藏羊肖氏隐孢子虫的分离鉴定

为了解隐孢子虫在藏羊中的感染情况,采用饱和蔗糖漂浮法检查34份藏羊新鲜粪便。通过巢式PCR扩增阳性分离株的18S rRNA、HSP70和CpA135位点基因,并采用限制性内切酶Ssp I和Vsp I对18S rRNA产物进行酶切分析。同时构建系统发育树,分析藏羊感染隐孢子虫类别。结果显示仅1只藏羊感染隐孢子虫,18S rRNA与Cryptosporidium xiaoi山羊分离株(GU553016)相似率为99.9%;HSP70与C.xiaoi绵羊分离株(FJ896041)相似率为98.7%;CpA135与C.ubiquitum人源分离株(HM358023)相似率为99.3%。18S rRNA产物经Ssp I酶切后获得3条条带493 bp、262 bp和103 bp,经Vsp I酶切后获得3条条带508 bp、181 bp和104 bp,与C.bovis酶切条带相似。基于18S rRNA和HSP70的系统发育分析显示,该分离株与C.xiaoi亲缘性最近。这是首次在藏羊体内发现肖氏隐孢子虫。     

贝氏隐孢子虫在珍珠鸡体内发育的扫描电镜观察

采用扫描电镜观察了贝氏隐孢子虫在珍珠鸡体内的发育。大量球形虫体镶嵌于气管和法氏囊微绒毛丛中。气管纤毛消失,微绒毛生发生融合。法氏囊粘膜表面可观察到宿主细胞突起,在突起的表面有数个虫体寄生。滋养体呈球状,平均大小为１．７μｍ。裂殖体拥有４个或８个香蕉状裂殖子。成熟大配子体大小为 ４.２ × ３．３μｍ,在其侧面可观察到锯齿状突起。偶尔能观察到卵囊,其表 面有一明显裂缝。虫体逸出后所留下的带虫空泡似弹坑状,根据其结构可将其分为两类,其中一类为裂殖子或小配子的形成场所,另一类为卵囊的形成场所。     

鸡隐孢子虫在安徽的首次定种及感染情况的数学分析

本次调研共采集合肥地区五个大型鸡场的150个新鲜鸡粪便样品,结果在62个粪样中检出了隐孢子虫,总阳性检出率为1.3％。因五个鸡场均发现有隐孢子虫,说明该虫感染在合肥地区较为普遍。不过,各鸡场之间的阳性检出率从10.0%到83.3％高低不等。本研究同时剖检了其中四个鸡场的38只病死鸡尸,发现隐孢子虫的有11只,死鸡感染率为28.9％。通过鉴定,首次认为安徽省有火鸡孢子虫（C.Meleagridis）和贝氏隐孢子虫（C.baileyi)两个虫种。统计分析得知：隐孢子虫的阳性检出率与鸡群的日龄呈一种极显著的抛物线形相关关系;与球虫感染相比,虽然阳性检出率略低,但差异不显著,均可达到同（第）一位（原虫）感染水平;不过,二者之间不存在有直线相关关系。     

贝氏隐孢子虫在鸡体内发育的形态学特征及其对寄生器官的影响

取 2日龄海兰雏鸡 5 0只 ,分为 5组 ,分别接种 0、 0 8× 10 6、 1 6× 10 6、 3 2× 10 6、 6 4× 10 6个鸭源贝氏隐孢子虫 (Cryptosporidiumbaileyi)卵囊。接种后在不同的时间间隔内剖杀雏鸡 ,取法氏囊、气管和喉头。扫描电镜观察发现贝氏隐孢子虫主要寄生在鸡的喉头、法氏囊、气管。裂殖体有 2种类型 :Ⅰ型裂殖体含 8个裂殖子 ,Ⅱ型裂殖体含 4个裂殖子。子孢子或裂殖子在钻入过程中 ,虫体逐渐由香蕉形过渡到鼓槌形 ,最后形成球形的滋养体。带虫空泡分为有球形残体的带虫空泡和无球形残体的带虫空泡。观察到小配子的释放和虫体寄生于杯状细胞的现象。贝氏隐孢子虫寄生于气管引起纤毛倒伏、排列紊乱、纤毛融合、脱落 ;致使法氏囊上皮肿胀 ,法氏囊粘膜表面形成皱褶 ,微绒毛脱落、融合、排列紊乱 ,粘液性分泌物增多 ,炎性细胞渗出     

粉纹夜蛾微孢子虫的分离及其初步分类鉴定

从粉纹夜蛾(Trichoplussia ni,Tn)幼虫中分离纯化得到一种微孢子虫,孢子形状为卵圆形,大小为(2.832±0.486)μm×(1.947±0.317)μm.它能寄生粉纹夜蛾的大多数组织器官,在虫体内的发育过程具有微粒子属Nosema的分类特征,并初步命名为Nosema sp..     

安徽省土壤绿僵菌分离株的分子鉴定

目的进一步验证ITS序列的系统发育分析可为绿僵菌属种的鉴定提供重要的参考依据。方法对分离自安徽土壤的13株绿僵菌菌株的内转录间隔区（ITS）片段进行PCR扩增和序列测定,采用Blast方法将测序结果在GenBank中进行同源搜索,依据邻接法构建获得与其相关菌株的ITS序列系统发育树。结果供试菌株分别位于系统发育树的3个分支上,分支I包括8个菌株和金龟子绿僵菌小孢变种,1个菌株和金龟子绿僵菌鳞鳃金龟变种形成分支III,另外4个菌株和黄绿绿僵菌棉蚜变种聚为分支X。结论结合同源比较的数据,将这8个、4个和1个绿僵菌菌株分别鉴定为金龟子绿僵菌小孢变种、黄绿绿僵菌棉蚜变种和金龟子绿僵菌鳞鳃金龟变种。     

几种放射孢子虫的形态特征和分子鉴定

为阐明黏孢子虫病的传播途径,在2018-2019年期间作者调查了异育银鲫养殖池塘,从底栖寡毛类苏氏尾鳃蚓中共检测出10种放射孢子虫.基于形态特征鉴定,10种放射孢子虫分别属于6个集合类群,其中雷氏放射孢子虫1种、桔瓣放射孢子虫2种、新放射孢子虫2种、三突放射孢子虫2种、棘放射孢子虫2种和匈牙利放射孢子虫1种;这些物种中...     

一株耐铝隐球酵母菌株5-2的分离鉴定及耐铝特性分析

目的分离高耐铝的微生物菌株,为耐铝基因克隆和耐铝机制研究奠定基础。方法用含5 mmol/L铝的平板逐级筛选和纯化,PCR扩增ITS序列和26S r DNA D1/D2序列,用菌株在不同铝浓度的固体培养基和液体培养基中的生长状况鉴定耐铝能力,用ICP-AES测量菌液上清中剩余活性铝的含量。结果通过ITS序列和26S r DNA D1/D2序列比对及形态观察,初步鉴定该菌株为Cryptococcus podzolicus,该菌株的最大耐铝能力达到100 mmol/L,而且该菌株能够吸附溶液中的活性铝,这可能是其耐铝的原因之一。结论该菌种是首次发现具有耐铝能力,从而为土壤微生物耐铝机制的研究及克隆耐铝基因提供了很好的实验材料。     

几株乳酸菌的分离鉴定

通过厌氧分离技术,从鸡肠道和西红柿花面分离得到五株产乳酸细菌,根据《伯杰细菌鉴定手册》（第八版）［１］鉴定ＳＢ１、ＳＢ２４５１、ＳＢ３１５１均为干酪乳杆菌（Ｌ．ｃａｓｅｉ）,Ａ、ＳＡ则可能是乳酸菌的一个新种。五株菌均为同型发酵,乳酸产量均达到９６％以上．对抗生素等药物及低ｐＨ有一定耐受性,是益生素饲料添加剂的优良菌种［２］。     

Cryptosporidium andersoni n. sp. (Apicomplexa: Cryptosporiidae) from cattle, Bos taurus

A new species of Cryptosporidium is described from the feces of domestic cattle, Bos taurus. Oocysts are structurally similar to those of Cryptosporidium muris described from mice but are larger than those of Cryptosporidium parvum. Oocysts of the new species are ellipsoidal, lack sporocysts, and measure 7.4 x 5.5 microm (range, 6.0-8.1 by 5.0-6.5 microm). The length to width ratio is 1.35 (range, 1.07-1.50). The colorless oocyst wall is < 1 microm thick, lacks a micropyle, and possesses a longitudinal suture at one pole. A polar granule is absent, whereas an oocyst residuum is present. Oocysts were passed fully sporulated and are not infectious to outbred, inbred immunocompetent or immunodeficient mice, chickens or goats. Recent molecular analyses of the rDNA 18S and ITS1 regions and heat-shock protein 70 (HSP-70) genes demonstrate this species to be distinct from C. muris infecting rodents. Based on transmission studies and molecular data, we consider the large form of Cryptosporidium infecting the abomasum of cattle to be a new species and have proposed the name Cryptosporidium andersoni n. sp. for this parasite.     

The identification and characterisation of an unusual genotype of Cryptosporidium from human faeces as Cryptosporidium meleagridis

An unusual genotype of Cryptosporidium was identified in the faeces of six human patients by PCR/RFLP analysis of the Cryptosporidium oocyst wall protein (COWP) gene. Conventional microscopy showed oocysts indistinguishable in size from those of Cryptosporidium parvum, which reacted with two different commercially available anti-oocyst monoclonal antibodies. The isolates were further characterised by PCR/RFLP analysis of the thrombospondin-related adhesive protein of Cryptosporidium-1 (TRAP-C1) genes as well as by DNA sequencing of the COWP and the TRAP-C1 gene fragments and of two regions of the 18S rRNA gene. Sequence analysis of the COWP, TRAP-C1, and 18S rRNA gene fragments confirmed that this genotype is genetically distinct from C. parvum. 18S rRNA gene sequences were found to be identical to those published for Cryptosporidium meleagridis.     

Phenotypic and genotypic characterization of Cryptosporidium species and isolates

Recent outbreaks of cryptosporidiosis from contaminated water supplies have led to a need for the detection of Cryptosporidium oocysts from various hosts and contaminating sources. The presence of nonpathogenic species or strains of Cryptosporidium is important for diagnostic purposes as there is a potential for false-positive detection of pathogenic parasites. The present review focuses on phenotypic differences and recent advances in genotypic analyses of the genus Cryptosporidium with an emphasis on detecting various isolates and identifying differences in Cryptosporidium parvum and other species in this genus. The information currently available demonstrates important patterns in DNA sequences of Cryptosporidium, and our understanding of macro- and microevolutionary patterns has increased in recent years. However, current knowledge of Cryptosporidium genetic diversity is far from complete, and the large amount of both phenotypic and genotypic data has led to problems in our understanding of the systematics of this genus. Journal of Industrial Microbiology & Biotechnology (2001) 26, 95–106. Received 18 March 2000/ Accepted in revised form 13 August 2000     

Generation of whole genome sequences of new Cryptosporidium hominis and Cryptosporidium parvum isolates directly from stool samples

Background

Whole genome sequencing (WGS) of Cryptosporidium spp. has previously relied on propagation of the parasite in animals to generate enough oocysts from which to extract DNA of sufficient quantity and purity for analysis. We have developed and validated a method for preparation of genomic Cryptosporidium DNA suitable for WGS directly from human stool samples and used it to generate 10 high-quality whole Cryptosporidium genome assemblies. Our method uses a combination of salt flotation, immunomagnetic separation (IMS), and surface sterilisation of oocysts prior to DNA extraction, with subsequent use of the transposome-based Nextera XT kit to generate libraries for sequencing on Illumina platforms. IMS was found to be superior to caesium chloride density centrifugation for purification of oocysts from small volume stool samples and for reducing levels of contaminant DNA.

Results

The IMS-based method was used initially to sequence whole genomes of Cryptosporidium hominis gp60 subtype IbA10G2 and Cryptosporidium parvum gp60 subtype IIaA19G1R2 from small amounts of stool left over from diagnostic testing of clinical cases of cryptosporidiosis. The C. parvum isolate was sequenced to a mean depth of 51.8X with reads covering 100 % of the bases of the C. parvum Iowa II reference genome (Bioproject PRJNA 15586), while the C. hominis isolate was sequenced to a mean depth of 34.7X with reads covering 98 % of the bases of the C. hominis TU502 v1 reference genome (Bioproject PRJNA 15585).The method was then applied to a further 17 stools, successfully generating another eight new whole genome sequences, of which two were C. hominis (gp60 subtypes IbA10G2 and IaA14R3) and six C. parvum (gp60 subtypes IIaA15G2R1 from three samples, and one each of IIaA17G1R1, IIaA18G2R1, and IIdA22G1), demonstrating the utility of this method to sequence Cryptosporidium genomes directly from clinical samples. This development is especially important as it reduces the requirement to propagate Cryptosporidium oocysts in animal models prior to genome sequencing.

Conclusion

This represents the first report of high-quality whole genome sequencing of Cryptosporidium isolates prepared directly from human stool samples.     

Direct evidence for cyanide-insensitive quinol oxidase (alternative oxidase) in apicomplexan parasite Cryptosporidium parvum: phylogenetic and therapeutic implications

Cryptosporidium parvum is a parasitic protozoan that causes the diarrheal disease cryptosporidiosis, for which no satisfactory chemotherapy is currently available. Although the presence of mitochondria in this parasite has been suggested, its respiratory system is poorly understood due to difficulties in performing biochemical analyses. In order to better understand the respiratory chain of C. parvum, we surveyed its genomic DNA database in GenBank and identified a partial sequence encoding cyanide-insensitive alternative oxidase (AOX). Based on this sequence, we cloned C. parvum AOX (CpAOX) cDNA from the phylum apicomplexa for the first time. The deduced amino acid sequence (335 a.a.) of CpAOX contains diiron coordination motifs (-E-, -EXXH-) that are conserved among AOXs. Phylogenetic analysis suggested that CpAOX is a mitochondrial-type AOX, possibly derived from mitochondrial endosymbiont gene transfer. The recombinant enzyme expressed in Escherichia coli showed quinol oxidase activity. This activity was insensitive to cyanide and highly sensitive to ascofuranone, a specific inhibitor of trypanosome AOX.     

Detection and identification by real time PCR/RFLP analyses of Cryptosporidium species from human faeces

AIMS: To detect a wide range of Cryptosporidium species from human faeces by analysis of the Cryptosporidium oocyst wall protein gene by PCR. METHODS AND RESULTS: The nested-assay comprised an initial amplification using a conventional thermocycler followed by real time PCR using a LightCycler with SYBR Green I for the characterization of the amplicons. The technique uses four sets of primers composed of five to six oligonucleotides with one to six base differences corresponding to the inter-species sequence differences of the gene fragment. Restriction fragment length polymorphism analysis identified Cryptosporidium hominis and C. parvum. The assay was evaluated using DNA extracted from purified material and faecal specimens containing a range of potential pathogens (including Cryptosporidium). The assay was specific, sensitive, reproducible and rapid. CONCLUSIONS: This unique technique enables the rapid detection of a range of polymorphic COWP gene sequences directly from faeces using real time PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates a novel approach to identification of Cryptosporidium species and the identification of C. hominis and C. parvum. The technique may be especially useful for the analysis of environmental samples which are likely to contain heterogeneous mixtures of Cryptosporidium species.     

Molecular epidemiology of Cryptosporidium in humans and cattle in The Netherlands

The protozoan parasite Cryptosporidium is found world-wide and can cause disease in both humans and animals. To study the zoonotic potential of Cryptosporidium in The Netherlands we isolated this parasite from the faeces of infected humans and cattle and genotyped those isolates for several different markers. The overall genotyping results showed: for humans isolates, 70% Cryptosporidium hominis, 19% Cryptosporidium parvum, 10% a combination of C. hominis and C. parvum, and 1% Cryptosporidium felis; and for cattle isolates 100% C. parvum. Analysis of the genetic variants detected for the HSP70, ML1 and GP60 markers showed: for human isolates, one C. hominis and two C. parvum variants (C. parvum and C. parvum NL) for HSP70, one C. hominis and five C. parvum variants (C1, C2, C3, and C2 NL1 and C2 NL2) for ML1, four C. hominis (mainly IbA10G2) and four C. parvum variants (mainly IIaA15G2R1) for GP60; and the cattle isolates only C. parvum (not C. parvum NL1) for HSP70, C1 and C2 for ML1, and 17 different IIa sub-types (mainly IIaA15G2R1) for GP60. Molecular epidemiological analysis of the human data showed a C. hominis peak in autumn. The majority (80%) of the human cases were children aged between 0 and 9 years and >70% of these were caused by C. hominis. Patients >25 years of age were infected mainly with C. parvum. We conclude that C. hominis IbA10G2 is found at high frequencies in autumn in humans and not in cattle. The high prevalence of C. parvum IIaA15G2R1 in both humans and cattle indicates that cattle may be a reservoir for this sub-type in The Netherlands.     

Molecular identification and genetic diversity within species of the genera Hanseniaspora and Kloeckera

Three molecular methods, RAPD-PCR analysis, electrophoretic karyotyping and RFLP of the PCR-amplified ITS regions (ITS1, ITS2 and the intervening 5.8S rDNA), were studied for accurate identification of Hanseniaspora and Kloeckera species as well as for determining inter- and intraspecific relationships of 74 strains isolated from different sources and/or geographically distinct regions. Of these three methods, PCR-RFLP analysis of ITS regions with restriction enzymes DdeI and HinfI is proposed as a rapid identification method to discriminate unambiguously between all six Hanseniaspora species and the single non-ascospore-forming apiculate yeast species Kloeckera lindneri. Electrophoretic karyotyping produced chromosomal profiles by which the seven species could be divided into four groups sharing similar karyotypes. Although most of the 60 strains examined exhibited a common species-specific pattern, a different degree of chromosomal-length polymorphism and a variable number of chromosomal DNA fragments were observed within species. Cluster analysis of the combined RAPD-PCR fingerprints obtained with one 10-mer primer, two microsatellite primers and one minisatellite primer generated clusters which with a few exceptions are in agreement with the groups as earlier recognized in DNA-DNA homology studies.