18S rRNA序列差异在马梨形虫分类学中的应用

为揭示18S rRNA V4高变区在物种分类学的本质意义,及进一步阐述马泰勒虫(之前称马巴贝斯虫)的分类学地位,本试验参考马泰勒虫(DQ287951)和驽巴贝斯虫(FJ209026) 18S rRNA基因序列,在其V4高变区设计引物.将获得的片段克隆至pMD19-T进行测序,正确的结果与其他种梨形虫的相应序列进行分析.系统发生树结果显示,泰勒虫和巴贝斯虫处于明显的两个分支,而称之为马巴贝斯虫的物种和泰勒虫有着较为密切的关系,他们处于同一分支,其亲缘关系较巴贝斯虫更远.序列对齐分析表明,巴贝斯虫核苷酸序列相对于泰勒虫种序列存在多个位点的缺失和突变.而马巴贝斯虫和泰勒虫18S rRNA V4高变区核苷酸序列的相似程度较高,与巴贝斯虫在该序列上的差异较大.以上结果表明,泰勒虫种和巴贝斯虫种18S rRNA V4高变区核苷酸序列间的这种缺失和突变可作为泰勒虫和巴贝斯虫分类依据的本质因素之一.马巴贝斯虫应隶属于泰勒虫科,泰勒虫属.     

牛的巴贝斯虫18S rRNA基因序列比较研究

对中国已报道的8株牛的巴贝斯虫（包括1株牛巴贝斯虫、1株双芽巴贝斯虫、1株大巴贝斯虫、3株卵形巴贝斯虫和2株巴贝斯虫未定种）的18S rRNA基因序列进行了测定与比较。自感染动物的血液中纯化虫体，提取基因组DNA，PCR扩增靶基因，然后将其连接到pGEM—T Easy载体上，进行克隆测序。研究结果显示：牛的巴贝斯虫18S rRNA基因大小在1653～1699bp之间；用所测得的和自GenBank下载的各种动物的巴贝斯虫18S rRNA基因序列构建了系统发生树，发现由刻点血蜱传播的大巴贝斯虫伊犁株与由长角血蜱传播的3株卵形巴贝斯虫存在明显差别，应属于2个独立种；由小亚璃眼蜱传播的牛巴贝斯虫未定种不同于目前已报道的任何种类，在中国应为一个新种。因而，中国存在5种牛的巴贝斯虫，即：牛巴贝斯虫，双芽巴贝斯虫、大巴贝斯虫，卵形巴贝斯虫和巴贝斯虫未定种。     

牛瑟氏泰勒虫吉林株18S rRNA基因的扩增及系统发育研究

本研究根据GenBank上发表的牛瑟氏泰勒虫18S rRNA基因的核苷酸序列设计并合成1对特异性引物,对寄生于牛体内的瑟氏泰勒虫基因组DNA进行扩增,得到1 356 bp的18S rRNA基因片段,测序后blast分析表明该虫种属牛瑟氏泰勒虫。将该基因片段序列与GenBank中8种已知泰勒虫的相应序列进行比较分析,建立系统发育树。结果表明,牛瑟氏泰勒虫吉林分离株与水牛泰勒虫亲缘关系最近,与小泰勒虫亲缘关系较远。这一结果说明宿主因素对泰勒虫的基因型影响较大。     

牛卵形巴贝斯虫不同靶基因PCR检测方法的比较

旨在筛选出检测牛卵形巴贝斯虫特异、敏感的PCR方法。本试验以牛卵形巴贝斯虫18S rRNA、AMA-1和CCTη基因为靶基因进行PCR检测,从敏感性、特异性和临床检出率方面进行比较。结果显示,以18S rRNA基因的PCR方法敏感性最高,最小检出率为16 fg/μL;以CCTη为靶基因的PCR方法敏感性最低,检测量为1.6 pg/μL;而以顶膜抗原(AMA-1)为靶基因的PCR方法的最低检测量为160 fg/μL。三种靶基因均扩增不出牛瑟氏泰勒虫、牛巴贝斯虫和双芽巴贝斯虫基因片段。通过60份临床血液样本的检测结果表明,以18S rRNA基因设计引物的检出率最高,为30%(18/60),明显高于以AMA-1基因25%(15/60)和CCTη基因21.67%(13/60)。本试验为卵形巴贝斯虫病的诊断提供了更为敏感、特异的检测技术。     

一例犬巴贝斯虫病的诊断及虫种的分子鉴定

为了诊断犬的巴贝斯虫病并鉴定巴贝斯虫的种类,本研究对病犬进行临床检查、生理生化检查、血涂片检查,并应用分子生物学方法对虫种进行鉴定。采取病犬血液做血涂片染色后于显微镜下观察到梨籽形状虫体,初步怀疑是巴贝斯虫,随后进行分子生物学鉴定。结果患病犬血液样本中扩增获得的18S rDNA序列长度为1560 bp与GenBank登录的Babesia gibsoni(MN928823.1)相似度为99.94%。并将该序列与GenBank中不同国家和地区的巴贝斯虫18S rDNA序列构建进化树,通过比对发现获得的序列与各地区B.gibsoni处于同一进化分支中,并与中国西安株序列(MN928823.1)亲缘关系最近。经临床检查、血常规检查和血涂片检查确定该犬患有巴贝斯虫病,经分子生物学方法最终确定感染的巴贝斯虫病原为吉氏巴贝斯虫(B.gibsoni)。     

甘肃省河西区域牛羊梨形虫病病原及媒介蜱分子流行病学调查

巴贝斯虫（Babesia spp.）和泰勒虫（Theileria spp.）是世界范围内流行的蜱传播梨形虫病病原体。为评价梨形虫病传播情况,为甘肃省河西区域梨形虫病防治提供流行病学资料,采集该区域部分县区的牛羊抗凝血样品和环境游离蜱虫进行梨形虫病病原检测,分析样品中病原体的存在和分布情况,利用MEGA 6.06软件和NCBI GenBank数据库的BLASTn工具,对阳性样品中的巴贝斯虫和泰勒虫18S rRNA基因进行序列分析和遗传进化树构建。通过基于18S rRNA的巢氏PCR方法,检出梨形虫病病原1目2科9种,包括莫氏巴贝斯虫、双芽巴贝斯虫和隐藏巴贝斯虫3种巴贝斯虫,东方泰勒虫、中华泰勒虫、分离泰勒虫、吕氏泰勒虫、狍泰勒虫和环形泰勒虫6种泰勒虫。牛羊抗凝血样品梨形虫病病原总感染率为8.84%（16/181）,阳性样品分布于武威市（感染率14.94%）和张掖市（感染率3.45%）;检出携带梨形虫病病原的蜱9只,蜱病原携带率为2.52%（9/357）。检出的梨形虫病病原分属泰勒虫属和巴贝斯虫属2大类,每种病原跟国内外检出虫株的同源性均较高,处于各虫株相应分支上,提交序列相似率达99%～...     

奶牛泰勒虫病的诊断及病原体18S rDNA分析

采用常规血涂片镜检和PCR技术对新疆塔城地区疑似原虫感染的荷斯坦病牛进行检测。通过扩增18S rRNA目的基因并克隆测序,将测序结果与Gen Bank上的牛泰勒虫和巴贝斯虫18S r DNA参考序列对比分析同源性,并构建系统进化树,确定血液原虫的种属。血涂片鉴定结果显示,红细胞中发现圆环形或卵圆形虫体,具有牛环形泰勒虫的典型特征。同源性分析发现,塔城地区牛泰勒虫18S r DNA序列与法国牛源环形泰勒虫、欧洲南部牛源环形泰勒虫核苷酸序列同源性为99%,且遗传演化分析发现三者亲缘关系较近,证实该牛感染的虫种为环形泰勒虫。     

驽巴贝斯虫病PCR检测方法的建立和应用

根据驽巴贝斯虫(Babesia caballi)18S rRNA基因序列设计1对特异性引物,扩增出452 bp核苷酸片段,建立了检测驽巴贝斯虫病的PCR方法。敏感性试验结果表明,该方法最低能检出0.01 fg/μL驽巴贝斯虫DNA模板。特异性试验结果显示,在被检测的6个巴贝斯虫株中,仅驽巴贝斯虫株能扩增出特异性片段,马泰勒虫、双芽巴贝斯虫、莫氏巴贝斯虫、卵形巴贝斯虫、大巴贝斯虫的扩增结果均为阴性。对45份马属动物血样进行检测,本研究建立的PCR方法测得驽巴贝斯虫病的阳性率为26.67%(12/45),与显微镜检测方法进行了比较,结果显示PCR检测方法可显著提高驽巴贝斯虫的检出率。     

牛中华泰勒虫新种(梨形虫亚目:泰勒虫科)2.分子分类学研究

分离自我国甘肃中部地区土种黄牛的一种形态特异的泰勒虫，采用传统分类学研究鉴定为新种，被定名为中华泰勒虫（Theileria sinensis sp.nov）。又利用对泰勒虫属特异的PCR引物（92/93）对该种基因组DNA扩增，结果表明该种属泰勒虫属原虫确定无疑，并对代表虫种进化和分类的18S rRNA基因序列进行了测定，对已测出该基因1067bp长片断序列与基因库中9种巴贝斯虫，7种已知牛泰勒虫的相应序列进行比较、分，建立起系统发生树。树图表明，瑟氏泰勒虫和水牛泰勒虫的亲缘关系非常近， 中华泰勒虫与这两个种的亲缘关系较近，与附膜泰勒虫、小泰勒虫、环形泰勒虫、斑羚泰勒虫、突变泰勒虫差异较大。     

牛瑟氏泰勒虫HSP70 cDNA片段的克隆与系统发育分析

从血液涂片检查为牛瑟氏泰勒虫阳性的病牛血液中提取总RNA,通过RT-PCR技术扩增出牛瑟氏泰勒虫HSP70基因,将其重组到pMD-18TSimple载体后进行克隆、序列测定及分析。结果表明该片段长1 966 bp,编码620个氨基酸残基,将该基因片段序列与GenBank中13种已知梨形虫的相应序列进行比较分析,牛瑟氏泰勒虫吉林分离株与已报道的牛瑟氏泰勒虫亲缘关系最近,其次是环形泰勒虫,与马巴贝斯虫亲缘关系较远。     

Occurrence of Theileria and Babesia species in water buffalo (Bubalus babalis, Linnaeus, 1758) in the Hubei province, South China

The presence and prevalence of tick-borne haemoparasites in water buffalo from the Hubei province, south China was investigated using the reverse line blot (RLB) hybridization assay and phylogenetic analysis of the parasite 18S rRNA gene. Theileria buffeli (19.1%) was the most frequently found species in all of the locations, followed by Babesia orientalis (8.9%), Babesia bovis (1.0%) and Babesia bigemina (0.7%). Only 12 (3.9%) of the samples had mixed infections. Eleven samples with single infections were selected for further characterization using 18S rRNA gene sequence analysis. Phylogenetic analysis showed that the eight T. buffeli 18S rRNA gene sequences obtained grouped into four clusters, of which three grouped with the known T. buffeli types B and D. The remaining five grouped separately from the previously describe T. buffeli types, constituting new T. buffeli types. The two B. bigemina 18S rRNA gene sequences obtained grouped closely with B. bigemina Kunming; this serves as the first report of B. bigemina in the Hubei province. The B. orientalis Daye 18S rRNA gene sequence obtained grouped closely with the previously reported B. orientalis Wuhan strain and with Babesia sp. Kashi 1 and Kashi 2.     

A quantitative PCR assay for the detection and quantification of Babesia bovis and B. bigemina

The haemoparasites Babesia bovis and Babesia bigemina affect cattle over vast areas of the tropics and temperate parts of the world. Microscopic examination of blood smears allows the detection of clinical cases of babesiosis, but this procedure lacks sensitivity when parasitaemia levels are low. In addition, differentiating between similar haemoparasites can be very difficult. Molecular diagnostic procedures can, however, overcome these problems. This paper reports a quantitative PCR (qPCR) assay involving the use of SYBR Green. Based on the amplification of a small fragment of the cytochrome b gene, this method shows both high sensitivity and specificity, and allows quantification of parasite DNA. In tests, reproducible quantitative results were obtained over the range of 0.1 ng to 0.1 fg of parasite DNA. Melting curve analysis differentiated between B. bovis and B. bigemina. To assess the performance of the new qPCR procedure it was used to screen for babesiosis in 40 cows and 80 horses. B. bigemina was detected in five cows (three of these were also found to be positive by standard PCR techniques targeting the 18S rRNA gene). In addition, B. bovis was detected in one horse and B. bigemina in two horses using the proposed method, while none was found positive by ribosomal standard PCR. The sequences of the B. bigemina cytochrome b and 18S rRNA genes were completely conserved in isolates from Spain and Argentina, while those of B. bovis showed moderate polymorphism.     

Study on some molecular characterization of Babesia orientalis

The study on buffalo babesiosis indicated that its pathogen was different from other Babesia on many aspects such as morphology, transmission and pathogenicity. Therefore, it was named as a new species—Babesia orientalis. In order to prove the validity of this taxon, molecular taxonomic study on the pathogen was done in this experiment. The complete 18S rRNA gene sequence of B. orientalis was determined by PCR. It was sequenced and blasted. The results indicated that the classification of the parasite belonged to the genus Babesia. The 1700 bp complete sequence was compared with 15 other Babesia sp. available in GenBank. The data were analyzed and a phylogenetic tree was established. The results indicated that the hereditary distance of the parasite was close to that of Babesia sp. from South Africa and Babesia ovis, and the hereditary distance was far from Babesia bigemina and B. bovis.     

Detection of Babesia equi (Laveran, 1901) by nested polymerase chain reaction

We describe a nested polymerase chain reaction (PCR) for the detection of Babesia equi in equine infected erythrocytes using oligonucleotides designed on the published sequence of a B. equi merozoite antigen gene (ema-1). A 102bp DNA fragment is specifically amplified from B. equi but not from Babesia caballi, Babesia bovis or Babesia bigemina DNA. In a mock infection we were able to detect down to six infected cells in 10(8) equine erythrocytes or to detect the parasite in blood with an equivalent parasitemia of 0.000006%. Furthermore, gene polymorphism was found by performing a PCR-RFLP (PCR combined with restriction fragment length polymorphism) on both the 102bp and the entire ema-1 gene DNA amplified from two B. equi isolates, Florida (USA) and Pelotas (Southern Brazil) isolates. The polymorphism was confirmed by sequencing the entire ema-1 gene from the B. equi isolate Pelotas. Our results demonstrate that the ema-1 based nested PCR is a valuable technique for routine detection of B. equi in chronically infected horses. It may be used for epidemiological and phylogenetic studies of the parasite as well as monitoring B. equi infected horses in chemotherapeutic trials.     

Detection of Theileria and Babesia species in ticks collected from cattle

The present study was carried out to detect tick species that infest cattle, and Theileria and Babesia species transmitted by these ticks in Kayseri province (Turkey). A total of 300 cattle were examined for tick infestations. Of the 300 cattle, 117 (39%) were infested with ticks. A total of 1160 ticks belonging to 11 Ixodid genera were collected from the infested animals and their shelters. The most prevalent tick species was Boophilus annulatus 26.37% (306/1160) followed by Hyalomma marginatum marginatum 21.12% (245/1160) and Rhipicephalus turanicus 18.7% (217/1160). The collected ticks were separated into 43 tick pools, according to their species. These pools were examined for bovine Theileria and Babesia species (Theileria sp., Babesia sp., Theileria annulata, T. buffeli/orientalis, Babesia bigemina, B. bovis and B. divergens) by using the reverse line blotting method (RLB). Of the 43 tick pools examined, 6 (14%) were infected with B. bigemina, 4 (9.3%) with T. annulata, and 1 (2.3%) with Babesia sp., whereas 1 (2.3%) displayed mixed infection with T. annulata + B. bigemina. The sequence and phylogenetic analyses of Babesia sp., which could not be identified to the species level by RLB, were performed. In the phylogenetic tree, Babesia sp. (Kayseri 1) grouped with Babesia sp. (Kashi 2), Babesia sp. (Kashi 1), Babesia sp. (Xinjiang) and B. orientalis with 96.8-100% identity.     

双芽巴贝斯虫体外培养技术初探

应用双芽巴贝斯虫染虫血和液氮保藏株,进行体外培养技术初步研究,获得了生长、增殖和保种效果。双芽巴贝斯虫在牛红细胞内带虫率达５％,低染虫率的连续培养已超过２５ｄ。奠定了双芽巴贝斯虫培养技术应用基础。     

The effect of neutrophils, tumor necrosis factor, and granulocyte macrophage/colony stimulating factor on Babesia bovis and Babesia bigemina in culture.

Bovine neutrophils, human recombinant tumor necrosis factor-alpha (TNF), and bovine recombinant granulocyte macrophage/colony stimulating factor (GM/CSF) were added to microaerophilic cultures of Babesia bovis and Babesia bigemina to determine if those substances could inhibit growth. Incorporation of [3H]hypoxanthine by the Babesia spp. was utilized as an indirect measure of parasite growth. When neutrophils were added to cultures of B. bovis and B. bigemina, the highest percentage inhibition of growth was attained. There was no significant enhancement of neutrophil killing when TNF or GM/CSF or both were added to either Babesia spp. Addition of TNF or GM/CSF or both substances (without neutrophils) resulted in an increase in growth of B. bovis and B. bigemina. For B. bovis, the group that contained neutrophils only and the group that contained neutrophils and TNF resulted in significantly higher growth inhibitions than the treatment group which contained neutrophils and GM/CSF or the group that contained neutrophils, TNF, and GM/CSF. No significant differences in inhibition were observed for the same treatment groups between B. bovis and B. bigemina.     

Progression towards endemic stability to bovine babesiosis in cattle introduced onto a game ranch

An opportunity to study progression toward endemic stability to Babesia bigemina arose when cattle were reintroduced onto a game ranch in 1999 after an absence of three years. The study was conducted between August 2000 and June 2001. The unvaccinated breeding cows were sampled only once. Calves born during October 1999 were initially vaccinated against B. bigemina and Babesia bovis at the age of 4 months and were then bled at 10, 17 and 20 months of age. Calves born during 2000 were bled at 7 and 8 months of age. Sera were collected from all the cattle sampled and later tested for antibodies against B. bigemina and B. bovis using the indirect fluorescent antibody (IFA) test. Although endemic stability to B. bigemina had not been achieved at Nooitgedacht 2 years after resumption of cattle ranching, the high seroprevalence in the unvaccinated 8-month-old calves suggested that the situation was approaching stability and that calf vaccination against bovine babesiosis was not required. Tick control should therefore be restricted to prevent excessive tick worry. Only vaccinated cattle were positive to B. bovis and it was concluded that the parasite was absent from the ranch.     

Bovine piroplasms in Minorca (Balearic Islands, Spain): a comparison of PCR-based and light microscopy detection

The present study provides the first epidemiological data regarding infection by Theileria and Babesia piroplasms in cattle in Minorca. More than 94% of the studied animals were positive for the presence of Theileria sp., and of those, 41.3% were positive for the presence of Theileria annulata. These results indicate that the prevalence of Mediterranean theileriosis caused by T. annulata is very high in Minorcan dairy farms and that other Theileria sp. are also present in the area. The prevalence of infection was similar throughout the study indicating an endemic situation in this island. The use of PCR resulted in significantly higher efficacy of detection of Theileria sp. compared to microscopical observation (MO) of blood smears and allowed the specific discrimination between pathogenic and non-pathogenic theilerias which cannot be accomplished by traditional diagnosis by MO. Babesia infection in the area was mainly due to Babesia bigemina (6.0% of the studied animals were infected), while one animal (0.75%) was found to be infected by Babesia bovis. It was observed that 31% of animals infected with B. bigemina had a concurrent infection of T. annulata. PCR also resulted in a significantly higher efficacy of detection of Babesia sp. compared to MO when infection levels were higher, towards the end of the study period. The results clearly demonstrate that parasitic infection by piroplasms, especially Theileria sp. is common and endemic in the island of Minorca and that PCR is the optimal approach for the detection and discrimination of these important parasites.     

Genetic detection of Babesia bigemina from Mongolian cattle using apical membrane antigen-1 gene-based PCR assay

We developed a new nested PCR (nPCR) assay based on the Babesia bigemina apical membrane antigen-1 (AMA-1) gene sequence for parasite-specific detection. The primers were designed to amplify 738-bp and 211-bp fragments of the AMA-1 gene by primary and nested PCRs, respectively. The assay was proven to be specific for the B. bigemina, whereas the previously established SpeI-AvaI nPCR assay amplified not only the target fragment of B. bigemina but also a homologous one from Babesia ovata. The AMA-1 nPCR assay was also evaluated using field DNA samples extracted from 266 bovine blood samples collected from Mongolia in 2010. In a comparative evaluation, 90 (33.8%) and 25 (9.4%) of the blood samples showed positive reactions for B. bigemina by the SpeI-AvaI nPCR and AMA-1 nPCR assays, respectively. The sequencing analysis of the nPCR products confirmed that the AMA-1 nPCR method had specifically detected the target B. bigemina DNA. However, 4 different kinds of sequences were determined among the SpeI-AvaI nPCR amplicons. Two of them were derived from B. bigemina and B. ovata, while the origins of the others were unknown. In the current study, the presence of B. bigemina was clearly demonstrated among Mongolian cattle populations by the current nPCR assay for the first time. Furthermore, our findings also indicate that the AMA-1 nPCR assay may be a useful diagnostic tool for the specific detection of B. bigemina.