表达序列标签的应用现状及分析方法研究

表达序列标签是由大规模随机挑取的cDNA克隆测序得到的组织或细胞基因组的表达序列标签。1个表达序列标签(EST)代表生物某一时期的某种组织或细胞的1个表达基因。数量迅速增加的表达序列标签已经成为开发分子标记的重要资源。介绍了EST原理、基因表达分析的方法比较、基因测序聚类分析的3个数据库比较及详细方法,表明EST在发现新基因及基因组研究中的应用具有良好的前景。     

Initiation of a Sarcocystis neurona expressed sequence tag (EST) sequencing project: a preliminary report

To accelerate genetic and molecular characterization of Sarcocystis neurona, the primary causative agent of equine protozoal myeloencephalitis (EPM), a sequencing project has been initiated that will generate approximately 7000-8000 expressed sequence tags (ESTs) from this apicomplexan parasite. Poly(A)(+) RNA was isolated from culture-derived S. neurona merozoites, and a cDNA library was constructed in a unidirectional lambda phage cloning vector. Sixty phage clones were randomly picked from the library, and the cDNA inserts were amplified from these clones using the T3 and T7 primers that flank the multi-cloning site of the lambda vector. This analysis demonstrated that 100% (60/60) of the clones selected from this library contained recombinant cDNA inserts ranging in size from 0.4 to 4.0 kilobases (kb) with an average size of 1.23kb. Single-pass sequencing from the 5' end of the 60 amplified cDNAs produced high-quality nucleotide sequence from 53 of the clones. Comparison of these ESTs to the current gene databases revealed significant matches for 10 of the ESTs, six of which are similar to sequences from other Apicomplexa (i.e., Toxoplasma gondii). Importantly, none of the ESTs were of obvious mammalian origin, thus indicating that the cDNAs in this library were derived primarily from parasite mRNA and not from mRNA of the bovine turbinate host cells. Collectively, these data indicate that the described cDNA library will provide an excellent substrate for generating a portion of the ESTs that are planned from S. neurona. This sequencing project will greatly hasten gene discovery for this protozoan pathogen thereby enhancing efforts towards the development of improved diagnostics, treatments, and preventatives for EPM. In addition, the S. neurona ESTs will represent a significant contribution to the extensive database of sequences from the Apicomplexa. Comparative analyses of these apicomplexan sequences will likely offer a multitude of important information about the biology and evolutionary history of this phylogenetic grouping of parasites.     

Bovine babesiosis (Babesia bovis infection) in Zambia

Two cases of Babesia bovis, a parasite associated with the tick Boophilus microplus, are reported for the first time from the central part of Zambia. It is concluded that infected B. microplus ticks are occasionally introduced into central Zambia by tick-infested cattle from the north-eastern part of the country where B. bovis is endemic. The spread of B. microplus in Southern Africa in a westward direction is discussed and related to the epidemiology of bovine babesiosis in Zambia.     

Bovine babesiosis (Babesia bovis infection) in Zambia

Summary

Two cases of Babesia bovis, a parasite associated with the tick Boophilus microplus, are reported for the first time from the central part of Zambia. It is concluded that infected B. microplus ticks are occasionally introduced into central Zambia by tick‐infested cattle from the north‐eastern part of the country where B. bovis is endemic. The spread of B. microplus in Southern Africa in a westward direction is discussed and related to the epidemiology of bovine babesiosis in Zambia.     

Continuous in vitro culture of erythrocytic stages of Babesia gibsoni and virulence of the cultivated parasite

Babesia gibsoni infected erythrocytes were collected from the blood of an experimentally infected dog. The parasite isolated could be continuously cultivated in vitro, with an average parasitemia of 18.2 +/- 2.4% on day 3 of culture, in RPMI-1640 medium supplemented with 7.5% normal dog serum in a humidified atmosphere containing 5% CO(2) at 37 degrees C. The parasites in the original culture were morphologically similar to those found in the peripheral blood of dogs, however, on the 4th generation of subculture, the large oval parasites, erythrocytes including many parasites and extracellular parasites were frequently observed. The B. gibsoni isolate was injected to the dog to test its infectivity after maintained in vitro for 738 days at the 214th subculture. The cultivated parasite did not cause a severe clinical sign in the dog.     

Immune control of Babesia bovis infection

Babesia bovis causes an acute and often fatal infection in adult cattle, which if resolved, leads to a state of persistent infection in otherwise clinically healthy cattle. Persistently infected cattle are generally resistant to reinfection with related parasite strains, and this resistance in the face of infection is termed concomitant immunity. Young animals are generally more resistant than adults to B. bovis infection, which is dependent on the spleen. Despite the discovery of B. bovis over a century ago, there are still no safe and effective vaccines that protect cattle against this most virulent of babesial pathogens. Immunodominant antigens identified by serological reactivity and dominant T-cell antigens have failed to protect cattle against challenge. This review describes the innate and acquired immune mechanisms that define resistance in young calves and correlate with the development of concomitant immunity in older cattle following recovery from clinical disease. The first sections will discuss the innate immune responses by peripheral blood- and spleen-derived macrophages in cattle induced by B. bovis merozoites and their products that limit parasite replication, and comparison of natural killer cell responses in the spleens of young (resistant) and adult (susceptible) cattle. Later sections will describe a proteomic approach to discover novel antigens, especially those recognized by immune CD4+ T lymphocytes. Because immunodominant antigens have failed to stimulate protective immunity, identification of subdominant antigens may prove to be important for effective vaccines. Identification of CD4+ T-cell immunogenic proteins and their epitopes, together with the MHC class II restricting elements, now makes possible the development of MHC class II tetramers and application of this technology to both quantify antigen-specific lymphocytes during infection and discover novel antigenic epitopes. Finally, with the imminent completion of the B. bovis genome-sequencing project, strategies using combined genomic and proteomic approaches to identify novel vaccine candidates will be reviewed. The availability of an annotated B. bovis genome will, for the first time, enable identification of non-immunodominant proteins that may stimulate protective immunity.     

Use of cerebellar brain smears in the diagnosis of babesiosis (Babesia bovis) in cattle

Summary Cerebral and cerebellar smears were made from 4 animals acutely reacting toBabesia bovis and 94 animals free from clinical babesiosis. The brain smears were stained by the Giemsa method and examined for the presence ofB. bovis parasites. In animals showing clinical babesiosis capillaries congested with parasitised erythrocytes were abundant in cerebral and cerebellar smears. Results obtained from both types of brain smears in animals free from clinical babesiosis agreed closely (83% conformity) as to the presence or absence of parasites. A third group of 39 animals from which cerebral and cerebellar smears were taken was also examined serologically by the indirect immunofluorescent antibody test (IFAT); about 69% of the IFAT positive and doubtful animals showed parasites in the cerebellar brain smears. The existence of false negatives in the IFAT test has been shown and discussed. It has been concluded that cerebellar samples obtained through the foramen occipitale can be used for the microscopic detection ofB. bovis parasites in latently infected bovines. This method can also be used in field cases suspected of cerebral babesiosis permitting brain sampling without resorting to the opening of the skull. Such an approach might prove particularly useful in areas where rabies occurs and the animal's head has to be sent to a diagnostic centre.

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El Empleo De Frotis De Cerebelo En El Diagnostico De Babesiosis (Babesia Bovis) En Bovinos

Resumen Se hicieron frotis de cerebro y cerebelo de 4 animales con síntomas agudos de babesiosis porBabesia bovis y de 94 animales clínicamente sanos. Los frotis se tiñeron con Giemsa y se examinaron por la presencia deB. bovis. En animales enfermos, los capilares del cerebro y cerebelo se encontraron congestionados y repletos de eritrocitos parasitados. Los resultados obtenidos en ambos tipos de frotis en animales libres de la enfermedad clínica, estuvieron de acuerdo (83% de conformidad) en cuanto a la presencia o ausencia de parásitos. Un tercer grupo de 39 animales de los cuales se tomaron frotis de cerebro y cerebelo, se examinaron también serológicamente mediante la técnica indirecta de anticuerpos fluorescentes (TIAF); cerca del 69% de animales positivos y dudosos por immunofluorescencia revelaron parásitos en los frotis de cerebelo. La existencia de falsos positivos se demostró mediante TIAF. Se concluye que los frotis de cerebelo tomados a través del agujero occipital pueden usarse para detectarB. bovis microscópicamente en animales con infecciones latentes. Este método se puede utilizar en el campo para no tener que abrir el craneo, sobre todo en áreas en donde existe rabia y las cabezas se envían al laboratorio.

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Examen De Frottis De Matière Cervelette Pour Le Diagnostic De La Babésiose (babesia bovis) Du Bétail

Résumé Des frottis de cerveau et de cervelet ont été effectués à partir de 4 animaux atteints de babésiose aiguë et de 94 autres n'ayant présenté aucun symptôme de la maladie. Les frottis ont été colorés au Giemsa et examinés pour mettre en évidence la présence deB. bovis. Chez les animaux atteints de babésiose clinique, les capillaires étaient congestionnés avec présence abondante d'érythrocytes parasités. Il existe une excellente concordance (83 p.100) entre les résultats des examens des deux types de frottis, qu'il s'agisse d'absence ou de présence des parasites. Un troisième groupe de 39 animaux dont les frottis de cerveau et de cervelet ont été faits ont également fait l'objet d'une étude sérologique pour déceler des anticorps par le test de l'immunofluorescence indirecte (I.F.A.T.); environ 69 p.100 des animaux reconnus positifs et douteux par l'I.F.A.T. ont montré des parasites dans les frottis du cervelet. L'existence de cas négatifs faux dans le test IFAT a été montrée et discutée. Il en a été conclu que les échantillons de cervelet obtenus à travers le foramen occipital peuvent être utilisés pour la détection microscopique deB. bovis chez des animaux en état d'infection latente. Cette méthode peut être également utilisée en brousse lors de suspicion de babésiose cérébrale car elle permet d'obtenir les prélèvements de tissus cérébraux nécessaires sans ouverture de la boite cranienne. Une telle approche peut être particulièrement utile dans les régions où sévit la rage et où il est nécessaire d'expédier la tête des animaux suspects à un centre de diagnostic.

     

An electron microscopic study of intra-erythrocytic stages of Babesia bovis in the brain capillaries of infected splenectomized calves.

Splenectomized vaccine donor calves undergoing primary reactions to Babesia bovis infections may develop cerebral babesiosis which leads to death if not treated in time. A brain biopsy was performed on an artificially-infected animal showing nervous symptoms and the tissue was immediately processed for electron microscopic examination. Virtually every erythrocyte in the brain capillaries sectioned was infected with B. bovis. Intra-erythrocytic merozoites, trophozoites and dividing trophozoites were indentified. Important features of the piriform merozoites included a reduced apical complex consisting of the anterior polar ring, microtubules, rhoptries and micronemes. Unidentified membrane-bound bodies, mostly spherical in shape, were observed anterior to the nucleus. The trophozoites showed very little structural differentiation and no food vacuoles or micropores could be detected. Each trophozoite produced 2 identical merozoites and the parent cell became totally incorporated in the daughter merozoites in the multiplication process. Projections were seen radiating from the surface of infected erythrocytes which appeared to adhere to other surfaces on contact. This probably resulted in the sludging of infected erythrocytes in the capillaries. The latter observations coincide with those described for Babesia argentina.     

Proteomic profiling of Rhipicephalus (Boophilus) microplus midgut responses to infection with Babesia bovis

Differences in protein expression in midgut tissue of uninfected and Babesia bovis-infected southern cattle ticks, Rhipicephalus (Boophilus) microplus, were investigated in an effort to establish a proteome database containing proteins involved in successful pathogen transmission. The electrophoretic separation of midgut membrane proteins was greatly improved by using liquid-phase isoelectric focusing combined with one-dimensional or two-dimensional (2-D) gel electrophoresis. A selection of differentially expressed proteins were subjected to analysis by capillary-HPLC-electrospray tandem mass spectrometry (HPLC-ESI-MS/MS). Among the identified Babesia-affected tick midgut proteins were six proteins that are implicated in signaling processes, including three Ca(2+)-binding proteins, a guanine nucleotide-binding protein, a protein with signal peptide activity and a translocon-associated receptor protein. Up-regulation of five metabolic enzymes indicated parasite-induced changes in electron and proton transport, protein processing and retinoic acid metabolism. Among the down-regulated proteins were a molecular chaperone, a cytoskeletal protein and a multifunctional protein of the prohibitin family. Identification of these proteins may provide new insights into the molecular interactions between B. bovis and its tick vector, and could lead to identification of anti-tick and transmission-blocking vaccine candidates.     

Serological survey of Babesia bovis and Babesia bigemina in cattle in South Africa

A total of 719 serum samples collected from clinically healthy cattle from eight provinces located in different districts of South Africa were examined by the indirect enzyme-linked immunosorbent assay (ELISA) and the standard indirect fluorescent antibody test (IFAT) to determine the serological prevalence of Babesia bovis and Babesia bigemina. The results showed that 35.3% and 39.7% of cattle were positive for B. bovis and 30% and 36.5% were positive for B. bigemina antibodies on ELISA and IFAT, respectively. Mixed infections were detected in 18.2% and 26.3% of the samples using ELISA and IFAT, respectively. Consequently, the ELISAs with recombinant B. bovis spherical body protein-4 (BbSBP-4) and B. bigemina C-terminal rhoptry-associated protein-1 (BbigRAP-1/CT) were proven to be highly reliable in the serological diagnoses of bovine babesiosis in South African cattle, as evidenced by the significant concordance rates when the results were compared to those of IFAT. Moreover, the serological prevalence was significantly different among the tested provinces, in which the ranges exhibited between 15% and 73% for B. bovis infection and between 13% and 54% for B. bigemina infection. High sero-positive rates were present in Mpumalanga and KwaZulu-Natal provinces, while the lowest rate was in the North West province. Our data provide important information regarding the current seroprevalence of bovine babesiosis in South Africa, which might be beneficial in developing rational strategies for disease control and management.     

PCR-based detection of the transovarial transmission of Uruguayan Babesia bovis and Babesia bigemina vaccine strains

Bovine babesiosis is responsible for serious economic losses in Uruguay. Haemovaccines play an important role in disease prevention, but concern has been raised about their use. It is feared that the attenuated Babesia bovis and Babesia bigemina vaccine strains may be transmitted by the local tick vector Boophilus microplus, and that reversion to virulence could occur. We therefore investigated the possibility that these strains could be transmitted via the transovarial route in ticks using a Babesia species-specific polymerase chain reaction (PCR) assay. DNA was extracted from the developmental stages of the tick vector that had fed on calves immunized with the haemovaccine. It was possible to detect Babesia DNA not only in adult ticks, but also in their eggs and larvae. In addition, it was shown that calves infested with larvae derived from eggs laid by ticks fed on acutely infected calves, were positive for Babesia using PCR. Caution should therefore be shown with the distribution of the haemovaccine in marginal areas. It is still advisable that suitable tick control measures be used to prevent transovarial transmission and the potential risk of attenuated Babesia reverting to virulence.     

Molecular detection of Babesia bovis and Babesia bigemina in white-tailed deer (Odocoileus virginianus) from Tom Green County in central Texas

Serologic and molecular evidence suggest that white-tailed deer in South Texas and North Mexico carry the agents of bovine babesiosis, Babesia bovis and Babesia bigemina. To determine if white-tailed deer in central Texas, which is outside the known occurrence of the vector tick at this time, harbor these parasites, blood samples from free-ranging and captive white-tailed deer (Odocoileus virginianus) in Tom Green County were tested by polymerase chain reaction (PCR) assays for B. bovis and B. bigemina 18S rDNA. Of the 25 samples tested, three (12%) were positive by nested PCR for B. bovis. This identity was confirmed by sequence analysis of the cloned 18S rDNA PCR product. Further confirmation was made by sequence analysis of the rRNA internal transcribed spacer (ITS) 1, 5.8S rRNA gene, and ITS 2 genomic region in two (representing samples from two different ranches) of the B. bovis positive samples. Three samples were positive by B. bigemina nested PCR, but sequencing of the cloned products confirmed only one animal positive for B. bigemina; Theileria spp. DNA was amplified from the other two animal samples. In addition to Theileria spp., two genotypically unique Babesia species sequences were identified among the cloned sequences produced by the B. bigemina primers in one sample. Phylogenetic analysis showed no separation of the deer B. bovis or B. bigemina 18S rDNA, or deer B. bovis ITS region sequences from those of bovine origin. Clarification of the possible role of white-tailed deer as reservoir hosts in maintaining these important pathogens of cattle is critical to understanding whether or not deer contribute to the epidemiology of bovine babesiosis.     

微嗜氧稳定期体外培养法分离巴贝西虫试验

自然感染的牛巴贝西虫（Babesiaboris）通常用来制作活疫苗和进行寄生虫的生物学研究，上述两个目的需要纯的B．boris，不能混有其他的血液寄生虫，但是B．boris的分布范围和其他的以节肢动物为媒介的血液寄生虫（包括牛泰勒焦虫（Theileria buffeli）、     

Attenuation of Babesia bovis by in vitro cultivation

A virulent strain of Babesia bovis was adapted to grow in erythrocyte culture in the presence of equine serum and in lieu of bovine serum. Four splenectomized calves inoculated with the adapted strain, 429, developed hematologic signs of infection and a low grade fever, but remained free of central nervous system (CNS) signs and recovered. All of six control animals inoculated with a virulent strain reacted severely and five showed CNS signs and died. The calves injected with the attenuated strain were solidly immune when challenged with the virulent strain at 44 or 78 days after vaccination.     

中华鳖表达序列标签资源中的微卫星信息分析

从NCBI下载中华鳖表达序列标签(Expressed sequence tag,EST)序列178条及浙江省水产农业新品种选育重大科技专项课题组构建的中华鳖腐皮病肝脏cDNA文库测序得到的4 146条EST序列中,通过去除片段长度过短和冗余的序列,得到全长为1 630.26 kb的2644条无冗余EST。采用MISA软件从这些序列中搜索到197个SSR,分布于137条EST序列中,出现频率为7.45%,平均分布频率为8.28 kb。2和3碱基重复是中华鳖主要的重复类型,分别占EST-SSR总数的60.91%和35.53%。AC/GT和AGC/CTG是2、3碱基重复中的优势类型,分别占2、3碱基重复的44.17%和62.86%。结果显示,中华鳖EST数据库中SSR序列出现频率较高,类型较丰富,根据中华鳖EST数据发掘SSR标记是一条可行的途径。     

牛巴贝斯虫巢式PCR诊断方法的建立

根据GenBank发表的XJ-MSA-2c核苷酸序列(登录号:EU328267)设计的2对特异性引物MS-1、MS-2、MS-3以及MS-4,建立牛巴贝斯虫病巢式PCR快速检测方法。在特异性检测试验中,仅从MSA-2c质粒样本中扩增出622、350bp2条目的片段,与预期片段大小相符,而作为对照样本的双芽巴贝斯虫、牛环形泰勒虫、东方巴贝斯虫基因组DNA均无此扩增目的条带出现。第1次和第2次扩增的敏感性分别为1.75、1.75×10-2μg/L。在对46份全血的DNA样本巢式PCR和显微镜检测中,阳性检出率分别为34.8%(16/46)和23.9%(11/46)。结果表明,所建立的巢式PCR方法准确、敏感、特异,作为牛巴贝斯虫病的快速检测和小范围的流行病学调查,具有重要的临床意义。