人兽共患隐孢子虫种类及基因型

隐孢子虫(Cryptosporidium)目前已经成为一种重要的肠道病原体,备受关注。该病原具有广泛的宿主类型,可寄生于人和禽类、哺乳类、爬行类和两栖类等240多种动物。目前,人兽共患隐孢子虫共有8个种和1个基因型,即,人隐孢子虫(C.hominis)、微小隐孢子虫(C.parvum)、犬隐孢子虫(C.canis)、猫隐孢子虫(C.felis)、火鸡隐孢子虫(C.meleagridis)、小鼠隐孢子虫(C.muris)、猪隐孢子虫(C.suis)、安氏隐孢子虫(C.andersoni)和微小隐孢子虫(C.parvum)鹿基因型。隐孢子虫的传播主要是粪-口途经或间接通过污染的环境、食物和水传播。发展中国家和发达国家腹泻病人的隐孢子虫感染率分别为4%～20%和0.6%～20%,我国腹泻病人的隐孢子虫感染率为1.4%～13.3%,儿童的感染率显著高于成年人。本文分别叙述了人兽共患隐孢子虫的各个种类和基因型的生物学特征和分子学特征,为进一步研究虫株的分布、进化特性以及隐孢子虫在人兽之间的传播途径提供了坚实的理论基础。     

五株隐孢子虫18S rRNA部分基因克隆和序列分析

设计Nested PCR引物扩增牛源微小隐孢子虫Cryptospordium parvum、羊源微小隐孢子虫C. parvum、牛源安氏隐孢子虫C. andersoni、鸡源贝氏隐孢子虫C. baileyi及猪源隐孢子虫C. suis 18S rRNA基因突变区,PCR产物经克隆测序,其片段大小分别为212、213、213、213和210 bp.将测得的序列用DNAStar软件分析并与NCBI数据库中相同与相近种株序列进行相似性比较, 进行相似性分析并用TREECON软件绘制系统发育进化树.结果表明测得的5株隐孢子虫与各自相同种相似性为98.1%～100%,与其他种相似性为90.1%～98.6%.分析显示在此突变区设置特异酶切位点能区分开C. parvum, C. andersoni, C. baileyi与C. suis,位点分别是TaqI、BstUI、MseI.本研究为我国隐孢子虫分类、分子流行病学研究提供了新的方法.     

隐孢子虫体外培养研究进展

隐孢子虫是一种重要的人兽共患寄生性原虫,可引起儿童和免疫低下人群以及幼龄动物发生严重胃肠道疾病.长期以来围绕建立隐孢子虫体外培养模型国外学者做了大量探索,国内则在该领域尝试不多.为了研究隐孢子虫详细的内生发育过程,快速筛选抗隐孢子虫的有效药物、研究功能基因表达和外源基因的有效表达等,需要建立有效的体外培养模型.本文就20多年来国内外隐孢子虫培养概况进行了综述.     

广东和云南部分地区艾滋病患者中隐孢子虫感染的调查分析

目的 了解艾滋病患者中隐孢子虫的感染状况.方法 收集广东和云南省部分地区艾滋病患者的粪便,采用改良的抗酸染色法和免疫荧光染色法检测隐孢子虫卵囊;并同时检测患者的CD4细胞计数.结果 212例艾滋病患者粪便标本中9例标本为阳性,感染率为4.25%.广东和云南的艾滋病患者隐孢子虫感染率分别为4.00%(5/126)和4.65%(4/86),两地之间差异无统计学意义(P＞0.05);稀便与软便中的隐孢子虫卵囊检出率分别为12.00%(3/25)和3.21%(6/187),其差异无统计学意义(校正χ2=2.31,P＞0.05);男性与女性艾滋病患者隐孢子虫感染率分别为5.07%(7/138)和2.70%(2/74),其差异无统计学意义(校正χ2=0.21,P＞0.05);50～59岁组隐孢子虫感染率高于30～39岁组的感染率(校正χ2=7.15,P＜0.01);接受抗病毒治疗组与未接受治疗组艾滋病患者隐孢子虫感染率分别为1.12%(2/179)和21.21%(7/33),两者差异有统计学意义(校正χ2=18.54,P=0.0000);艾滋病晚期,尤其是CD4细胞计数少于100个/μl的患者其发病率明显提高.结论 我国南方艾滋病患者中存在着隐孢子虫感染,但发病率明显低于国外报道;艾滋病患者的便形、性别及所处地区不能预示隐孢子虫感染率;抗病毒治疗能降低隐孢子虫病感染率,艾滋病患者的隐孢子虫感染多发生在疾病的终末期.     

当归补血汤对隐孢子虫感染小鼠肠道局部免疫的影响

目的 探讨当归补血汤及其多糖对免疫低下小鼠感染隐孢子虫后的免疫调节作用.方法 用微小隐孢子虫卵囊感染免疫抑制的BALB/c小鼠,建立隐孢子虫感染的动物模型,再给该模型小鼠灌服当归补血汤及其多糖10天后,ELISA法检测小鼠肠道局部IL-2、IFN-γ、IL-4及sIgA的水平,HE染色检查肠道病理学改变.同时设模型组和正常组作对照.结果 当归补血汤及其多糖能不同程度促进隐孢子虫感染小鼠肠道粘膜IL-2、IFN-γ、IL-4及sIgA的分泌;肠道粘膜病理改变明显好转.结论当归补血汤及其多糖可调节小鼠肠道局部免疫并抑制隐孢子虫感染.     

犬隐孢子虫和隐孢子虫病的研究进展

隐孢子虫病（Cryptosporidiosis）是由隐孢子虫（Cryptosporidium）起的一种重要的人畜共患病。近年来，隐孢子虫病的检出率逐年增高，已被列为世界最常见的6种腹泻病之一。犬隐孢子虫病世界各地均有报道，严重威胁着人类和动物的健康。本文对犬隐孢子虫病的病原分类、虫体形态和寄生部位、流行病学特点、诊断、防治等方面研究状况进行了综述，为犬隐孢子虫病的有效防制提供参考。     

基于PNO基因序列分析隐孢子虫种系发育关系

本文以核基因组的功能蛋白丙酮酸∶NADP^＋氧化还原酶（pyruvate∶NADP^＋ oxidoreductase,PNO）编码基因作为研究对象,对本实验室分离保存的隐孢子虫虫株进行扩增测序,用ClustalX 1.81对扩增序列与GenBank相关参考序列进行比对,用Paup4.0程序中邻接法（Neighbor-joining method,NJ）、最大简约法（Parsimony,MP）和最大似然法（Maximum Likelihood,ML）进行聚类分析,以确定不同隐孢子虫分离株之间的遗传进化关系,并以18S rRNA和HSP70基因构僵的基因树作参照,进而评价PNO这一基因座是否适合作为隐孢子虫基因分型和进化关系分析的基因座。结果表明通过PNO构建的进化树将隐孢子虫分为2大类：Cryptosporidium bailey和C.meleagridis处于一个分枝,C.hominis、C.parvum牛基因型和C.parvum鼠基因型处于另一个分枝上。不同隐孢子虫之间的同源性介于95.0%-100%,能有效区分隐孢子虫不同基因型。因此,PNO基因序列也适合作为隐孢子虫分离株种系发育的遗传标记。     

应用巢式PCR方法诊断微小隐孢子虫感染的实验研究

目的应用巢式PCR扩增方法诊断微小隐孢子虫感染。方法用昆明种小鼠通过免疫抑制方法建立动物模型，通过不连续蔗糖密度梯度离心法分离、纯化隐孢子虫卵囊。抽提DNA后，用巢式PCR扩增隐孢子虫的18S核糖体DNA，电泳、割胶回收后测序。结果成功建立隐孢子虫的小鼠免疫抑制动物模型，纯化后的隐孢子虫卵囊形状均一，呈圆形或椭圆形，大小在3-6um左右。经巢式PCR扩增，在840bp左右有一条特异性条带。通过测序和生物信息学分析，证实该基因序列与微小隐孢子虫18S具有高度同源性。结论巢式PCR方法扩增18S基因是诊断微小隐孢子虫感染的敏感方法。     

十堰地区恶性肿瘤患者隐孢子虫感染情况调查

目的调查十堰地区恶性肿瘤患者的隐孢子虫感染情况及流行特点,为恶性肿瘤患者隐孢子虫感染的防治提供依据。方法采集十堰市太和医院217例恶性肿瘤患者粪便,采用金胺酚＋改良抗酸染色法检查隐孢子虫卵囊。结果 217例肿瘤患者隐孢子虫感染率为48.85%,男性患者与女性患者隐孢子虫感染率分别为49.58%和47.96%,差异无统计学意义（P〉0.05）;化疗、放疗、化疗＋放疗患者隐孢子虫感染率分别为38.46%、37.88%和69.86%,差异有统计学意义（P〈0.01）;不同类别的肿瘤患者的隐孢子虫感染率分别是：呼吸系统为37.50%、消化系统为60.78%、乳腺为38.89%、其他（包括脑瘤、肾上腺瘤、甲状腺瘤等）为40.00%,差异有统计学意义（P〈0.01）。结论恶性肿瘤患者易发生隐孢子虫感染,可能与其机体的免疫功能下降有关。     

贾第鞭毛虫和隐孢子虫的流行病学研究进展

蓝氏贾第鞭毛虫和隐孢子虫在我国的流行状况还不是很清楚,本文简单介绍了贾第鞭毛虫和隐孢子虫的特性及分类,综述了国外贾第鞭毛虫和隐孢子虫的流行状况及其分子流行病学方面的内容。     

Genetic diversity at the hinge region of the unique immunoglobulin heavy gamma (IGHG) gene in leporids (Oryctolagus, Sylvilagus and Lepus)

Unlike other species, European rabbit (Oryctolagus cuniculus) possesses only one immunoglobulin gamma class. Allelic diversity at the Ig (immunoglobulin) gamma constant region encoded by the unique IGHG (immunoglobulin heavy gamma) gene is moreover much reduced. In the European rabbit, the genetic variation at IGGH hinge region is limited to a single nucleotide substitution, which causes a Met-Thr interchange at amino acid position 9 (IMGT hinge numbering). We have analysed the diversity at this region more in-depth by, (1) analysing the allelic variation in 11 breeds of domestic European rabbit (Oryctolagus cuniculus cuniculus), and (2) sequencing the gamma hinge exon in wild specimens of six species of rabbit (Oryctolagus and Sylvilagus) and hares (Lepus), including the two Oryctolagus subspecies (O. cuniculus cuniculus and O. cuniculus algirus). It appeared that among leporid species, amino acid changes occur exclusively at positions 8 and 9. However, while position 8 is occupied by either Pro or Ser residues, four different residues can occur at position 9 (Met, Thr, Pro and Leu). This variation concerns sites of potential O-glycosylation and/or proteolytic cleavage, suggesting that the underlying genetic diversity could be the outcome of selection. Preservation of the gamma hinge polymorphism in domestic stocks could therefore be important. We report here a polymerase chain reaction/restriction fragment length polymorphism protocol that has allowed the monitoring of the heterozygosity levels at the gamma hinge in 11 breeds of domestic European rabbit.     

Molecular characterisation of Cryptosporidium isolates from humans in Slovenia

Twenty-nine faecal specimens from Slovenian patients in which Cryptosporidium oocysts had been identified were studied. A fragment of the Cryptosporidium 18S rRNA gene and a fragment of the Cryptosporidium COWP gene were amplified by PCR and sequenced. Cryptosporidium parvum was identified in 26 of the 29 specimens, Cryptosporidium hominis in two, and Cryptosporidium cervine genotype in one. The fact that C. parvum, which is associated traditionally with animals, was identified in the majority of human faecal specimens suggests that cryptosporidiosis may have primarily a zoonotic origin in Slovenia.     

Molecular characterization of Cryptosporidium isolates obtained from human immunodeficiency virus-infected individuals living in Switzerland, Kenya, and the United States

A total of 22 Cryptosporidium isolates from human immunodeficiency virus-infected patients from Kenya, Switzerland, and the United States were examined at three genetic loci: the 18S ribosomal DNA, HSP-70, and acetyl coenzyme A synthetase genes. Four distinct Cryptosporidium genotypes were identified: (i) the Cryptosporidium parvum "human" genotype, (ii) the C. parvum "cattle" genotype, (iii) Cryptosporidium felis, and (iv) Cryptosporidium meleagridis. This is the first report of C. meleagridis in a human host. These results and those of others indicate that immunocompromised individuals are susceptible to a wide range of Cryptosporidium species and genotypes. Future studies are required to understand the full public health significance of Cryptosporidium genotypes and species in immunocompromised hosts.     

Real-time PCR assay targeting the actin gene for the detection of Cryptosporidium parvum in calf fecal samples

Cryptosporidium parvum infection is very important with respect to public health, owing to foodborne and waterborne outbreaks and gastrointestinal illness in immunocompetent and immunocompromised persons. In cattle, infection with this species manifests either as a subclinical disease or with diarrheal illness, which occurs more often in the presence of other infectious agents than when alone. The aim of this study was to develop a real-time polymerase chain reaction (PCR) assay for the detection of C. parvum in calf fecal samples and to compare the results of this assay with those of the method routinely used for the diagnosis of Cryptosporidium spp., nested PCR targeting the 18S rRNA gene. Two hundred and nine fecal samples from calves ranging in age from 1 day to 6 months were examined using real-time PCR specific for the actin gene of C. parvum and by a nested PCR targeting the 18S rRNA gene of Cryptosporidium spp. Using real-time PCR detection, 73.2% (153 out of 209) of the samples were positive for C. parvum, while 56.5% (118 out of 209) of the samples were positive for Cryptosporidium spp. when the nested PCR amplification method was used for the detection. The analytical sensitivity of the real-time PCR was approximately one C. parvum oocyst. There was no significant nonspecific DNA amplification of any of the following species and genotype: Cryptosporidium andersoni, Cryptosporidium baileyi, Cryptosporidium bovis, Cryptosporidium canis, Cryptosporidium galli, Cryptosporidium ryanae, Cryptosporidium serpentis, or avian genotype II. Thus, we conclude that real-time PCR targeting the actin gene is a sensitive and specific method for the detection of C. parvum in calf fecal samples.     

A pox on thee! Manipulation of the host immune system by myxoma virus and implications for viral-host co-adaptation

The poxviruses have evolved a diverse array of proteins which serve to subvert innate and adaptive host responses that abort or at least limit viral infections. Myxoma virus and its rabbit host are considered to represent an ideal poxvirus-host system in which to study the effects of these immunomodulatory proteins. Studies of laboratory rabbits (Oryctolagus cuniculus) infected with gene knockout variants of myxoma virus have provided compelling evidence that several myxoma virus gene products contribute to the pathogenic condition known as myxomatosis. However, myxomatosis, which is characterized by skin lesions, systemic immunosuppression, and a high mortality rate, does not occur in the virus' natural South American host, Sylvilogus brasiliensis. Moreover, in Australia where myxoma virus was willfully introduced to control populations of O. cuniculus, myxomatosis-resistant rabbits emerged within a year of myxoma virus introduction into the field. In this review I discuss the characterized immunomodulatory proteins of myxoma virus, their biochemical properties, their pathogenic effects in laboratory rabbits, the role of the host immune system in the susceptibility or resistance to myxomatosis, and the evidence that immunomodulatory genes may have been attenuated during the co-adaptation of myxoma virus and O. cuniculus in Australia.     

New Insights into Human Cryptosporidiosis

Cryptosporidium parvum is an important cause of diarrhea worldwide. Cryptosporidium causes a potentially life-threatening disease in people with AIDS and contributes significantly to morbidity among children in developing countries. In immunocompetent adults, Cryptosporidium is often associated with waterborne outbreaks of acute diarrheal illness. Recent studies with human volunteers have indicated that Cryptosporidium is highly infectious. Diagnosis of infection with this parasite has relied on identification of acid-fast oocysts in stool; however, new immunoassays or PCR-based assays may increase the sensitivity of detection. Although the mechanism by which Cryptosporidium causes diarrhea is still poorly understood, the parasite and the immune response to it probably combine to impair absorption and enhance secretion within the intestinal tract. Important genetic studies suggest that humans can be infected by at least two genetically distinct types of Cryptosporidium, which may vary in virulence. This may, in part, explain the clinical variability seen in patients with cryptosporidiosis.     

Molecular characterization of <Emphasis Type="Italic">Cryptosporidium</Emphasis> isolates from pigs at slaughterhouses in South Bohemia,Czech Republic

A total of 123 fecal samples of slaughtered finisher pigs and 21 sows from 14 farms were screened for Cryptosporidium spp. infection using the aniline-carbol-methyl violet staining method. Positive samples were molecularly characterized by direct sequencing of partial small subunit ribosomal RNA (SSU rRNA) and GP60 partial genes and polymerase chain reaction restriction fragment length polymorphism of SSU rRNA. Cryptosporidium oocysts were microscopically identified in 36 finishers (29%) and two sows (10%). Twenty-one mono-infections of Cryptosporidium pig genotype II and 15 mixed-infection of Cryptosporidium pig genotype II and Cryptosporidium suis in finishers were found. Both sows were infected with the Cryptosporidium parvum subgenotype IIaA16G1R1, which is reported from pigs for the first time.