沈阳昆明(KM)小鼠源和长春牛源隐孢子虫卵囊形态的数字图像分析

目的建立并分析沈阳KM小鼠源和长春牛源隐孢子虫卵囊的形态学参数,分析两种隐孢子虫卵囊的形态学差异。方法应用计算机摄像系统采集沈阳KM小鼠源和长春牛源隐孢子虫卵囊图像,计算机数字图像分析系统测量隐孢子虫卵囊的长径、横径等参数,计算卵形指数并进行分析比较。结果测量沈阳KM小鼠源隐孢子虫卵囊1190个,卵囊长径均值为5.93μm,横径的均值为4.96μm,卵形指数为1.19。测量长春牛源隐孢子虫卵囊922个,卵囊长径均值为5.19μm,卵囊横径的均值为4.17μm,卵形指数为1.25。结论沈阳KM小鼠源隐孢子虫卵囊的长径、横径均比长春牛源隐孢子虫卵囊的大,且两者存在显著差异(P<0.01)。     

人源隐孢子虫卵囊形态学参数的测定和分析

目的建立并分析人源隐孢子虫卵囊的形态学数据和参数。方法取阳性粪便涂片,金胺酚-改良抗酸染色法检测隐孢子虫卵囊。应用显微摄像系统采集1 138个人源隐孢子虫卵囊图像,使用计算机图像处理系统测量卵囊的长径、短径、等效直径、面积、体积及卵形指数。应用Excel数据处理软件对采集到的数据进行统计学分析。结果1 138个人源隐孢子虫卵囊长径均值为5.25μm,95%可信区间为3.79～6.72μm;卵囊短径均值为4.00μm,95%可信区间为2.72～5.28μm;卵囊面积均值为15.44μm~2;卵囊体积均值为46.55μm~3;卵囊等效直径均值为4.39μm;卵囊卵形指数1.33。结论建立了人源隐孢子虫卵囊形态学数据和参数,为量化及评价虫体的形态奠定了基础。     

粗球孢子菌病原学鉴定及形态学参数分析

目的 建立粗球孢子菌的形态学参数并对相关数据进行分析,为粗球孢子菌病原学鉴定提供数字化资料。方法 取粗球孢子菌肺感染患者痰液进行巴氏染色,应用显微镜摄像系统采集粗球孢子菌图像,通过计算机数字图像分析系统测量粗球孢子菌的长径,短径,周长,面积,计算其圆球度等一系列形态学参数。应用SPSS数据处理软件对采集到的数据进行统计学分析。结果 测量粗球孢子菌297个,长径均值为70.91 μm,最大值为110 μm,最小值为33 μm;短径均值为54.46 μm,最大值为92 μm,最小值为27 μm;周长最大值为382 μm,最小值 116 μm,平均值为227.60 μm;面积最大值85.93 mm²,最小值8.49 mm²,平均值为30.75 mm²。结论 利用计算机数字图像分析系统和SPSS数据处理软件获得粗球孢子菌长径、短径、周长、面积、圆球度等的累加、均值、标准差、最大值、最小值及95％可信区间等一系列形态学参数。粗球孢子菌形态学参数和数据的建立为量化及鉴别该菌提供了可靠的资料。     

隐孢子虫牛源分离株的分离和鉴定

目的 分离和鉴定采自徐州自然感染奶牛粪便中的隐孢子虫卵囊。 方法 在徐州某奶牛场采集经改良抗酸染色镜检确认为隐孢子虫感染的奶牛粪便，用不连续Sheather′s蔗糖密度梯度离心法纯化卵囊。采用Chelex-100方法提取卵囊基因组DNA，设计引物扩增小亚基核糖体RNA（SSU rRNA）基因和卵囊囊壁蛋白（COWP）基因，分别克隆到pGEM-T和pGEM-T Easy载体，测定核苷酸序列，运用BLAST和MEGA软件进行序列同源性和种系发生分析。 结果 分离的隐孢子虫卵囊个体大小为（7.4±0.32） μm×（5.4±0.21） μm，长宽比为1.37±0.07（n=20）。徐州牛源隐孢子虫与Gen-Bank公布的安氏隐孢子虫比较，SSU rRNA和COWP基因序列同源性分别为100%和99%。种系发生分析显示该株隐孢子虫与安氏隐孢子虫处于同一分支。 结论 由徐州自然感染奶牛粪便中分离获得的隐孢子虫是安氏隐孢子虫。     

合肥地区奶牛源隐孢子虫的分离与鉴定

目的分离合肥地区奶牛源隐孢子虫,并鉴定其种类。方法采集安徽省合肥市某奶牛场285头成年母牛粪样,采用饱和蔗糖溶液漂浮法和改良抗酸染色法检测牛粪中隐孢子虫卵囊,并分离纯化隐孢子虫阳性粪样中的卵囊,抽提其基因组DNA作为模板,根据隐孢子虫18S rRNA和HSP70基因设计引物,用PCR和巢式PCR方法扩增目的片段,对巢式PCR产物进行克隆、测序,并运用DNAStar软件对序列进行同源性比对,构建系统进化树。结果两种方法检测均为阳性的粪样有5份,感染率为1.8%(5/285)。隐孢子虫卵囊呈椭圆或卵圆形,饱和蔗糖溶液漂浮法分离的隐孢子虫卵囊大小为7.37μm×6.13μm,形状指数(长/宽)为1.20;改良抗酸染色法检获的隐孢子虫卵囊大小为7.58μm×6.20μm,形状指数为1.22。巢式PCR扩增出的18S rRNA和HSP70基因片段分别为250 bp和325 bp。合肥奶牛源隐孢子虫5株分离株18S rRNA基因与安氏隐孢子虫(Cryptosporidium andersoni)DQ989573序列一致性最高,两者在系统进化树上为同一分支。这5株分离株的HSP70基因序列与安氏隐孢子虫AY954892和D...     

不同免疫状态小鼠感染隐孢子虫的阈剂量研究

目的求出健康小鼠和免疫抑制小鼠感染隐孢子虫的ID5（5%感染剂量）,为原虫卫生标准的制定提供参考依据。方法以地塞米松为免疫抑制剂,建立免疫抑制小鼠模型,感染不同剂量的隐孢子虫卵囊,粪检法观察感染率,对感染剂量和感染率作线性回归分析,计算相应的ID5,并与非免疫抑制组比较。结果免疫抑制组的胸腺指数、脾脏指数、血清IgG、IgM含量与相应的非免疫抑制组比较差异均具有统计学意义（P〈0.05）;免疫抑制组小鼠感染隐孢子虫的ID5为4个卵囊,非免疫抑制小鼠的ID5为12个卵囊。结论免疫抑制小鼠对隐孢子虫的易感性增加,制定卫生标准需要对此情况加以考虑。     

重组酶介导的隐孢子虫属特异性等温核酸扩增 方法的建立及评价

目的　建立一种可用于隐孢子虫检测的重组酶介导的等温核酸扩增（RAA）方法，并对其检测敏感性和特异性进行评价。方法　以隐孢子虫属特异性18S rRNA核酸序列作为检测靶标，采用Amplfix软件设计、合成引物及荧光检测探针，构建并优化RAA荧光反应体系；分别以不同拷贝数的含18S rRNA靶序列的重组质粒、不同浓度隐孢子虫卵囊基因组DNA及不同数量隐孢子虫卵囊基因组DNA为模板进行RAA荧光法检测，以评价其敏感性；分别以隐孢子虫卵囊、蓝氏贾第鞭毛虫包囊、日本血吸虫虫卵、似蚓蛔线虫虫卵、华支睾吸虫虫卵、沙门氏菌、志贺氏菌基因组DNA为模板进行RAA荧光法检测，以评价其特异性。结果　成功建立了隐孢子虫检测RAA法，该方法可在39 ℃、20 min内得到隐孢子虫属18S rRNA基因片段的有效扩增。以不同拷贝数的含18S rRNA靶序列的重组质粒、不同浓度隐孢子虫卵囊基因组DNA及不同数量隐孢子虫卵囊基因组DNA为模板，该方法最低检出限分别为102拷贝/μL、1 pg/μL和1个/50 μL；以蓝氏贾第鞭毛虫包囊、日本血吸虫虫卵、似蚓蛔线虫虫卵、华支睾吸虫虫卵、沙门氏菌、志贺氏菌基因组DNA为模板的荧光RAA扩增结果均为阴性。结论　成功建立了一种可用于检测隐孢子虫卵囊DNA的RAA法，该方法操作简便、反应快速，敏感性和特异性均较好。     

从感染小鼠获得微小隐孢子虫卵囊方法的改进

目的　建立从感染C5 7BL/ 6N小鼠获得大量微小隐孢子虫卵囊的方法。方法　在饮水中添加不同浓度的地塞米松对人工感染小鼠进行免疫抑制 ,探讨获得最多卵囊数的药物添加量 ;比较不同纯化方法得到的卵囊回收率和纯度 ;探讨来自不同动物、不同纯化方法的卵囊在牛输卵管上皮细胞培养系统的感染情况。结果　地塞米松加入量为 2 0 μg/ml的第 3组获得的卵囊量高达 4 .16× 10 9/ 30个小鼠 ;用饱和盐水漂浮法与用蔗糖密度梯度离心法纯化得到的卵囊具有相同的回收率和纯度 ;从感染小鼠与犊牛中获得的卵囊在感染性上差异不显著。结论　成功地建立了从感染小鼠获得大量微小隐孢子虫卵囊的方法 ,具有简单、方便、快速等优点 ,并且不影响活力。     

隐孢子虫卵囊攻击免疫抑制小鼠的剂量反应关系研究

目的求出小鼠尤其是免疫抑制小鼠感染隐孢子虫的ID5、ID50、ID95,为水质原虫卫生标准的制定提供依据。方法用地塞米松饮水喂饲小鼠建立免疫抑制模型,并对攻击剂量和感染率做线性回归分析,计算相应的ID5、ID50、ID95。结果免疫抑制组小鼠的胸腺指数、脾脏指数、血清IgG、IgM含量与相应的非免疫抑制组小鼠比较,均具有统计学差异(P<0.05);免疫抑制组小鼠感染隐孢子虫的ID5、ID50、ID95分别是4、44、445个卵囊,而非免疫抑制小鼠分别是12、360、10580个。免疫抑制条件下,小鼠感染隐孢子虫ID5、ID50、ID95的剂量降低。结论地塞米松使小鼠免疫功能受到抑制后,小鼠对隐孢子虫的易感性增加,制定水质原虫卫生标准时对免疫抑制群体应予以高度重视。     

犊牛隐孢子虫卵囊的分离与鉴定

采集南京地区三个奶牛场2～4周龄犊牛新鲜粪便，应用不连续蔗糖密度梯度离心法分离纯化隐孢子虫卵囊，然后通过用光学显微镜观察，单克隆抗体间接免疫荧光试验和小鼠感染试验进行鉴定。结果显示，该地区犊牛感染的隐孢子虫主要为小型隐孢子虫（Cryptosporidium parvum），这一结果为该地区隐孢子虫病的防治提供了科学依据，同时为开展隐孢子虫病的研究提供了新的分离株。这是国内首次应用单克隆抗体技术和免疫荧光技术鉴定小隐孢子虫卵囊。     

Prevalence of Cryptosporidium parvum infections in weaned piglets and fattening porkers in Kanagawa Prefecture, Japan

Fecal samples from 232 weaned piglets (1 and 3 months old) and 252 fattening porkers (6 months old) in 8 stock-raising farms located in Kanagawa Prefecture, Japan, from June 1998 to June 2000 were examined to determine the prevalence of Cryptosporidium infection. Detection of oocysts was performed using the ethyl acetate fecal concentration method and immunofluorescent staining. C. parvum oocysts were identified in 77 (33.2%) 1-3 months old weaned piglets from four farms. The odds of excreting among 1-3 months old piglets were more than 100 times greater than among 6 months old porkers (95% confidence interval: 17-902). This strongly suggests that weaned piglets are important reservoirs of pathogenic microbes whose potential contamination of drinking water has epidemiological implications for human health.     

Short report: possible Cryptosporidium muris infection in humans

Oocysts of cryptosporidia whose morphology resembled that of Cryptosporidium muris were found in the stool of 2 healthy girls in Surabaya, Indonesia. The oocysts were predominantly oval and measured 7.75+/-0.17 x 5.55+/-0.13 microm (mean+/-SD). The number of oocysts excreted were more than 10(5) per gram of stool. The oocysts were well stained with fluorescein-conjugated monoclonal antibody to Cryptosporidium. The specimens from both girls containing the oocysts showed a positive result by the polymerase chain reaction (PCR) using primers specific for the genus Cryptosporidium, but a negative result by the PCR using primers specific for C. parvum. The 2 girls passed oocysts for 5 and 6 days, respectively. They did not complain of any symptoms during the passage of oocysts.     

微小隐孢子虫卵囊的形态结构与染色观察

目的研究隐孢子虫卵囊的形态与染色特点。方法应用图像分析系统,观察隐孢子虫卵囊的形态与内部结构,并应用Adobe Photoshop CS4软件绘制各种卵囊形态的模拟图。卵囊经改良抗酸染色法、姬姆萨染色法、阿利新蓝染色法和苏木精-伊红染色法染色后观察其染色特点。结果隐孢子虫卵囊呈圆形或椭圆形,外层有一薄的卵囊壁,内部含有0~4个月牙形子孢子和圆点状残留体,个别卵囊呈空泡状。隐孢子虫卵囊经改良抗酸染色、姬姆萨染色、阿利新蓝染色和HE染色后,分别呈现玫瑰红色、蓝色、绿色和红色。结论隐孢子虫卵囊形态和内部结构多样,改良抗酸染色法染色效果较好。     

Protozoal contamination of water used in Thai frozen food industry

This study evaluated the prevalence of contamination of water that was used for food preparation. Since protozoal cysts can be found in small numbers in water, 1,000 liters of either untreated or treated water were filtered through activated carbon block filters (1 microm nominal porosity). Identification of protozoa was performed using specific monoclonal antibodies against Giardia and Cryptosporidium parasites followed by fluorescence microscopy. Twelve of 20 untreated water samples (60%) were found to be contaminated by Giardia cysts, with an average of 53.33 cysts/1,000 liters (geometric mean 39.43), whilst 7 samples (35%) were contaminated by Cryptosporidium oocysts, with an average of 28.57 oocysts/1,000 liters (geometric mean 26.92). Three samples of untreated water (15%) were positive for both organisms. In contrast, none of the treated water samples were contaminated.     

Evaluation of usefulness of different methods for detection of Cryptosporidium in human and animal stool samples

There are many methods for detection of Cryptosporidium oocysts. Most of them (more than 20) enable the microscopic detection of Cryptosporidium oocysts in faecal smears. Such a great variability of diagnostic methods may lead to confusion as far as the choice of an appropriate technique by a given laboratory is concerned. This study evaluated the diagnostic usefulness of Cryptosporidium oocysts and coproantigen detection methods in the diagnosis of cryptosporidiosis in human (266 stool specimen) and animals (205 from cattle, 160 from sheep, 30 from horses, 80 from cats, 227 from dogs and 11 from wild animals). The total number of human and animal stool specimens processed was 266 and 713, respectively. In this study the usefulness of several diagnostic methods was compared. The following techniques were taken into account: wet mounts, hematoxylin staining, four different specific methods (modified Zeihl-Neelsen, Kinyoun's, safranin-methylene blue, as well as carbol-methyl violet and tartrazyne) and commercially available kit based on enzyme-linked immunoassay (ProspecT(r) Cryptosporidium Microplate Assay). The final number of positive specimens was 123. Out of them 77 were positive in all specific methods. The oocysts found in stool specimens were measured. Humans were infected with C. parvum and animals with C. parvum, C. andersoni or C. felis. The statistical analysis has shown that EIA test was a better than microscopy method for identification of Cryptosporidium in faecal samples in human and wild animal. Sensitivity and specificity are important factors for the choice of a proper diagnostic method for Cryptosporidium detection, however other factors such as cost, simplicity and ease of interpretation of results are also important considerations.     

计算机图像分析系统对含空泡阴道毛滴虫形态的定量研究

目的　定量研究细胞质中有空泡阴道毛滴虫滋养体的形态学改变。　方法　用计算机图像分析系统获取姬氏染色阴道毛滴虫滋养体原始二值图像 ,分别测量 2 12个细胞质中有空泡及无空泡两组滋养体及相应细胞核的长径、短径、周长、长宽比、等效圆直径、圆度、面积 ;测量有空泡滋养体的空泡面积 ;计算滋养体、细胞核和细胞质的体积。　结果有空泡滋养体的长径、短径、周长、长宽比、等效圆直径、圆度、面积和体积的数值比无空泡滋养体相应的数值大 ,其差异有极显著性意义 (P <0 .0 1)。有空泡滋养体的细胞核仅在长径、周长、圆度上与无空泡滋养体细胞核的差异有极显著性(P <0 .0 1)。相关分析显示有空泡滋养体的空泡面积与细胞质面积间的相关性有极显著性意义 (r =0 .43 0 ,P <0 .0 1) ,而与细胞核面积之间无相关性 (r =0 .12 1,P >0 .0 5 )。　结论　含空泡阴道毛滴虫滋养体细胞质增加造成虫体变大 ,形态变圆 ,相应的细胞核长径、周长、圆度也发生了变化。计算机图像分析系统的形态定量分析为研究滋养体形态的变化提供了一个敏感的方法。