-day intervals until 60 days post immunization. Thereafter, the sera were tested for antibodies by indirect hemagglutination test in microtiter plate.Results:After two immunizations, all the infected animals in test group showed substantial increases in antibody titer. Statistical analysis showed a significant difference between the titer obtained in the test and negative control groups in both phases of immunization (p<0.05).Conclusion:The results showed that the PSTSA is a promising immunogenic compound for immunization of sheep against CE.

Protective efficacy by various doses of a new brucellosis vaccine candidate based on Salmonella strains expressing Brucella abortus BSCP31, Omp3b and superoxide dismutase against brucellosis in murine model.

Brucella species are important etiological agents of zoonotic diseases. Attenuated Salmonella strains expressing Brucella abortus BCSP31, Omp3b and superoxide dismutase proteins were tested as vaccine candidates in this study. In order to determine the optimal dose for intraperitoneal (IP) inoculation required to obtain effective protection against brucellosis, mice were immunized with various doses of a mixture of the three vaccine strains. Fifty BALB/c mice were divided into five equal groups (groups A–E). Group A mice were intraperitoneally inoculated with 100 μL of sterile phosphate-buffered saline. Group B, C, D and E mice were intraperitoneally immunized with approximately 1.2 × 105 colony-forming units (CFU) mL−1 of Salmonella containing pMMP65 in 100 μL and with 1.2 × 104 CFU mL−1, 1.2 × 105 CFU mL−1 and 1.2 × 106 CFU mL−1 of the mixture of the three strains in 100 μL, respectively. Serum IgG, tumor necrosis factor alpha and interferon gamma concentrations were significantly higher in group E than in groups A–D. Following challenge with B. abortus 544, the challenge strain was not detected in the spleen of any mouse from group E. Thus, IP immunization with 1.2 × 106 CFU mL−1 of the mixture of the three vaccine strains induced immune responses and provided effective protection against brucellosis in mice.

Reproductive hormonal variations and adenohypophyseal lesions in pre-pubertal buffalo heifers inoculated with Pasteurella multocida type B: 2 and its immunogens

BackgroundHemorrhagic septicemia is a fatal disease of cattle and buffaloes caused by P. multocida. Although the pathogenesis of the bacteria has been well established in literature, there is a paucity of information on the possible role of the bacteria and its immunogens; lipopolysaccharide (LPS) and outer membrane proteins (OMPs) on the reproductive capacity of buffalo heifers.MethodsIn this study, twenty one healthy prepubertal female buffaloes aged 8 months were divided into seven groups of 3 buffaloes each (G1-G7). Group 1 (G1) served as the negative control group and were inoculated orally with 10 mL sterile Phosphate Buffer Saline (PBS), groups 2 (G2) and 3 (G3) were inoculated orally and subcutaneously with 10 mL of 1012 colony forming unit (cfu) of P.multocida type B: 2, while groups 4 (G4) and 5 (G5) received 10 mL of bacterial LPS orally and intravenously, respectively. Lastly, groups 6 (G6) and 7 (G7) were orally and subcutaneously inoculated with 10 mL of bacterial OMPs. Whole blood was collected in EDTA vials at stipulated time points (0, 2, 4, 6, 8, 10, 12, 24, 36, 48, 72, 120, 168, 216, 264, 312, 360, 408, 456 and 504 h), while tissue sections of the pituitary glands were collected and transported to the histopathology laboratory in 10% buffered formalin for processing and Hematoxylin and eosin staining. Plasma levels of luteinizing hormone (LH), follicle stimulating hormone (FSH), progesterone (PG), estradiol (EST) and gonadotrophin releasing hormone (GnRH) were determined.ResultsThe histopathological lesions observed in the pituitary gland included hemorrhage, congestion, inflammatory cell infiltration, hydropic degeneration, necrosis and edema. These changes were higher (p < 0.05) in distribution and severity in G3, G6 and G7. Hormonal concentrations of LH, FSH, PG, EST and GnRH declined in all inoculation groups as time elapsed and were lower (p < 0.05) than that of the control group.ConclusionBased on these findings, P.multocida B: 2 and its immunogens can be said to negatively affect the hypothalamic-pituitary-gonadal axis, resulting in decreased levels of reproductive hormones which may predispose to infertility in buffalo heifers.

Bone marrow mesenchymal stem cells modulated the inflammatory response by regulating the expression of IL-4 and RAGE products in the rats with MODS

To investigate the underlying mechanism of bone marrow mesenchymal stem cells (BMSC) modulating the inflammatory response during the multiple organ dysfunction syndrome (MODS), especially the expression of inflammatory cytokines, which will provide new theoretical and experimental basis of MODS in clinic. BMSC of Sprague-Dawley (SD) rat (female, 4 weeks) was extracted and cultivated, and the 4th passage were used in experimental study. According to the random number table, 60 female SD rats were divided into three groups (n = 20 per group): sham group, MODS group, BMSC group. MODS model in rats was induced by lipopolysaccaride (LPS, 1 mg/kg) via femoral vein injection. Sham group was injected with the sterile phosphate buffer saline (PBS) in the same volume. BMSC group, in which BMSC infusion was started at 2 hours after 0.5 mL LPS stimulation (1×106/cells) through the tail vein. The survival rate was observed after 72 hours in each group. Abdominal aortic blood was collected for routine blood and biochemical examination at 72 hours after operation. Protein microarray was used to detect the related 34 inflammatory cytokines. Signal ratio was defined as the differentially expressed factors when it was more than 2.0 or less than 0.5. And enzyme linked immunosorbent assay (ELISA) was be applied to validate the significant inflammation factor. Meanwhile, the heart, kidney, intestine tissue was harvested, then their pathological changes were observed by hematoxylin eosin (HE) staining. 20, 12, 16 rats lived in sham group, MODS group and BMSC group respectively at 72 hours after operation. Compared with the sham group, the indicators (routine blood, liver and kidney function, myocardial enzyme) were apparently unusual, and the heart, kidney, intestine tissue were injured obviously in the MODS group. After BMSC administration, the organ function was improved and tissue damaged was alleviated significantly. Protein microarray showed that interleukin-4 (IL-4) and receptor for advanced glycation end products (RAGE) were significantly different in 34 goal cytokines. The signal ratio change of IL-4 was 0.397, 1.124, 2.826 respectively, and the signal ratio of RAGE was 6.197, 1.552, 0.250, respectively in MODS/sham group, BMSC/sham group, BMSC/MODS group. ELISA validated the result that the expression level of IL-4 decreased significantly (ng/L: 3.59±1.21 vs. 29.10±5.78) and the expression level of RAGE increased significantly (ng/L: 1.09±0.04 vs. 0.11±0.03) in MODS group as compared with sham group (both P < 0.05). Compared with the MODS group, the level of IL-4 was obviously higher than that in BMSC group (ng/L: 9.59±2.21 vs. 3.59±1.21, P < 0.01), and RAGE decreased significantly (ng/L: 0.29±0.07 vs. 1.09±0.04, P < 0.05). BMSC administration can regulate the expression of IL-4 and RAGE in the rats subjected to MODS. Moreover, BMSC can promote the restoration of tissue and organ function, thus improve the survival rate. BMSC may be the target in cell therapy for the inflammatory disease.

RETRACTED: Dietary Allium hookeri reduces inflammatory response and increases expression of intestinal tight junction proteins in LPS-induced young broiler chicken

We undertook a study to assess the effects of Allium hookeri (AH) root and fermented root on inflammation and intestinal integrity of lipopolysaccharide (LPS)-challenged broiler chickens. Birds were assigned to six groups (n = 25 birds/treatment) and fed with basal diets (CON) or basal diets supplemented with AH root or fermented root at two concentrations (1 or 5%). At 7 d of age, five groups (n= 125) in each dietary treatment were injected with LPS (1 mg/kg body weight), and the remaining 25 birds were injected with sterile phosphate-buffered saline (PBS) as a negative control. LPS challenge significantly reduced average body weight gain at 24 h post-injection compared with PBS control. Fermented root supplementation increased average body weight gain by 1% compared with the LPS-challenged control. Serum α- 1-AGP levels, interleukin (IL)-1β, IL-8, tumor necrosis factor superfamily member 15 (TNFSF15), and LPS-induced tumor necrosis factor-α factor (LITAF) transcript levels were significantly higher in the small intestine in LPS-injected chickens. However α-1-AGP levels were reduced by AH root or fermented root (1 and 5%) supplementation and IL-1β, IL-8, and LITAF were also down-regulated by root and fermented root (1 and 5%) supplementation. The reduced expression of tight junction proteins (junctional adhesion molecule 2 (JAM2) and occludin) and intestinal mucin 2 (MUC2) by LPS challenge was reversed by root or fermented root (1 and 5%) supplementation. These findings demonstrate that dietary AH root and fermented root influence antiinflammatory activity and tight junction protein expression in LPS-induced chickens.

Intraperitoneal adoptive transfer of mesenchymal stem cells enhances recovery from acid aspiration acute lung injury in mice

BackgroundMesenchymal stem cells (MSCs) might act as fine-tuners of inflammation during acute lung injury. We assessed the effects of adoptive transfer of MSCs in acid aspiration acute lung injury and explored the role of long pentraxin PTX3.MethodsWe conducted a prospective experimental interventional study on wild-type (WT) and PTX3-deficient (PTX3−/−) mice. Acute lung injury was induced in WT and PTX3−/− mice by instillation of hydrochloric acid into the right bronchus. One hour later, animals received intraperitoneal sterile phosphate-buffered saline (PBS), WT-MSCs (1 × 106) or PTX3−/−-MSCs (1 × 106). Twenty-four hours after injury, we measured the effects of treatments on arterial blood gases, wet/dry lung weight (W/D), CT scan analysis of lung collapse, neutrophils, TNFα and CXCL1 in bronchoalveolar lavage, and plasma PTX3. d-dimer was assayed in 1 week and OH-proline in 2 weeks to track the fibrotic evolution.ResultsIn 24 h, in comparison to PBS, WT-MSCs improved oxygenation and reduced W/D and alveolar collapse. These effects were associated with decreased concentrations of alveolar neutrophils and cytokines. WT-MSCs increased d-dimer concentration and decreased OH-proline levels, too.Treatment with PTX3−/−-MSCs ameliorated oxygenation, W/D, and alveolar TNFα, though to a lesser extent than WT-MSCs. PTX3−/−-MSCs did not improve lung collapse, neutrophil count, CXCL1, d-dimer, and OH-proline concentrations. The protective effects of WT-MSCs were dampened by lack of endogenous PTX3, too.ConclusionsIn acid aspiration acute lung injury, MSCs improve pulmonary function and limit fibrosis by fine-tuning inflammation. The role of PTX3 in determining MSCs’ effects might merit further scrutiny.

Inhibitory effects of vancomycin and fosfomycin on methicillin-resistant Staphylococcus aureus from antibiotic-impregnated articulating cement spacers.

ObjectivesVancomycin and fosfomycin are antibiotics commonly used to treat methicillin-resistant Staphylococcus aureus (MRSA) infection. This study compares the in vitro inhibitory effects against MRSA of articulating cement spacers impregnated with either vancomycin or fosfomycin.MethodsVancomycin-impregnated articulating cement spacers and fosfomycin-impregnated articulating cement spacers were immersed in sterile phosphate-buffered saline (PBS) solutions and then incubated. Samples were collected for bioactivity evaluation. The aliquots were tested for MRSA inhibition with the disc diffusion method, and the inhibition zone diameters were measured. The inhibition zone differences were evaluated using the Wilcoxon Rank Sum Test.ResultsThe vancomycin group had significantly larger inhibition zones than the fosfomycin group from day three through to completion of the fourth week of incubation (p < 0.001). The vancomycin group exhibited a MRSA inhibition zone up to four weeks but the fosfomycin group showed an inhibition zone for only three days and after that did not show the the potential to inhibit MRSA.ConclusionThis in vitro study found that the inhibitory effect of vancomycin-impregnated articulating cement spacers against MRSA outperformed fosfomycin-impregnated articulating cement spacers. Further comparing our results to other published reports suggests there might be a limitation of the disc diffusion bioassay to show a large inhibitory zone in a high concentration of a highly soluble antibiotic.Cite this article: V. Yuenyongviwat, N. Ingviya, P. Pathaburee, B. Tangtrakulwanich. Inhibitory effects of vancomycin and fosfomycin on methicillin-resistant Staphylococcus aureus from antibiotic-impregnated articulating cement spacers. Bone Joint Res 2017;6:132–136. DOI: 10.1302/2046-3758.63.2000639.

Development and clinical application of decellularized porcine SIS and cornea for the repair of corneal defects in animals

Porcine small intestine sub-mucosa (SIS) and cornea were decellularized using ionic biological detergent (1% SDS) and stored at –20°C in sterile phosphate buffer saline (PBS) solution containing mixture of antibiotics. The prepared biomaterials were subjected to histological and scanning electron microscopic observations to ascertain the decellularization status before their clinical application. Both the biomaterials were evaluated for the repair of corneal defects in 11 animals. Five animals having corneal defects repaired with SIS and 4 repaired with cornea demonstrated successful healing without clinical signs of infections or reoccurrence during the 8 months follow up period. Results of study support the use of decellularized porcine SIS and cornea as an alternative to traditional implantation materials to treat different corneal defects in animals.

Effect of quercetin regulating the expression of B cell lymphoma/leukemia-2, B cell lymphoma/leukemia-2 associated X protein on apoptosis of fibroblast-like synoviocytes in human rheumatoid arthritis

Objective To observe the effect of Quercetin regulating the expression levels of B cell lymphoma/leukemia-2 associated X protein (bax), B cell lymphoma/leukemia-2 (bcl-2) on the apoptosis of human rheumatoid arthritis fibroblasts-like synoviocytes (RAFLSs). Methods The synovial tissues were collected from patients with rheumatoid arthritis (RA). The standard of diagnosis of RA was the criteria revised by American College of Rheumatology (ACR). Fresh synovial tissues were chopped into fragments, washed in sterile phosphate-buffered saline (PBS), and digested with collagenase to obtain synoviocytes. Human RAFLSs morphology was observed under light microscope. Flow cytometry was used to measure the expression of cluster of differentiation 3 (CD3), CD14, CD19 and CD90 on the cell surface of RAFLSs. Human RAFLSs were treated with different concentrations of quercetin (0, 100, 200, and 300 μmol/L) for 24 h. Western blotting was used to detect the expression of anti-apoptotic protein bcl-2 and pro-apoptotic protein bax. The effect of quercetin through bcl-2/bax gray ratio on the apoptosis of fibroblast-like synoviocytes (FLSs) was analyzed. Results As compared with control group (the bcl-2/bax value was 8.26±0.08), the bcl-2/bax in the quercetin-treated groups (100, 200, and 300 μmol/L) (bcl-2/bax values were 3.23±0.11, 0.26±0.05 and 0.12±0.03 respectively) significantly decreased (P<0.01). The apoptosis rate of RAFLSs in the control group (6.5%±0.9%) was significantly lower than in 100 μmol/L quercetin-treated group (19.9%±3.8%) (P<0.05), and 200, 300 μmol/L quercetin-treated groups (34.3%±6.7% and 42.5%±4.8% respectively) (P<0.01). Conclusion Quercetin can regulate the expression of bax and bcl-2 to influence RAFLSs apoptosis, and the apoptosis rate of RAFLSs is inversely proportional to the bcl-2/bax value. Key words: Quercetin; Rheumatoid arthritis; Fibroblastlike synoviocytes; B cell lymphoma/leukemia 2 associated X protein; B cell lymphoma/leukemia-2

Milk acidification to control the growth of Mycoplasma bovis and Salmonella Dublin in contaminated milk

Bacterial contamination of milk fed to calves compromises calf health. Several bacterial pathogens that infect cows, including Mycoplasma bovis and Salmonella enterica ssp. enterica serovar Dublin, are shed in milk, providing a possible route of transmission to calves. Milk acidification lowers the milk pH so that it is unsuitable for bacterial growth and survival. The objectives of this study were to (1) determine the growth of M. bovis and Salmonella Dublin in milk, and (2) evaluate the efficacy of milk acidification using a commercially available acidification agent (Salstop, Impextraco, Heist-op-den-Berg, Belgium) to control M. bovis and Salmonella Dublin survival in milk. For the first objective, 3 treatments and a positive control were prepared in 10 mL of milk and broth, respectively, and inoculated with M. bovis or Salmonella Dublin to an approximate concentration of 104 cfu/mL. Each treatment was retained at 5, 23, or 37°C with the positive control at 37°C. Aliquots were taken at 4, 8, 24, 28, 32, 48, 52, and 56 h after inoculation and transferred onto agar medium in triplicate following a 10-fold dilution series in sterile phosphate-buffered saline. All plates were incubated and colonies counted. For the second objective, 4 treatments and a positive control were prepared with 100 mL of milk and inoculated with M. bovis or Salmonella Dublin to an approximate concentration of 106 cfu/mL. With the use of Salstop, treatments were adjusted to an approximate pH of 6, 5, 4, or 3.5. The positive control was left untreated. At 1, 2, 4, 6, 8, and 24 h after treatment, triplicate aliquots were taken, the pH measured, and then the aliquots were transferred onto agar medium and into broth for enrichment. Following incubation, agar colonies were counted, while broths were plated and incubated prior to colonies being counted. All trials were repeated. Mycoplasma bovis did not grow in milk, but Salmonella Dublin proliferated. The pH of all acidification treatments remained stable for 24 h. No viable M. bovis organisms were detected at 1 h of exposure to pH 3.5 and 4 or at 8 h of exposure to pH 5. Following 24 h of exposure to pH 6 M. bovis remained viable. No viable Salmonella Dublin organisms were detected at 2 and 6 h of exposure to pH 3.5 and 4, respectively. Salmonella Dublin remained viable following 24 h of exposure to pH 5 and 6. These results demonstrate that milk acidification using Salstop is effective at eliminating viable M. bovis and Salmonella Dublin organisms in milk if the appropriate pH and exposure time are maintained.

Comparison of the Detection Limits of the Culture and PCR Methods for the Detection of Clostridium difficile, Clostridium perfringens, Campylobacter jejuni, and Yersinia enterocolitica in Human Stool

Background: Detection of fastidious enteropathogenic bacteria in fecal samples of patients with gastroenteritis is a challenge in clinical microbiological laboratories. Objectives: The aim of this study was to compare the detection limits of the PCR and culture methods for the diagnosis of Campylobacter spp., Yersinia spp., Clostridium perfringens, and Clostridium difficile in human stool samples. Methods: Healthy human stool and sterile phosphate-buffered saline (PBS) samples were separately spiked with 10-fold dilutions of C. jejuni, C. difficile, Y. enterocolitica, and C. perfringens reference strains to obtain final concentrations of 101 - 108 colony forming units (CFU) per gram. Dilutions of each suspension were inoculated onto specific culture media and colony counts were determined. Polymerase chain reaction (PCR) was carried out on DNA extracts of each dilution using specific primers. All of the assays were performed in two separate replicas. Results: In the cases of the culture and PCR assays, detection limits of 101 and 102 CFU/g for C. difficile, 2 × 104 and 2 × 104 CFU/g for C. perfringens, 104 and 102 CFU/g for C. jejuni, and 102 and 104 CFU/g for Y. enterocolitica, respectively, were obtained. In the cases of the spiked PBS samples, a detection limit of 101 for C. jejuni and Y. enterocolitica was obtained using the culture method. While 102 -fold higher sensitivity was observed for C. jejuni via PCR compared with the culture assay, equal (C. perfringens) or lower sensitivity limits (C. difficile and Y. enterocolitica) were detected for the spiked stool samples with other bacteria. Conclusions: These results showed differences in the bacterial culture and PCR methods for quantitative detection of fastidious bacteria in human stool samples. However, a bacterial load of 104 CFU per gram of stool was measured as a sufficient amount for detection of the fastidious bacteria by either culture or PCR assays. More suitable PCR methods could be used for rapid diagnosis of the slow-growing bacteria in the patients’ stool samples.

Root canal disinfection comparing conventional irrigation vs photon-induced photoacoustic streaming (PIPS) using a buffered 0.5 % sodium hypochlorite solution

Abstract Background The purpose of this study was to assess the antimicrobial effect of a buffered 0.5 % sodium hypochlorite solution activated by photon-induced photoacoustic streaming compared to conventional irrigation. Methods The canals on 48 single canal lower bicuspids were cleaned and shaped using rotary instrumentation. All roots were autoclaved for 20 min. Thirty-six of the roots were placed in glass flasks with blood heart infusion media and Enterococcus faecalis (ATCC 4082) for 4 weeks. The remaining 12 roots were placed in a sterile environment and served as negative controls. The contaminated roots were irrigated by conventional means using a buffered 0.5 % sodium hypochlorite solution with or without photon-induced photoacoustic streaming (PIPS) activation (n = 12 each group). The remaining 12 roots did not receive any treatment and served as positive controls. The apical 3 mm of each tooth was sectioned and pulverized. The pulverized samples were collected and placed in Eppendorf micro-centrifuge tubes with sterile phosphate-buffered saline. Thirty MicroLiters of the collected samples was then placed in the blood heart infusion media and incubated for 24 h at 37 °C. Colony forming units (CFU) were compared with Wilcoxon signed ranked test. Mann-Whitney U test was used to assess PIPS effectiveness in comparison with conventional irrigation. Results Both regimens reduced significantly the number of CFU; however, reduction was significantly higher for the PIPS group (p = 0.002). Conclusion Buffered 0.5 % sodium hypochlorite delivered by conventional method was effective in removing E. faecalis from contaminated root canals; however, activation of a buffered 0.5 % sodium hypochlorite solution by PIPS significantly increased its antimicrobial capacity.

Relationship between the Corynebacterium pseudotuberculosis, phospholipase D inoculation and the fertility characteristics of crossbred Boer bucks

The aim of this study was to investigate the effects of Corynebacterium pseudotuberculosis and phospholipase D (PLD) on buck's fertility, specifically, testosterone concentration, scrotal circumference and semen quality. Crossbred Boer goats (n=13) aged 12–14 months were divided into three groups. The first group (n=3) was inoculated with one ml sterile phosphate buffer saline subcutaneously into subaxillary area as the control. The second group (n=5) was inoculated with live C. pseudotuberculosis 1×109 cfu subcutaneously into subaxillary area. The third group (n=5) was inoculated with PLD 1ml/20kg BW intravenously into jugular vein. Blood collection was done twice a week over a period of three months for testosterone analyses. Semen was collected using electro-ejaculator once every two weeks whilst scrotal circumference was measured once every three weeks. Semen volume, semen pH and sperm progressive motility, concentration, live/dead percentage and morphology were evaluated. Bucks were euthanized three months' post inoculation and histopathological examination of the testicles and epididymis was performed. The results showed seven folds' significant decrease (P<0.05) in testosterone concentration in both C. pseudotuberculosis and PLD inoculated groups compared to the control. Semen volume, percentage of dead/live and abnormal sperm morphology showed significant increase (P<0.05) in both inoculated groups compared to the control. The following sperm defects were observed; tapered sperm, decapitated and knobbed head, thickened midpiece, distal and proximal droplet, sterilizing tail and folded tail. Scrotal circumference, semen pH, semen wave pattern, sperm motility and concentration showed significant decrease (P<0.05) in both inoculated groups compared to the control. The testicles showed varied degrees of degeneration and necrosis with shrunken seminferous tubules. In conclusion, testosterone concentration, scrotal circumference and semen quality were negatively affected in both inoculated groups. Moreover, percentages of live/dead and abnormal sperm morphology were also increased. The results suggest that both C. pseudotuberculosis and PLD had detrimental effects on buck's fertility.

Cardiomyocyte-Specific Human Bcl2-Associated Anthanogene 3 P209L Expression Induces Mitochondrial Fragmentation, Bcl2-Associated Anthanogene 3 Haploinsufficiency, and Activates p38 Signaling

The Bcl2-associated anthanogene (BAG) 3 protein is a member of the BAG family of cochaperones, which supports multiple critical cellular processes, including critical structural roles supporting desmin and interactions with heat shock proteins and ubiquitin ligases intimately involved in protein quality control. The missense mutation P209L in exon 3 results in a primarily cardiac phenotype leading to skeletal muscle and cardiac complications. At least 10 other Bag3 mutations have been reported, nine resulting in a dilated cardiomyopathy for which no specific therapy is available. We generated αMHC-human Bag3 P209L transgenic mice and characterized the progressive cardiac phenotype invivo to investigate its utility in modeling human disease, understand the underlying molecular mechanisms, and identify potential therapeutic targets. We identified a progressive heart failure by echocardiography and Doppler analysis and the presence of pre-amyloid oligomers at 1 year. Paralleling the pathogenesis of neurodegenerative diseases (eg, Parkinson disease), pre-amyloid oligomers-associated alterations in cardiac mitochondrial dynamics, haploinsufficiency of wild-type BAG3, and activation of p38 signaling were identified. Unexpectedly, increased numbers of activated cardiac fibroblasts were identified in Bag3 P209L Tg+ hearts without increased fibrosis. Together, these findings point to a previously undescribed therapeutic target that may have application to mutation-induced myofibrillar myopathies as well as other common causes of heart failure that commonly harbor misfolded proteins.

An experiment model of obstructive hydrocephalus in the rats: injecting the astrocytes into the lateral ventricle

Objective To establish the animal model of obstructive hydrocephalus in rat by injecting lateral ventricle astrocytes from rat. Methods Three different concentrations of 20 μl astrocytes suspension with 5×107, 5×108, 5×109 cells per milliliter were injected into the right lateral ventricle of group A, B, C through stereotactic puncture, respectively, while 20 μl sterile phosphate buffer saline (PBS) solution was injected into the right lateral ventricle of control group. The magnetie resonance imaging (MRI) was performed to obtain T2 weighted brain images at 7, 14, 21, 28, 35 days after injection. Measure the maximum transverse diameter of lateral ventricle through coronary position images to determine the degree of hydrocephalus. Results The mean value of maximum transverse diameter in the control group, group A, group B and group C was (0.11±0.01), (1.01±0.78), (1.83±0.23), (1.89±0.21) mm at 35th day after injection, respectively. Compared with group A, group B and group C, the maximum transverse diameter of control group was statistically different(P 0.05). Conclusion The animal model of obstructive hydrocephalus in rat can be successfully established by injecting lateral ventricle astrocytes. Key words: Hydrocephalus; Rats; Models, animal; Astrocytes

Group B streptococcus alters properties of vaginal epithelial cells in pregnant women

Group B streptococcus (GBS) infection in pregnancy is a major cause of maternal and neonatal morbidity. An understanding of the mechanisms responsible for GBS persistence in the genital tract, as well as recognition of host defenses employed to combat its presence, are crucial to our efforts to reduce maternal GBS colonization and prevent the acquisition of neonatal infections. However, alterations in vaginal immunity in response to GBS colonization in pregnant women remain incompletely defined. Whether GBS modulates autophagy, a major host defense mechanism and contributor to the control of intracellular microbial infections, also remains unclear. We sought to identify differences in the extent of autophagy as well as in the concentration of biomarkers previously shown to be involved in vaginal innate immunity between GBS-positive and GBS-negative pregnant women. We performed a prospective cohort study of healthy pregnant women, who had vaginal secretions obtained at 35-37 weeks of gestation, just prior to the standard GBS rectovaginal sample collection. The contents of the swabs were released into tubes containing 1 mL of sterile phosphate-buffered saline. Samples were centrifuged, and supernatant and cell pellet fractions were collected and stored separately at -80°C until used for analysis. Epithelial cells were then lysed, and the extent of autophagy was determined by measuring the residual level of p62 remaining in the cytoplasm. p62 is a protein that is consumed during autophagy, and so its concentration detectable in the cytoplasm is inversely related to the extent of autophagy induction. The intracellular level of the inducible 70-kDa heat shock protein (hsp70), an inhibitor of autophagy, was also measured. The cell-free fraction was assayed for D- and L-lactic acid, neutrophil gelatinase-associated lipocalin, extracellular matrix metalloproteinase inducer (EMMPRIN), matrix metalloproteinase (MMP)-8, alpha amylase, hyaluronan, and total protein. Laboratory personnel were blinded to all clinical data. There were 145 women included in the study, of which 45 (31%) were culture-positive for GBS. Vaginal cells from GBS-positive women had elevated intracellular levels of p62 (2.1 vs 0.7 pg/mL, P < .01) and hsp70 (16.9 vs 9.6 ng/mL, P = .03) as compared to GBS-negative women. The p62 and hsp70 levels were highly correlated in both groups of subjects (P < .01). In vaginal fluid, concentrations of neutrophil gelatinase-associated lipocalin (1.1 vs 0.7 ng/μg total protein, P = .01), MMP-8 (21.9 vs 11.1 pg/μg total protein, P = .01), and extracellular MMP inducer (8.8 vs 7.2 pg/μg total protein, P = .03) were highest in GBS-positive women. There were no differences in the concentrations of D- and L-lactic acid, alpha amylase, or hyaluronan between the 2 groups of women. The inhibition of autophagy in vaginal epithelial cells by GBS-induced hsp70 production is associated with its persistence. Concurrently, alterations in components known to influence vaginal bacterial colonization or facilitate microbial passage to the upper genital tract also occur in relation to GBS carriage.

Duck Tembusu Virus Exhibits Pathogenicity to Kunming Mice by Intracerebral Inoculation.

In this study, Kunming mice were used as the animal models to study the pathogenicity of TMUV. Three groups of 3-week-old female Kunming mice (n = 15 mice per group) were infected with the SDSG strain of TMUV in 50 μL allantoic fluid (104.8 ELD50/0.2 ml) respectively by the intracerebral (i.c.), subcutaneous (s.c.) and intranasal (i.n.) routes. The control group (n = 15 mice) was inoculated with 50 μL sterile phosphate-buffered saline. Clinical signs, gross, and microscopic lesions, viral loads in different tissues, and serum antibody titers were examined and recorded. Kunming mice infected intracerebrally showed typical clinical symptoms, including severe hindlimb paralysis, weight loss and death. Only dead mice presented severe intestinal mucosal edema. No gross lesions were observed in mice sequentially euthanized. However, microscopic lesions in the brain, spleen, liver, kidney, and lung were very typical including varying degrees of viral encephalitis, lymphocytes depletion, liver cell necrosis and nephritis, etc. Viral loads in different tissues were detected by the SYBR Green I real-time PCR assay. Viral loads in the brain, liver, and spleen were first detected and maintained a longer time, which indicated that these organs may be the target organs of TMUV. The level of viral loads was consistent with the severity of clinical signs and microscopic lesions in different tissues. The neutralizing antibody began to seroconvert at 8 dpi. Clinical signs, microscopic lesions, viral loads and serum neutralizing antibodies weren’t observed in other groups. In summary, TMUV can cause systemic infections and death in Kunming mice by i.c., which provides some experimental basis for further study of the significance of TMUV in public health.

Report of Mollicutes in the Ear Canal of Domestic Dogs in Brazil

Several microorganisms as bacteria, fungi, yeast and ectoparasites compose canine ear canal microbiota. These agents can be native or invasive pathogens that cause clinical otitis. The aim of this study was to associate Mollicutes in external ear canal with clinical otitis. 41 domestic dogs were examined, and a total of 82 otologic samples were collected using sterile swabs, placed in a plastic storage tube containing 2 milliliters of sterile phosphate buffer saline (PBS, pH 7.2) and appropriately labeled. The samples were processed for isolation of Mollicutes using Hayflick’s medium. The frequency of Mollicutes class in the isolation was 32.9% (27/82). 14.8% (4/27) of the dogs, which were positive for Mollicutes, had external otitis and 85.2% (23/27) were healthy dogs. The genus Mycoplasma spp. was detected in 7.4% (2/27) by digitonin test. It was the first isolation of Mollicutes from the external ear canal in dogs. Although there have been important findings in this study, the presence of these bacteria cannot indicate the onset and perpetuation of external otitis in dogs.