In vitro analysis of antiangiogenic activity of fungi isolated from clinical cases of equine keratomycosis

OBJECTIVE: The goal of this project was to explore the possibility that fungal organisms produce metabolites that inhibit angiogenesis. Procedures Fungal cultures were obtained from cases of keratomycosis, grown in Sabouraud's dextrose broth, and sterile filtered for use in experiments. The Matrigel assay was used to screen the filtrate samples for antiangiogenic activity. Matrigel is a basement membrane matrix that supports the differentiation of human umbilical vein endothelial (HUVE) cells into a capillary-like network of tubules. HUVE cells were cultured using standard techniques and passaged at confluence, with all cells being used at passage 3-6. HUVE cells (40 000 cells) were pipetted into each well of a 24-well tissue-culture plate coated with Matrigel. An aliquot of fungal media filtrate was added to each well and the plates allowed to incubate for 18 h, at which time they were evaluated for tubule formation. RESULTS: Two fungal isolates showed inhibition of tubule formation. The addition of 100, 200 and 400 &mgr;L of the fungal media filtrate from the first isolate (Fusarium sp. 99A34574) produced a consistent and dose-dependent inhibition of tubule formation. The second isolate (Aspergillus sp. 271599) did not show inhibition of tubule formation with 100 or 200 &mgr;L added to the wells, however, it did show inhibition at 400 &mgr;L/well. The remaining three isolates did not cause inhibition at any concentration. CONCLUSIONS: Our findings suggest that certain fungal organisms produce metabolites that inhibit tubule formation in vitro, and that these metabolites may play a significant role in altering the host vascular response to fungal infections of the cornea.     

In vitro susceptibility patterns of fungi associated with keratomycosis in horses of the northeastern United States: 68 cases (1987-2006)

OBJECTIVE: To determine in vitro susceptibility patterns of fungi associated with keratomycosis in horses in the northeastern United States and compare those patterns with results of studies from other geographic regions. DESIGN: Retrospective case series. ANIMALS: 68 horses with keratomycosis. PROCEDURES: Medical records of horses with a clinical diagnosis of keratomycosis, positive results of corneal fungal cultures, and susceptibility data were reviewed from the years 1987 to 2006. Fungal identification and in vitro antifungal susceptibility test results were recorded. The percentage of susceptible isolates was compared among antifungals for all isolates together and for the most common genera individually. RESULTS: 74 fungal isolates from 68 horses that met inclusion criteria were identified. Aspergillus, Candida, and Fusarium spp were the most frequent isolates. Grouped isolates had the highest percentage of susceptibility to nystatin (87.7%), natamycin (87.5%), and clotrimazole (80.6%). Grouped isolates had the lowest percentage of susceptibility to fluconazole (15.8%) and miconazole (27.5%). Aspergillus spp (> or = 81.0%) were most susceptible to nystatin, clotrimazole, itraconazole, and natamycin. Candida spp (100%) were most susceptible to ketaconazole, natamycin, and nystatin. Fusarium spp (100%) were only consistently susceptible to natamycin. CONCLUSIONS AND CLINICAL RELEVANCE: On the basis of in vitro susceptibility testing, nystatin, natamycin, or clotrimazole is recommended for initial topical treatment of keratomycosis in horses from the northeastern United States. Contrary to results of studies of ocular fungal isolates of horses from other regions, Candida spp were identified more frequently and miconazole had lower in vitro efficacy in the present study.     

In vitro efficacy of lufenuron against filamentous fungi and blood concentrations after PO administration in horses

Lufenuron is a benzoylphenyl urea-derived insecticide that has been recently introduced as a novel treatment for fungal infections in horses. The purposes of this study were to determine (1) the in vitro efficacy of lufenuron against Aspergillus and Fusarium spp. and (2) the ability of lufenuron to reach efficacious blood concentrations after PO administration in horses. Fungal colonies isolated from diseased equine corneas were tested against lufenuron solutions up to 700 microg/mL. Twenty-one adult horses received 1 of 3 PO lufenuron treatment regimens: 5 mg/kg body weight (BW) q24h for 3 days, 20 mg/kg BW q24h for 3 days, or 60 mg/ kg BW q24h for 1 day. Blood samples were collected up to 96 hours after drug administration and analyzed by high-performance liquid chromatography. Statistical analyses of lufenuron blood concentrations were performed by analysis of variance and Fischer's Least Significant Difference test, with statistical significance set at P < .05. Lufenuron showed no effect on the in vitro growth of Aspergillus or Fusarium spp. Lufenuron was detected in the blood of all but 1 horse and showed no adverse effects. The maximum blood lufenuron concentration (83.5 +/- 58.7 microg/L) was lower than the concentrations proven to be ineffective in vitro in this study. Further therapeutic use of lufenuron as an antifungal agent in horses should be based on proven efficacy against specific strains of clinically relevant fungi with pharmacokinetic data demonstrating sufficient lufenuron concentrations in target tissues.     

头孢喹诺对几种常见动物病原菌的体外抗菌作用

采用微量肉汤稀释法测定国产头孢喹诺对5种常见动物病原菌的最小抑菌浓度(MIC),并与头孢噻呋、氨苄西林及环丙沙星进行比较。结果显示头孢喹诺对金黄色葡萄球菌的MIC为1~2μg/mL,抗菌活性强于其他3种药物;对大肠杆菌的MIC≤0.031~0.25μg/mL,抗菌活性与环丙沙星相近,高于头孢噻呋和氨苄西林;对链球菌的MIC≤0.031~1μg/mL,抗菌活性与头孢噻呋和氨苄西林相近,高于环丙沙星;对多杀性巴氏杆菌以及胸膜肺炎放线杆菌的MIC分别≤0.031~0.5μg/mL和≤0.031μg/mL,抗菌活性与头孢噻呋和环丙沙星相近,强于氨苄西林。结果表明头孢喹诺对革兰氏阳性和阴性菌均具有强大的体外抗菌作用。     

In vitro fermentation of feces from normal and chronically diarrheal horses

Feces from 13 healthy horses and 8 horses with chronic diarrhea were subjected to an in vetro fermentation procedure that had been developed for rumen fluid. Fermentations were conducted over 6 hours in a closed system, with and without an essential amino acid (EAA) mixture being added to the basic starch-buffer medium. The addition of EAA caused no significant difference in results of fermentation of feces from healthy horses. For diarrheic animals, there was a significant (P less than 0.01) increase in gas and total volatile fatty acids production whether EAA were present or not, and alpha-amino nitrogen was utilized in significantly (P less than 0.01) greater amounts only if EAA was present. Fermentations were repeated on feces from five of the eight diarrheal horses after they had been treated with oral iodochlorhydroxyquin for 1 week, and had shown desirable clinical response. A significant difference was not shown between pre- and posttreatment fermentations, except for decreased butyrate production. The results are consistent with the hypothesis that chronic equine diarrhea is primarily a colonic disease and indicates that colonic maldigestion may, in part, be responsible for excess fecal water.     

奶牛乳房炎主要致病菌的分离与鉴定及体外抑菌试验

引起奶牛乳房炎的主要致病菌为金黄色葡萄球菌、停乳链球菌、无乳链球菌和大肠杆菌[1].对于乳房炎的治疗,一般都是采用抗生素等药物,但随着抗生素长期大剂量的广泛使用,细菌抗药性和抗生素乳中残留问题被普遍关注.     

In vitro evaluation of the ovistatic and ovicidal effect of the cosmopolitan filamentous fungi isolated from soil on Ascaris suum eggs

The ovicidal activity of seven fungal strains: Acremonium alabamense, Alternaria chlamydospora, Cladosporium herbarum, Fusarium solani, Paecilomyces variotii, Paecilomyces viridis and Penicillium verruculosum isolated from urban soil samples from Poland was determined in vitro. The fungal mycelium was co-cultured with Ascaris suum eggs on plates with 2% water–agar for 28 days. Eggs exposed and unexposed (control) to fungal mycelium were observed weekly by light microscopy and the percentage of malformed eggs were determined. The eggs were classified according to following parameters: type 1 – biochemical and physiological effect without morphological damage to the eggshell; type 2 – lytic effect with morphological alteration of the eggshell and embryo; type 3 – lytic effect with morphological alteration of eggshell and embryo with hyphal penetration and internal egg colonization. All examined species of fungi extended embryogenesis, but the retardation of embryonic development was varied and depended on the species. A. alabamense, A. chlamydospora and P. verruculosum exhibited very high inhibitory activity on A. suum egg development. The fungus-exposed eggs revealed morphological alternations in all stages of embryogenesis. Isolates of F. solani, P. variotii and P. viridis showed hyphal penetration and internal colonization of A. suum eggs (type 3 effect). No appressoria were produced and simple hyphal penetrations were most commonly observed. A. alabamense and P. verruculosum demonstrated morphological destruction, with eggshell destruction. The remaining fungi showed type 1 effect. The results demonstrated that examined strains of F. solani, P. variotii and P. viridis may be considered to be potential limiting factors of parasitic geohelminth populations.     

In vitro safety assessments and antimicrobial activities of Lactobacillus rhamnosus strains isolated from a fermented mare's milk

Safety and probiotic characteristics such as antimicrobial activities of three Lactobacillus rhamnosus strains, FSMM15, FSMM22 and FSMM26, previously isolated as potential probiotics from fermented mare's milk were investigated. The three FSMM strains were susceptible to ampicillin, gentamycin, kanamycin, streptomycin, tetracycline and chloramphenicol, whereas they were resistant to erythromycin (minimal inhibitory concentration (MIC) = 4?8 µg/mL) and clindamycin (MIC = 4 µg/mL); bioconversion of bile salts, hemolytic activity and mucin degradation activity were negative; enzymatic activities of α‐chymotrypsin and β‐glucosidase were detected, but those of α‐galactosidase, β‐glucuronidase and N‐acetyl‐β‐glucosaminidase, were undetectable. Among the strains, strain FSMM15 was chosen as a safer probiotic candidate due mainly to the lack of plasminogen binding ability. Despite lower acid production of strain FSMM15 than others, its cell‐free culture supernatant inhibited growths of Salmonella Typhimurium LT‐2, Shigella sonnei , Listeria monocytogenes , and Escherichia coli O157 with comparable levels of ampicillin, suggesting a favorable aspect of strain FSMM15 as a probiotic strain.     

马波沙星对猪临床分离病原菌的体外抑菌活性研究

考察马波沙星对猪临床分离病原菌的体外抑菌活性,以期为临床应用提供依据。采用琼脂二倍稀释法测定马波沙星对猪临床分离大肠杆菌、金黄色葡萄球菌和链球菌的最小抑菌浓度(MIC),并以土霉素、恩诺沙星、诺氟沙星、环丙沙星、庆大霉素和多西环素作为对照药物。结果表明:马波沙星对猪临床分离病原菌大肠杆菌、金黄色葡萄球菌和链球菌均有较低的最小抑菌浓度,对临床分离大肠杆菌、金黄色葡萄球菌和链球菌的抑菌活性明显高于诺氟沙星、环丙沙星、庆大霉素和强力霉素,对临床分离大肠杆菌的抑菌活性高于恩诺沙星。马波沙星对引起猪乳房炎-子宫炎-无乳综合征(MMA)的主要病原菌的抑菌活性优于常用抗菌药。     

In vitro susceptibility patterns of Aspergillus and Fusarium species isolated from equine ulcerative keratomycosis cases in the midwestern and southern United States with inclusion of the new antifungal agent voriconazole

Objective  To evaluate and compare the in vitro susceptibility of Aspergillus and Fusarium spp. isolated from horses with ulcerative keratomycosis, address regional differences in equine keratomycosis isolates, and provide susceptibility data to update prior studies.
Animal studied  Fourteen horses with ulcerative keratomycosis.
Procedures  Banked fungal isolates from equine corneal ulcers (eight Aspergillus spp. and six Fusarium spp.) were identified at The University of Texas Health Science Center at San Antonio. In vitro minimum inhibitory concentration and susceptibility to natamycin, fluconazole, itraconazole, voriconazole, ketoconazole, and miconazole were determined for each isolate.
Results  Fungi were significantly more susceptible to voriconazole than to natamycin, itraconazole, fluconazole, and ketoconazole, but miconazole susceptibility did not differ significantly from voriconazole. Aspergillus spp. were most susceptible to voriconazole, miconazole, and itraconazole, which were significantly better to fluconazole and ketoconazole. Fusarium spp. susceptibility was greatest to natamycin and voriconazole and lowest to itraconazole and ketoconazole. Fusarium spp. were significantly less susceptible to itraconazole and ketoconazole compared to natamycin. No significant differences in susceptibility were found when isolates from Florida were compared with isolates from other states.
Conclusions and clinical relevance  Based on in vitro evidence, voriconazole appears to be the most effective antifungal for initial treatment of equine keratomycosis in the midwestern and southern United States. Results are comparable with previous studies in that isolated fungi from equine keratomycosis cases showed consistently poor susceptibility to fluconazole. Organisms isolated in different geographic locations of the midwestern and southern United States appeared to have similar patterns of antifungal susceptibility.     

Disassociation of bactericidal and fungistatic activities from the oxidative burst of avian macrophages

Avian peritoneal exudate macrophages, when exposed to phagocytic stimuli, produced an appreciable oxidative burst as measured by production of chemiluminescence, superoxide anion, and hydrogen peroxide. Metabolic inhibitors of the oxidative burst and scavengers of oxygen radicals clearly inhibited macrophage chemiluminescence, but had no significant effect on macrophage bactericidal activity against Escherichia coli or fungistatic activity against Candida tropicalis. Therefore, avian macrophages were capable of oxygen-independent bactericidal and fungistatic activities.     

产后子宫内膜炎患牛致病性真菌的研究

为探讨致病性真菌感染在奶牛子宫内膜炎中的作用,试验选用产后7～10 d子宫内膜炎患牛,通过真菌分离培养和套式PCR反应检测子宫真菌感染情况,并确定致病性真菌种类。结果表明:子宫内膜炎患牛真菌检出率一年四季均不同,春季为10.6%、夏季为15.2%、秋季为21.1%、冬季为18.1%,分离到的致病性真菌经鉴定为念珠球菌。说明部分子宫内膜炎患牛由致病性真菌引发,以秋冬两季真菌性子宫内膜炎发病率最高,致病性真菌主要为念珠球菌。     

大环内酯类抗生素对畜禽病原菌体外抗菌敏感性试验研究

针对兽医临床的细菌耐药现象,为指导临床合理用药,采用微量肉汤稀释法研究了三代大环内酯类抗生素泰乐菌素、替米考星和泰地罗新对兽医临床常见的15种共74株病原菌的体外抑菌效果。结果显示:对于革兰阳性菌,泰乐菌素和替米考星对乳房炎常见的金黄色葡萄球菌、猪链球菌、无乳链球菌、停乳链球菌均有较强的抑菌活性,优于泰地罗新;泰地罗新对肺炎链球菌抑菌效果较强。对于革兰阴性菌,如支气管败血波氏杆菌、溶血性曼氏杆菌、胸膜肺炎放线杆菌、副猪嗜血杆菌等致病菌,替米考星的抑菌效果比泰乐菌素强1～64倍,泰地罗新比替米考星强2～32倍。对引起肠道感染的大肠杆菌,泰地罗新亦表现出很好的抑菌活性,效果优于泰乐菌素和替米考星。本试验为畜禽细菌性疾病的防控以及大环内酯类抗菌药物的合理使用提供了基础数据。     

In vitro responses of distal airways in horses with recurrent airway obstruction

Distal airway segments (ID, 3 to 4 mm; length, 5 mm) from 2 groups of horses were isolated and suspended in tissue baths filled with Krebs solution, aerated with 5% CO2 in oxygen and maintained at 37 degrees C. Responses to exogenous acetylcholine, isoproterenol, or electrical field stimulation were compared. Control horses (n = 30) had no history of recurrent airway obstruction, whereas principal horses (n = 15) had recurrent airway obstruction and were studied during an acute episode of airway obstruction. Although the distal airways contracted in response to the cumulative half-logarithmic addition of acetylcholine (10(-10) M to 10(-3) M) in both groups, bronchi obtained from principals were less sensitive to acetylcholine than were bronchi obtained from controls. Tetdrodotoxin-sensitive electrical field stimulation-induced contractions were observed in both groups of airways, but the tension achieved in principal bronchi was less than in controls. All electrical field stimulation-induced contractions were abolished by atropine, indicating that the only excitatory innervation of equine distal airway is through the parasympathetic system. To examine the effect of isoproterenol and determine inhibitory innervation, bronchi were precontracted with histamine. Electrical field stimulation did not cause relaxation of precontracted bronchi in either group, thus indicating that distal airways lack inhibitory innervation. Isoproterenol caused similar, dose-dependent relaxation in both groups.     

In vitro response to noradrenaline of small intestine taken from normal and grass sickness-affected horses

Small intestine was taken from the caudal flexure of the duodenum and the terminal ileum proximal to the ileocaecal fold of 25 horses, 9 with acute grass sickness (AGS), 12 with subacute grass sickness (SAGS) and 12 with chronic grass sickness (CGS). The motility in the samples was measured isometrically either within 1 h of death or after storage for 24 h at 4°C.In control tissue, noradrenaline produced contractions of muscle strips which did not involve a muscarinic cholinergic mechanism and which were unaffected by the 1 antagonist prazosin but were blocked by the 2 antagonist yohimbine. Pretreatment with the antagonist phentolamine prevented the contractile response to noradrenaline and the background contractions either continued at a reduced rate and amplitude or were abolished after a few minutes. Thus, following blockade, noradrenaline reduced the background contraction rate by an effect on inhibitory adrenoceptors. The rate of background contractions in duodenal preparations was significantly greater than that in control ileal preparations.Although cold storage for 24 h caused a reduction in the background contraction rates of the control preparations, there was no effect on the contractile responses to noradrenaline, the associated pharmacology being similar to that of fresh tissue. This suggests that noradrenaline-evoked contraction was not dependent on enteric neural elements.The response to noradrenaline by grass sickness-affected tissue was generally similar to that of tissue from control horses, with an immediate contraction which was 2 sensitive. The contractile response to noradrenaline after propranolol was significantly reduced in the CGS group and there were significant differences between the AGS, CGS and control groups. There was a significant difference between the ileal preparations from the control and SAGS groups in their response to noradrenaline following pretreatment with propranolol.