短棒状杆菌胞壁肽聚糖的实验研究

目的提取短棒状杆菌的有效成分,观察生物学效应,为研制新型治疗制剂奠定基础。方法将短棒状杆菌77—1株用冷热外压法获得细胞壁,再经苯酚及三氯甲烷萃取肤聚糖成分。结果提纯品经紫外吸收测定、淋巴细胞转化试验、抑瘤试验、脾激活试验、毒性试验证实,具有生物学活性及安全性。其主要成分经生化检测含有多肽及聚糖类物质。结论短棒状杆菌的有效成分是细胞壁上的肽聚糖类物质;实验采用的提纯工艺具有可操作性和实际应用价值,为新产品开发提供了可靠依据。     

短棒状杆菌体外抑菌作用的初步探讨

探讨短棒状杆菌体外用药的抑菌效果,为临床应用奠定基础。将短棒状杆菌稀释成6.0×109/mL的菌悬液,分别与已传代扩增的1.0×102/mL的供试菌菌悬液等量混合,再接种至培养皿中,计算菌落总数。同时设生理盐水、CP滤液和灭活CP的对照试验。大肠埃希菌、金黄色葡萄球菌、淋病奈瑟菌、铜绿假单胞菌、乙型溶血性链球菌较对照组同比长菌量明显减少,T检验与对照组均有显著性差异;白假丝酵母菌、黑曲霉与对照组长菌量均无显著性差异。短棒状杆菌外用给药对某些细菌具有一定的抑菌效果,对白假丝酵母菌及黑曲霉菌无抑菌作用。     

细胞壁缺陷白喉棒状杆菌致病作用的观察及其毒力基因检测

目的比较白喉棒状杆菌亲代细菌型及其稳定L型动物致病性的差异,了解白喉棒状杆菌稳定L型变异的特点,探讨其变异的性质及其与细胞壁缺陷突变的关系。方法用氨苄青霉素在非高渗培养基内人工诱导产毒性白喉棒状杆菌为稳定L型。采用白喉棒状杆菌稳定L型纯培养物及其代谢产物皮内感染家兔,观察局部感染部位皮肤或全身的病理改变。采用微量法提取白喉棒状杆菌稳定L型的染色体DNA,用Tox基因特异性引物进行PCR扩增以检测毒素蛋白结构基因,并进行序列测定和分析。结果白喉棒状杆菌在氨苄青霉素作用下可发生细胞壁缺陷而成为L型,白喉棒状杆菌稳定L型不能引起动物局部或全身发生异常表现,该稳定L型的传代培养物可仍然保留同其亲代细菌型一致的Tox基因及其核苷酸序列。结论提示细胞壁缺失将导致白喉棒状杆菌与产生毒素蛋白有关结构基因在宿主菌细胞内的表达受到抑制,以致使白喉棒状杆菌稳定L型丧失了产生外毒素致病的作用。     

细胞壁缺陷对白喉棒状杆菌致病性影响的研究

检测白喉棒状杆菌稳定L型对动物的致病性,探讨细胞壁缺陷对白喉棒状杆菌致病性的影响及其可能的分子机制。采用氨苄青霉素在非高渗培养基内诱导并获得产毒性白喉棒状杆菌稳定L型纯培养物。收集白喉棒状杆菌稳定L型纯培养物及其代谢产物,将收集的高于细菌型10 000倍浓度的白喉棒状杆菌稳定L型纯培养物及其代谢产物皮内注射家兔,观察局部注射部位皮肤或全身的病理改变。分别采用对流免疫电泳(CIEP)和SDS-不连续聚丙烯酰胺凝胶电泳(SDS-PAGE)检测白喉棒状杆菌稳定L型可溶性代谢产物中的白喉毒素蛋白质。结果显示,白喉棒状杆菌稳定L型不能引起动物局部或全身发生异常表现,在其可溶性代谢产物中并未检测到白喉毒素蛋白质。提示细胞壁缺陷变异可影响白喉棒状杆菌产生白喉毒素蛋白质,从而使其丧失了产生外毒素致病的作用。     

伪结核棒状杆菌pld基因无痕缺失株构建及其生物学特性及致病性研究

伪结核棒状杆菌是一种可以感染多种动物和人的兼性胞内寄生病原,主要引起被感染动物慢性化脓性炎症反应。[目的]为进一步评价磷脂酶D基因(pld)在伪结核棒状杆菌感染致病中的作用。[方法]本研究采用同源重组技术,在不引入外源基因情况下,构建伪结核棒状杆菌pld无痕缺失株。通过比较pld缺失株和野生株的菌落形态及生长曲线、体外对巨噬细胞乳酸脱氢酶(LDH)释放及其在胞内的繁殖情况以及体内感染小鼠致死率及促炎细胞因子分泌水平,研究pld与该病原感染致病之间的关系。[结果]无痕缺失pld对伪结核棒状杆菌的菌落形态及生长无明显影响。与野生株ATCC 19410和XH02相比,ATCC 19410Δpld和XH02Δpld失去了与马红球菌ATCC 6939的协同溶血功能,所感染巨噬细胞的LDH释放水平显著下降,胞内细菌数量显著降低;体内试验发现ATCC 19410Δpld对小鼠的致死率、肝脏和脾脏载菌量以及腹水及脏器促炎细胞因子水平均低于ATCC 19410。[结论]成功构建了伪结核棒状杆菌pld无痕缺失株。证实pld在该病原体外感染引发巨噬细胞死亡以及体内感染小鼠致病中具有重要作用。     

不同方法生产的短棒状杆菌生物学活性比较

提高短棒状杆菌疫苗临床疗效,解决接种后局部硬结,发烧等不良副反应。在菌体菌苗的基础上,采用超声、破碎、离心、胰酶消化脱脂的方法提取细胞壁做脾激活抑瘤试验生物学检定。结果显示,纯化的短棒状杆菌细胞壁可使小鼠脾激活指数达到4.06。能抑制艾氏腹水瘤的生长,而菌体脾指数3.18。实验证实,纯化的短棒杆菌细胞壁菌苗的活性好于菌体菌苗。     

嗜酸乳杆菌LA-G80的毒理学研究

目的对嗜酸乳杆菌的毒性进行研究。方法采用大、小鼠急性毒性试验、Ames试验、小鼠骨髓细胞微核试验、小鼠精子畸形试验和大鼠30 d喂养等对嗜酸乳杆菌进行安全性试验研究。结果急性经口毒性试验表明,大、小鼠灌胃给予嗜酸乳杆菌,最大耐受剂量雌雄两性别均大于20.0 g/kg体重,Ames试验、微核试验和精子畸形试验结果均为阴性。大鼠30 d喂养试验结果表明各项指标均未见明显毒性反应。结论在本次实验条件下,嗜酸乳杆菌未见遗传毒性。由此可初步判定,使用嗜酸乳杆菌是安全可靠的。     

厌氧棒状杆菌与双歧杆菌抗小鼠腹水瘤活性的比较

厌氧棒状杆菌和双歧杆菌分别制成死菌苗,在相同实验条件下观察两菌对小鼠腹水瘤的抗瘤效果。结果显示,两种菌苗均有显著的抗瘤活性,而厌氧棒状杆菌抗瘤作用更强于双歧杆菌,特别在延长观察期尤为显著。应用两菌苗后,脾指数均有不同程度升高,未见明显毒性作用。     

菜籽粕制备复合氨基酸螯合铜的工艺优化

发酵工艺已成功开发并用于生产大部分必需氨基酸,但蛋氨酸却是例外.尽管已经尝试利用微生物法生产蛋氨酸,但至今未能实现商业化生产.本文详细讨论了大肠杆菌、棒状杆菌和短杆菌等有潜在产蛋氨酸能力的菌株体内的蛋氨酸生物合成调节机制,阐述了微生物发酵法产蛋氨酸的研究进展,对蛋氨酸发酵生产的发展前景进行了展望.     

两种乳杆菌联用在食蟹猴阴道的定植效率及其毒性分析

研究乳杆菌鼠李糖乳杆菌-格氏乳杆菌联用制剂(LACT)的定植效率, 评估其有效剂量在动物体内的急性和长期毒性。

在食蟹猴中进行2轮阴道定植试验, 通过检测阴道分泌物pH值和清洁度, 并分离鉴定分泌物中的乳杆菌, 确定乳杆菌联用制剂的定植率和定植剂量; 通过单次和多次阴道给药, 检测联用制剂对食蟹猴体质量、食量、生殖器官及生化指标等的影响, 检测联用制剂在体内的急性和长期毒性。

联用制剂在食蟹猴中连续阴道给药5 d后能成功定植, 改善阴道的微生态; 甲硝唑预处理能够提高定植效率和定植时间; 联用制剂对食蟹猴不具有急性和长期的毒性作用。

在动物模型中, 2种乳杆菌联用制剂对细菌性阴道病具有良好的治疗潜质。

     

Die-off of Cryptosporidium parvum in soil and wastewater effluents

AIMS: To determine the effect of biotic and abiotic components of soil on the viability and infectivity of Cryptosporidium parvum, and evaluate the suitability of viability tests as a surrogate for oocyst infectivity under various environmental settings. METHODS AND RESULTS: The die-off of C. parvum in saturated and dry loamy soil was monitored over time by immunofluorescence assay (IFA) and PCR to estimate oocysts viability and by cell culture to estimate oocysts infectivity. Pseudomonas aeruginosa activity resulted in digestion of the outer layer of the oocysts, as demonstrated by loss of the ability to react in IFA. Whereas, P. aeruginosa activity did not affect the DNA amplification by PCR. A 1-log reduction in the oocysts infectivity was observed at 30 degrees C in distilled water and in saturated soil while oocysts viability was unchanged. Incubation for 10 days in dry loamy soil at 32 degrees C resulted in a 3-log(10) reduction in their infectivity while no change of oocysts viability was recorded. CONCLUSIONS: Under low temperature, C. parvum oocysts may retain their infectivity for a long time. Soil desiccation and high temperatures enhance the die-off rate of C. parvum. SIGNIFICANCE AND IMPACT OF THE STUDY: Previous die-off studies of C. parvum used viability tests that do not necessarily reflect the oocyst infectivity. Under low temperatures, there was an agreement observed between viability and infectivity tests and oocysts retained their infectivity for a long time. Desiccation and high temperatures enhance the loss of infectivity of C. parvum. The presented die-off data have significant implications on the management of wastewater reuse in warm environments.     

Immunohistochemistry based assay to determine the effects of treatments on Cryptosporidium parvum viability

Cell culture infectivity assays can provide an accurate means of detecting viable Cryptosporidium parvum oocysts from environmental samples or to test the effects of various treatments on oocyst infectivity. Cell culture assays can also be used to test candidate chemotherapeutic agents. The use of a human cell line provides a situation close to human infection. The present assay uses an anti-Cryptospordium primary antibody, combined with a biotinylated secondary antibody, and an immunoperoxidase detection system. Cryptosporidium parvum oocysts excysted in vitro when placed on monolayers of HCT-8 cells and developmental stages including schizonts and merozoites were visualized using light microscopy of the immunoperoxidase stained slides and by transmission electron microscopy of infected HCT-8 cell cultures. Because the immunoperoxidase system used gives a permanent preparation, the cell cultures can be retained and examined later. Dose titration of oocysts indicated that as few as 50 inoculated oocysts could be detected. The activity of paromomycin was evaluated in this system and 500 microg/ml produced a 97.8% reduction in infection.     

More productive in vitro culture of Cryptosporidium parvum for better study of the intra- and extracellular phases

The great difficulties in treating people and animals suffering from cryptosporidiosis have prompted the development of in vitro experimental models. Due to the models of in vitro culture, new extracellular stages of Cryptosporidium have been demonstrated. The development of these extracellular phases depends on the technique of in vitro culture and on the species and genotype of Cryptosporidium used. Here, we undertake the molecular characterization by polymerase chain reaction-restriction fragment length polymorphism of different Cryptosporidium isolates from calves, concluding that all are C. parvum of cattle genotype, although differing in the nucleotide at positions 472 and 498. Using these parasites, modified the in vitro culture technique for HCT-8 cells achieving greater multiplication of parasites. The HCT-8 cell cultures, for which the culture had not been renewed in seven days, were infected with C. parvum sporozoites in RPMI-1640 medium with 10% IFBS, CaCl2 and MgCl2 1 mM at pH 7.2. Percentages of cell parasitism were increased with respect to control cultures (71% at 48 h vs 14.5%), even after two weeks (47% vs 1.9%). Also, the percentage of extracellular stages augmented (25.3% vs 1.1% at 96 h). This new model of in vitro culture of C. parvum will enable easier study of the developmental phases of C. parvum in performing new chemotherapeutic assays.     

Complete development of Cryptosporidium parvum in rabbit chondrocytes (VELI cells)

Cryptosporidium parvum is an intracellular protozoan parasite that causes severe infection in humans and animals. The great difficulties in treating people and animals suffering from cryptosporidiosis have prompted the development of in vitro experimental models. The aim of this study was to demonstrate that C. parvum can complete its entire life cycle-from sporozoite to infective oocyst-in VELI cells (a line derived from primary culture of rabbit auricular chondrocytes). Successful infections were produced by inoculating cell cultures. Infection of MDCK, HTC-8 and VELI cells with C. parvum closely paralleled in vivo infections with regard to host cell location and chronology of parasite development. Oocysts which were produced in VELI cells were infective for infant NMRI mice. The growth of C. parvum in VELI cells provides a model, both simple and inexpensive, for testing anticryptosporidial drugs and studying host-parasite interactions.     

Pathogenicity of Ureaplasma urealyticum and Ureaplasma parvum in the lower genital tract of female BALB/c mice

The aim of this study was to establish a murine model of lower genital tract infection by Ureaplasma urealyticum and Ureaplasma parvum and evaluate differences in pathogenicity of five serotypes. BALB/c female mice were divided into seven groups (five mice in each group), including five groups infected in the lower genital tract after treatment with estradiol with U. urealyticum serotypes 4 and 8 and U. parvum serotypes 1, 3, and 6, respectively, and two control groups of untreated mice and estradiol treated mice. The presence of infection was determined on solid and liquid culture media. Tumor necrosis factor-alpha (TNF-α) expression in lower genital tract secretions was determined by PCR, and morphological and histological changes of the lower genital tract were observed. The genital secretions of all inoculated mice were positive for U. urealyticum and U. parvum on culture in both liquid and solid media. TNF-α expression at 7 and 14 days after infection was markedly increased as compared with that of the controls. Morphological changes of the external genitalia included hair loss and erosions, and histological examination revealed infiltration by inflammatory cells. The five serotypes tested were all found to be pathogenic, and the pathogenicity varied with serotype 4 showing the greatest pathogenicity.     

The utilization of sodium taurocholate in excystation of Cryptosporidium parvum and infection of tissue culture.

The present work deals with optimization of excystation of Cryptosporidium parvum oocysts and the infection process of tissue culture cells by the parasite. It was shown that presence of the bile salt sodium taurocholate in the incubation medium expedited excystation of the tested GCH1 isolate and enhanced it, as compared with bleaching of the oocysts. This bile salt had no effect on the viability of tissue culture cell lines MDBK and HCT-8 at the tested concentration of 0.375% for up to 2 hr of coincubation. Infection studies conducted on tissue culture cells showed higher infection rates in the presence of sodium taurocholate than with bleached oocysts in the absence of this bile salt. It may be concluded that, at least as regards the GCH1 strain of C. parvum, the whole infection process can be performed in the presence of sodium taurocholate, and does not require separation and cleaning of the excysted sporozoites before their application to tissue culture cells.     

Cryptosporidium parvum: in vitro cultivation in Madin-Darby canine kidney cells.

To facilitate studies of the biology of Cryptosporidium parvum, we have developed an in vitro culture system using Madin-Darby canine kidney (MDCK) cells as the host cell. Oocysts or free sporozoites were incubated 37 degrees C with monolayers of MDCK cells in supplemented RPMI 1640 medium and the cells were examined at various time intervals after initiation of the culture. High rates of infection (up to 90% of MDCK cells) were achievable. Sequential development of trophozoites, meronts, microgametocytes, and macrogametocytes was observed over a 72-h period of culture. Between 72 and 96 h we observed formation of oocyst walls, but fully sporulated oocysts were not observed. This culture system provides access to both the asexual and sexual intracellular stages of C. parvum.     

Quantitative-PCR assessment of Cryptosporidium parvum cell culture infection

A quantitative TaqMan PCR method was developed for assessing the Cryptosporidium parvum infection of in vitro cultivated human ileocecal adenocarcinoma (HCT-8) cell cultures. This method, termed cell culture quantitative sequence detection (CC-QSD), has numerous applications, several of which are presented. CC-QSD was used to investigate parasite infection in cell culture over time, the effects of oocyst treatment on infectivity and infectivity assessment of different C. parvum isolates. CC-QSD revealed that cell culture infection at 24 and 48 h postinoculation was approximately 20 and 60%, respectively, of the endpoint 72-h postinoculation infection. Evaluation of three different lots of C. parvum Iowa isolate oocysts revealed that the mean infection of 0.1 N HCl-treated oocysts was only 36% of the infection obtained with oocysts treated with acidified Hanks' balanced salt solution containing 1% trypsin. CC-QSD comparison of the C. parvum Iowa and TAMU isolates revealed significantly higher levels of infection for the TAMU isolate, which agrees with and supports previous human, animal, and cell culture studies. CC-QSD has the potential to aid in the optimization of Cryptosporidium cell culture methods and facilitate quantitative evaluation of cell culture infectivity experiments.     

Cryptosporidium parvum: the many secrets of a small genome

The coccidium Cryptosporidium parvum is an obligate intracellular parasite of the phylum Apicomplexa. It infects the gastrointestinal tract of humans and livestock, and represents the third major cause of diarrhoeal disease worldwide. Scarcely considered for decades due to its apparently non-pathogenic nature, C. parvum has been studied very actively over the last 15 years, after its medical relevance as a dangerous opportunistic parasite and widespread water contaminant was fully recognised. Despite the lack of an efficient in vitro culture system and appropriate animal models, significant advances have been made in this relatively short period of time towards understanding C. parvum biology, immunology, genetics and epidemiology. Until recently, very little was known about the genome of C. parvum, with even basic issues, such as the number and size of chromosomes, being the object of a certain controversy. With the advent of pulsed field gradient electrophoresis and the introduction of molecular biology techniques, the overall structure and fine organisation of the genome of C. parvum have started to be disclosed. Organised into eight chromosomes distributed in a very narrow range of molecular masses, the genome of C. parvum is one of the smallest so far described among unicellular eukaryotic organisms. Although fewer than 30 C. parvum genes have been cloned so far, information about the overall structure of the parasite genome has increased exponentially over the last 2 years. From the first karyotypic analyses to the recent development of physical maps for individual chromosomes, this review will try to describe the state-of-the-art of our knowledge on the nuclear genome of C. parvum and will discuss the available experimental evidence concerning the presence of extra-chromosomal elements.