摘要
羊梨形虫病(旧称羊焦虫病)是由顶复门、梨形虫纲、梨形虫目中的巴贝斯科(Babesiidae)巴贝斯属(Babesia)和泰勒科(Theileriidae)泰勒属(Theileria)的多种原虫寄生于羊的红细胞或网状内皮细胞内所引起的一类血液原虫病的统称,临床以高热、贫血、黄疸、血红蛋白尿或体表淋巴结肿大为主要特征,严重时可引起动物死亡。本病在我国分布广、危害严重,常常给疫区的养羊业造成巨大损失。
本研究利用实验室液氮保存的羊梨形虫病病原,包括2种(6株)羊的巴贝斯虫和2种(2株)羊的泰勒虫,采用核糖体RNA的两个内转录间隔区(ITS1和ITS2)基因组区域和5.8S rRNA基因序列分析方法,对我国的新疆喀什地区、河北承德以及甘肃省的临潭、宁县、天祝、麻当六个不同地区羊的巴贝斯虫和甘肃省的渭源、宁夏的隆德两个地区羊泰勒虫进行分子分类学及分子诊断研究。其主要研究结果包括两个方面:
1.利用实验室保存的8株羊梨形虫,提取基因组DNA,测得ITS1-5.8S-ITS2、ITS1、5.8S和ITS2序列,分别构建系统发生树,并进行同源性比较。结果显示我国羊的梨形虫可明显分为巴贝斯虫和泰勒虫两枝;其中,莫氏巴贝斯虫株之间的同源性明显高于新疆喀什株未定种巴贝斯虫。巴贝斯虫的ITS1和ITS2核苷酸区段变异较大,种间同源性分别在19.7%-99.4%,河北株、宁县株、临潭株、天祝株和麻当株的巴贝斯虫都是由血蜱属传播的,在遗传演化过程中的亲缘关系较近,同源性比较高,在ITS1-5.8S-ITS2、ITS1和ITS2序列的系统发生树上,共处一枝。新疆喀什株是由璃眼蜱属传播的,被其它的一些巴贝斯虫隔开,独立占有一枝,可以认为是一个独立的种。5.8S rRNA较保守,种间同源性在87.1%-100%之间,系统发生树上各种巴贝斯虫的分布与ITS1-5.8S-ITS2、ITS1和ITS2系统发生树基本一致。两种泰勒虫之间的同源性比较高,同已经测得的18S rRNA基因区段相似,其ITS1和ITS2区段种间变异较小,ITS1-5.8S-ITS2、ITS1、5.8S和ITS2序列的同源性分别为96.8%、96.4%、100%和95.2%。该结果与18S rRNA基因序列分类方法基本符合。
2.在已经确定的18S rRNA基因V4高变区段的基础上,利用反向线状印迹杂交技术(RLB)高特异性和高敏感性的特点,设计了梨形虫通用探针和属种特异性探针,用RLB技术检测以上8株羊梨形虫。所有种特异性探针都仅仅和它们各自的靶序列发生反应,无论是在不同虫种,还是不同种群之间都没有交叉反应,与绵羊/山羊基因组对照试验没有杂交信号。将两种羊巴贝斯虫(莫氏巴贝斯虫和巴贝斯虫未定种喀什株)和两种羊泰勒虫(吕氏泰勒虫渭源株,尤氏泰勒虫隆德株)基因组经分光光度计定量在100ng/ul,连续10倍稀释10-1-10-12,用RLB方法检测,结果显示泰勒虫较羊巴贝斯虫的敏感性高,莫氏巴贝斯虫、巴贝斯虫未定种喀什株、吕氏泰勒虫和尤氏巴贝斯虫的敏感性分别是0.01pg、0.1ng、0.0001pg、0.1pg,吕氏泰勒虫的敏感性明显高于尤氏泰勒虫。该方法还可以用来鉴别诊断吕氏泰勒虫和尤氏泰勒虫感染。对117份野外样品检测,并与ELISA血清检测和PCR结果相比较,发现反向线状印迹的检出率明显高于ELISA和PCR。所建立的方法可用于羊梨形虫病的诊断和流行病学调查。
Ovine piroplasmosis is caused by Apicomplexa, Piroplasmia, Babesiidae Babesia and Theileriidae Theileria. The disease is usually characterized by fever, hemolytic anemia, hemoglobinuria, and even death in severe case. It is widely distributed in China, and considered to be the most frequent and important tick-born disease of small ruminants and responsible for vast economic loss.
In this study, genomic DNA of the etiological agents (6 isolates of 2 Babesia species and 2 isolates of 2 Theileria species) of ovine piroplasmosis was extracted. The agents of Babesia were originated from Chengde County of Hebei Province, Kashi region of Xinjiang Province and Lintan, Ningxian, Tianzhu and Madang regions of Gansu Province, while agents of Theileria were from Weiyuan in Gansu province and Longde regions in Ningxia province. The molecular taxonomy was carried out based on the gene sequencing of ITS1, ITS2 and 5.8SrRNA of these isolates. The major results were presented as following: 1. The sequencing of ITS1-5.8S-ITS2 gene was sequenced, and then alignment of ITS1-5.8S-ITS2, ITS1, 5.8S and ITS2 was conducted by Clustal W method respectively and the corresponding phylogenetic trees were inferred. The method was successfully discriminating Babesia from Theileria. The phylogenetic trees illustrated that Babesia isolates from Hebei, Ningxian, Lintan, Tianzhu and Madang considered as Babesia motasi were placed onto one branch, whereas the isolate from Kashi remained onto another. The homology among Babesia motasi was obviously higher than that of Babesia sp. Kashi indicating the later is a new species of ovine Babesia. Moreover, B. motasi is transmitted by ticks of genus of Haemaphysalis, whereas B. sp Kashi is transmitted by ticks of Hyalomma genus. This supports that B. sp Kashi could be an independent species. B. motasi and Babesia sp. Kashi possessed unique size(s) of ITS1 and ITS2 and species specific nucleotide sequences. The 5.8S gene is conserved; the homology among the species was 87.1%-100%. The homology between two Theileria was high, which of ITS1-5.8S-ITS2、ITS1、5.8S and ITS2 was 96.8%、96.4%、100% and 95.2% respectively.
2. Based on the V4 hypervariable region of 18S rRNA sequences of ovine piroplasma, the general piroplasma probes and species-specific probes were synthesized for RLB technique. Eight isolates of ovine piroplasma were detected by applying this method, the species-specific probes could recognize only their corresponding piroplasma genomic DNA, while there was no hybridization signal with the genome of sheep/goat as the control. The sensitivity of RLB was detected by 10-fold dilution of genome of two ovine Babesia spp (B. motasi and B. sp Kashi strain ) and two ovine Theileria(T. luwenshuni Weiyuan strain and T. uilenbergi strain )from 100ng/ul on. The results indicated that the sensitivity of ovine Babesia spp is lower than the ovine Theileria, and the sensitivity of T. luwenshuni is higher than T. uilenbergi obviously. We found that the detection ability of RLB is higher than that of ELISA and PCR through detected 117 samples from field by these methods. The established RLB can be used to detect, diagnose and perform epidemiologic survey.
引文
[1]. Gajadhar A A, Marquardt W C, Hall R, et al. Ribosomal RNA sequences of Sarcocystis muris, Theileria annulata and Crypthecodinium cohnii reveal evolutionary relationships among apicomplexans, dinoflagellates, and ciliates[J]. Molecular and Biochemical Parasitology, 1991, 45(1):147~154.
[2]. Hashemi-Fesharki R, Uilenberg G. Babesia crassa n.sp. (Sporozoa, Babesiidae) of domestic sheep in Iran[J]. Veterinary Quarterly, 1981, 3(1): 1~8.
[3]. 殷宏. 羊的泰勒虫生物学及分子生物学特性研究:[博士学位论文].南京:南京农业大学,2002,12~15.
[4]. Weisburg W G, Barns S M, Pelletier D A. et al. 16S ribosomal DNA amplification for phylogenetic study.[J]. Bacteriol, 1991, 173(2): 697~703.
[5]. Ellis J, Hefford C, Baverstock P.R, et al. Ribosomal DNA sequence of Babesia and Theileria[J]. Molecular and Biochemical Parasitology, 1992, 54(1): 87~95.
[6]. Allsopp M.T, Cavalier-Smith T, De Waal D.T, et al. Phylogeny and evolution of the piroplasms[J]. Parasitology, 1994, 108(Pt2): 147~152.
[7]. Yin H, Liu G.Y, Luo J.X, et al. Phylogenetic analysis of Theileria species transmitted by Haemaphysalis qinghaiensis[J]. Parasitol Research, 2004, 92(1): 36~42.
[8]. Liu A.H, Yin H, Guan G.Q, et al. At least two genetically distinct large Babesia species infective to sheep and goats in China[J].Veterinary Parasitology 2007,147(3-4): 246~251.
[9]. 罗建勋,殷宏,刘光远, 等. 牛的巴贝斯虫 18S rRNA 基因序列比较研究[J]. 畜牧兽医学报, 2005, 36(9): 906~911.
[10]. 张龙现, 蒋金书, 刘群, 等. PCR-RFLP鉴定隐孢子虫种类研究. 畜牧兽医学报, 2004, 35 (5) , 555~559.
[11]. 张道远, 陈之端, 孙海英, 等. 用核糖体 DNA 的 ITS 序列探讨中国柽柳科植物系统分类中的几个问题[J]. 西北植物学报, 2000, 20 (3):421~431.
[12]. Gasser R.B, Zhu X.Q. Sequence-based analysis of enzymatically amplified DNA fragments by mutation detection techniques[J]. Parasitology Today, 1999, 15 (11):462~465.
[13]. Gasser R.B, Rossi L, Zhu X.Q. Identification of Nematodirus species (Nematoda: Molineidae) fromwild ruminants in Italy using ribosomal DNA markers[J]. International Journal for Parasitology, 1999,29(11): 1809~1817.
[14]. 翁亚彪, 谢德华,林瑞庆,等. 弓形虫 ITS 及 5.8S 序列的 PCR 扩增、克隆及分析[J].畜牧兽医学报, 2005, 36(1): 70~73.
[15].Gasser R.B, Jennifer R.M, Zhu X.Q, et al. Polymerase chain reaction linked singled strand conformation polymorphism of ribosomal DNA to fingprint parasites[J]. Electrophoresis. 1997, 18 (9): 1564~1566.
[16].吕召宏, 徐广庭, 林瑞庆, 宋慧群, 等. 鸡异刺线虫 ITS rDNA 的 PCR 扩增、克隆及序列分析[J].中国畜牧兽, 2005, 32 (8): 41~43.
[17].邓莉, 吴亚敏, 彭礼飞, 等. 蛲虫核糖体 DNA ITS1 的 PCR 扩增及序列分析[J]. 中国热带医学,2006, 6 (8): 1336~1337.
[18]. 董晓波, 李冬梅, 花丽茹, 等. 旋毛虫 ITS Ⅱ区基因的克隆及其在分类学上的应用[J]. 畜牧兽医学报, 2006 ,37(2): 173~175.
[19]. 宋慧群,李宾,林瑞庆,等.我国犬钩虫ITS及5.8SrDNA的克隆与序列分析[J].中国兽医科学,2007,37(9): 756~759.
[20]. Zahler M, Schein E, et al. Characteristic genotypes discriminate between Babesia canis isolates of differing vector specificity and pathogenicity to dogs[J]. Parasitol Research, 1998, 84(7): 544~548.
[21]. Schnittger L, Yin H, Bai Q, et al. Simultaneous detection and differentiation of Theileria and Babesia parasites infecting small ruminants by reverse line blotting[J].Parasitol Research, 2004, 92(3): 189~196.
[22]. Saiki RK, Chang CA, Levenson C H, et al. Diagnosis of sickle cell anemia and beta-thalassemia with enzymatically amplified DNA and nonradioactive allelespecific oligonucleotide probes[J]. N Engl J Med, 1988, 319(9): 537~541.
[23]. Kaufhold A, Podbieldski A, Baumgarten G, et al. Rapid typing of group A streptococci by the use of DNA amplification and non-radioactive allele-specific oligonucleotide probes[J]. FEMS Microbiol Lett, 1994, 119(1-2): 19~25.
[24]. Rijpkema S G, Molkenboer M J, Schouls L M, et al. Simultaneous detection and genotyping of three genomic groups of Borrelia burgdorferi sensu lato in Dutch Ixodus ricinus ticks by characterization ofthe amplified intergenic spacer region between 5S and 23S rRNA genes[J]. J Clin Microbiol, 1995, 33(12):3091~3095.
[25]. Kamerbeek J, Schouls L, Kolk A, et al. Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology[J]. J Clin Microbiol, 1997, 35(4): 907~914.
[26]. Schouls L M, Van D P, Rijpkema S G, et al. Detection and identification of Ehrlichia, Borrelia burgdorferi sensu lato and Bartonella species in Dutch Ixodes ricinus ticks[J].J Clin Microbiol, 1999, 37(7): 2215~2222.
[27]. Gubbels M J, De Vos A P, van der W M, et al. Simultaneous detection of bovine Theileria and Babesia species by reverse line blot hybridization[J]. J Clin Microbiol, 1999, 37(6): 1782~1789.
[28]. Sparagano O, Loria G R, Gubbels M J, et al. Integrated molecular diagnosis of Theileria and Babesia species of cattle in Italy[J]. Annals New York Academy Science 2000, 916: 533~539.
[29].Liu J.L, Yin H, Luo J.X, et al. Discrimination of Babesia major and Babesia ovata based on ITS1-5.8S-ITS2 region sequences of rRNA gene[J]. Parasitol Research, 2007, 102(4): 709~713.
[30]. Shayan P, Hooshmand E, Rahbari S, et al. Determination of Rhipicephalus spp. as vectors for Babesia ovis in Iran[J]. Parasitol Research, 2007, 101(4): 1029~1033.
[31]. 殷宏,罗建勋,吕文顺, 等. 莫氏巴贝斯虫和羊巴贝斯虫在我国的分离及形态学观察[J]. 中国兽医科技,1997, 27(10): 7~9.
[32]. 连灿,白启, 等. 在中国新发现的一种羊的巴贝斯虫(Babesia)未定种[J]. 中国兽医学报, 1997, 17(2): 116~119.
[33]. 刘光远, 连灿, 何虎成, 等. 绵羊巴贝虫单一种的分离及其繁殖特性观察[J].中国兽医科技,1999, 29(4): 12~13.
[34]. Markov A A, D’yakonov L P. Study of transmission of haemoparasites of sheep in the mammalian and arthropod host (in Ressian)[J]. Tr Vses Onst Eksp Vet, 1970, (38), 15.
[35]. Pegrffi G, Mura D, Sulla piroplasmosi delle capre[J]. Atti Soc Ital Sci Vet, 1948,(2):72.
[36]. Uilenberg G, Rombach M C, Perie N M et al. Blood parasites of sheep in the Netherlands. II.Babesia motasi (Sporozoa, Babesiidae)[J]. Tijdschrift voor diergeneeskunde, 1980,15, 105(2): 3~14.
[37]. 蒋金书. 动物原虫病学.(第一版),北京,中国农业大学出版社,2000, 7.
[38]. Ristic M. Babesiosis of Domestic Animal and Man[J]. Boca Ratom, Flarida : CRC Press ,1988. 23~52 ,99~118.
[39]. Lestoquard F. Les piroplasmosis du mouton en Algerie[J]. Bulletin de la societe de Pathologie Exotique, 1926, 4: 222~315.
[40]. Yakimoff W. Endoglobulare parasiten der schafe im Nordkaukasus[J]. Achive fur Protisenkunde, 1929, 65: 151~164.
[41]. Baumann R. Die kleinasiatische schaftheileriose[J]. Berlin und Munchener Tierarzliche Wochenscrift, 1939, 30: 469~474.
[42]. Khayyat S M, Gilder A A. Ovine piroplasmosis in Iraq[J]. Transaction of the Royal Society of Tropical Medicine and Hygiene, 1947, 41: 119~126.
[43]. Mazlum Z. Vector of Theileria hirci in south and southern Iran[J]. Annale de Parasitologie Humaine Comparee, 1970, 45: 523~525.
[44]. Sisodia R S. Gautam O P. Experimental cases of Theileria hirci infection in sheep and goats[J]. Indian Journal Animal Science, 1983, 53: 162~166.
[45]. Hooshmand-Rad P, Hawa N J. Malignant theileriosis of sheep and goats[J]. Tropical Animal Health and Production, 1973, 5: 97~102.
[46]. Sasmal N K, Biswas S S, Bhattacharyya B, et al. Theileria hirci-studies on transmission from Sahabadi sheep to black Bengal goat[J]. Indian Veterinary Journal, 1983, 60: 599~602.
[47]. Friedhoff K T. Tick-borne diseases of sheep and goats caused by Babesia, Theileria or Anaplasma spp.[J]. Parassitologia, 1997, 39(2): 99~109.
[48]. Leemans I. Coparative aspect of Theileria lestoquardi and Theileria annulata infections of sheep and cattle[J]. Ph D thesis, Swedish University of Agricultural Sciences, Uppsala, 2001, pp13~23.
[49]. Levine N D. Protozoan parasites of domestic animals and of man. Minneapolis, Minnesota: Burgess Publishing Company, 1973, pp 342~343.
[50]. Uilenberg G, Andreasen M P. Haematoxenus separatus sp. n. (sporozoa, Theileriidae) of sheep in Tazania[J]. Revue d'Elevage et de Medecine Veterinaire des Pays Tropicaux. 1974, 27: 459~465.
[51]. Uilenberg G, Schreuder B E. Further studies on Haematoxenus separatus (Sporozoa, Theileriidae) of sheep in Tanzania[J]. Revue d'Elevage et de Medecine Veterinaire des Pays Tropicaux, 1976, 29: 119~126.
[52]. Young A S, Mchinja S J. Observations on Haematoxenus separatus Uilenberg & Andreasen 1974 in the erythrocytes of Kenyan sheep[J]. Research in Veterinary Science, 1977, 23: 387~388.
[53]. Alani A J, Herbert I V. Morphology and transmission of Theileria recondita (Theileriidae: Sporozoa) isolated from Haemaphysalis punctata from north Wales[J].Vet Parasitol. 1988, 28(4): 283~291.
[54]. Alani A J, Konrad J, Herbert I V. Serodiagnosis of Babesia motasi (Wales), Theileria recondita (Wales) and Cytoecetes phagocytophila infection in sheep[J]. Research in Veterinary Science 1987, 43(1): 104~108.
[55]. Alani A J, Herbert I V. Pathogenesis of infection with Theileria recondita (Wales) isolated from Haemaphysalis punctata from north Wales[J]. Veterinary Parasitology, 1988, 28(4): 293~301.
[56]. Enigk K Das Auftreten der Schafpiroplasmose in Deutschland[J]. Zeitschrift fur Tropenmedizin und Parasitolologie, 1953, 4: 175~183.
[57]. 李有全.我国羊泰勒虫的生物学特性研究.[博士学位论文],北京,中国农业科学院,2007,48~51.
[58]. Vawter L, Brown W M. Rates and patterns of base change in the small subunit ribosomal RNA Gene[J]. Genetics Society of America. 1993, 134(2): 597~608.
[59]. Schlegel M, Elwood H J, Sogin M L. Molecular evolution in hypotrichous ciliates: sequence of the small subunit ribosomal RNA genes from Onychodromus quadricornutus and Oxytricha granulifera (Oxytrichidae, Hypotrichida, Ciliophora)[J]. J Mol Evol. 1991, 32(1):64~69.
[60]. Germot A, Philippe H.Critical analysis of eukaryotic phylogeny: a case study based on the HSP70 family[J]. J Eukaryot Microbiol. 1999, 46(2):116~124.
[61]. Mattsson J G, Guss B, Johansson K E. The phylogeny of Mycoplasma bovis as determined by sequence analysis of the 16S rRNA gene[J]. FWMS Microbiol Lett.1994, 115(2-3):325~328.
[62]. Chae J, Lee J, Kwon O, et al. Nucleotide sequence heterogeneity in the small subunit ribosomal RNA gene variable (V4) region among and within geographic isolates of Theileria from cattle, elk and white-tailed deer[J]. Veterinary Parasitology, 1998, 75: 41~52.
[63]. Gubbels M J, Yin H, Bai Q et al. The phylogenetic position of the Theileria buffeli group in relation to other Theileria species[J]. Parasitol Research 2002, 88(13 suppl 1): S28~32.
[64]. Schnittger L, Yin H, Gubbels M J. Phylogeny of sheep and goat Theileria and Babesia parasites. Parasitol Research. 2003, 91(5):398~406.
[65]. Adam J. Birkenheuer, Julia Whittington,et al. Molecular characterization of a Babesia species identified in a North American Raccoon[J]. Journal of Wildlife Diseases, 2006, 42(2): 375~380.
[66]. 谢明权, 李国清. 现代寄生虫学 .广东:科技出版社,2003,18~19.
[67]. Troell K, Mattsson J G, Alderborn, et al. Pyrosequencing analysis identifies discrete populations of Haemonchus contortus from small ruminants [J]. Int J Parasitol, 2003, 33 (7): 765~771.
[68]. Hung G C, Chilton N B, Beveriddge I, et al. Molecular evidence for cryptic species within Cylicostephanus munutus (Nematoda: Strongylidae ) [J]. Int J Parasitol, 1999, 29(2): 285~291.
[69]. Zhu X Q, D ’Amelio S, Paggi L, et al. Assessing sequence variation in the internal transcribed spacers of ribosomal DNA within and among members of the Contracaecum osculatum comp lex (Nematoda: Ascaridoidea:Anisakidae) [J]. Parasitol Research, 2000, 86: 677~683.
[70]. Zhu X Q, Chilton N B, Jacobs D E, et al. Characterisation ofAscaris from human and p ig hosts by nuclear ribosomalDNA sequence [J]. Int J Parasitol, 1999, 29: 469~478.
[71]. Navajas M, Lagnel J, Gutierrez J, Species-wide homogeneity of nuclear ribosomal ITS2 sequences in the spider mite Tetranychus urticae contrasts with extensive mitochondrial COI polymorphism[J]. Heredity, 1998, 80 (6): 742~752.
[72]. Morgan J A T, Blair D. Nuclear rDNA ITS sequence variation in the trematode genus Echinostoma : an aid to establishing relationships within the 372 collar-spine group[J]. Parasitology ,1995, 111 (5) : 609~615.
[73]. Bowles J, Blair D , McManus D P. A molecular phylogeny of the human schistosomes[J]. Mol Phylogenet Evol, 1995, 4(2) :103~109.
[74]. 胡友兰,李国清. 荒川库蠓 rDNA ITS2 的序列测定与分析[J]. 动物学报, 2003,19(2):277~280.
[75]. Heise M, Epe C, Schnieder T. Differences in the second internal transcribed spacer (ITS-2) of eight species of gastrointestinal nematodes of ruminants[J]. J parasitol 1999, 85(3): 431~435.
[76]. Jacobs, D E, Zhu X Q, Gasser R B et al. PCR-based methods for identification of protentially zoonotic ascaridoid parasites of the dog, fox and cat[J]. Acta Tropica.1997, 68 : 191~200.
[77]. Kjemtrup A M, Conrad P A. A review of the small canine piroplasms from California: Babesia conradae in the literature[J]. Veterinary Parasitology, 2006, 138 (1-2):112~117.
[78]. Paskewitz S M, Wesson D M, Collins F H. The internal transcribed spacers of ribosomal DNA in five members of the Anopheles gambiae species complex[J]. Insect Mol Biol, 1993, 2(4) : 247~257.
[79]. Fenton B , Malloch G, Germa F. A study of variation in rDNA ITS regions shows that two haplotypes coexist within a single aphid genome[J]. Genome, 1998 , 41(3) : 337~344.
[80]. van Herwerden L , Blair D , Agatsuma T. Intra- and inter-individual variation in ITS1 of Paragonimus westermani (Trematoda : Digenea) and related species : implications for phylogenetic studies[J]. Mol Phylogenet Evol, 1999, 12(1) : 67~73.
[81]. Kuhls K, Mauricio I L, Pratlong F. et al. Analysis of ribosomal DNA internal transcribed spacer sequences of the Leishmania donovani complex[J]. Microbes and Infection, 2005, 7(11-12): 1224~1234.
[82]. Anonymous. Ticks and Tick-borne Disease Control, A Practical Field Manual[J]. Tick borne Disease Control, Vol 2, FAO, Rome, 1984, pp 300~621.
[83]. Tsur I. Adler S. Growth of Theileria annulata schizonts in monolayer tissue cultures[J]. Journal Protozoology, 1963, 10: 36~37.
[84]. 吕文顺, 李观娣, 吕文祥. 环形泰勒焦虫裂殖体细胞培养的研究[J]. 动物学报, 1975, 21(3): 243~256.
[85]. Erp E E, Gravely S M, Smith R D. et al. Growth of Babesia bovis in bovine erythrocyte cultures[J]. Am J Trop Med Hyg. 1978, 27(5):1061~1064.
[86]. Levy M G, Ristic M. Babesia bovis: continuous cultivation in a microaerophilous stationary phase culture[J]. Science, 1980, 207 (14): 1218~1220.
[87]. Thomford JW, Conrad PA, Boyce WM. Isolation and in vitro cultivation of Babesia parasites from free-ranging desert bighorn sheep (ovis Canadensis nelsen) and mule deer (odocoileus hemionus) in California[J]. Journal of Parasitology, 1993, 79 (1): 77~84.
[88]. Hirato K, Ninomiya N, Uwano Y, et al. Studies on the complement fixation reaction for equinepiroplasmosis. Japanese Journal of Veterinary Science, 1945, 1: 204; as quoted by Weiland G, Reiter I, In: Ristic M. Babesiosis of domestic animals and man, Beca Raton: CRC Press, 1988, pp146.
[89]. Mahoney D F. Bovine babesiosis: diagnosis of infection by a complement fixation test[J]. Australian Veterinary Journal, 1962, 38: 48.
[90]. Mayer S, Tongio M M. A study of maternal-fetal anti-HL-A immunization Extension of immunization[J]. Vox Sang, 1973, 25 (6): 546~551.
[91]. Pipano E, Cahana M. Fluorescent antibody test for the serodiagnosis of Theileria annulata[J]. Journal of Parasitology, 1969, 55: 765.
[92]. Schindler R, Wokatsch. Versuche zur differenzierung der Theilerien-spezies des rinders durch serologische Untersuchungen, Zeitschrift fur Tropenmedizin und Parasitologie, 1965, 166: 17-23.
[93]. Konyukohov M P, Poboluboyarinova GV. The role of CFT and prolonged CFT in revealing the immunity in animal having had theileriosis[J]. Veterinariya, 1967, 10: 56~58.
[94]. Hawa N, Latif B M, Bakir F A. Application of the indirect fluorescent antibody test for diagnosis of Theileria hirci infection of sheep: using cell culture schizont antigen[J].Tropical Animal Health Production, 1976, 8(2): 97~101.
[95]. Kiurtov N, Immunofluorescent test in the diagnosis of babesiasis[J].Vet med Nauki 1976, 13(7): 61~66.
[96]. Leemans I, Hooshmand-Rad P, Uggla A. The indirect fluorescent antibody test based on schizont antigen for study of the sheep parasite Theileria lestoquardi[J]. Veterinary Parasitology, 1997, 69(1-2): 9~18.
[97]. David F, Joaquim Castella Juan F. Gutierrez. Santiago LavIn, and Ignasi Marco Seroprevalence of Babesia ovis in Mouflon Sheep in Spain[J]. Journal of wildlife Diseases.1998, 34(3): pp 637~639.
[98]. Engvall E, Perlmann P. Enzyme linked immunosorbent assay, ELISA. Ⅲ. Quantitation of specific antibodies by enzyme labeled anti-immunoglobulin in antigen coated tubes[J]. Journal of Immunology, 1972, 109: 129.
[99]. Bidwell D E, Turp P, Joyner L P, et al. Comparisons of serological tests for Babesia in British cattle[J]. Vet Rec. 1978, 103(20): 446~449.
[100]. Barry D N., Rodwell B J, Timms P, et al. A microplate enzyme immunoassay for detecting and measuring antibogies to Babesia bovis in cattle serum[J]. Australian Veterinary Journal, 1982, 59:136.
[101]. Duzgun A, Wright I G, Waltisbuhl D J, et al. An ELISA for the diagnosis of Babesia ovis infection utilizing a synthetic, Babesia bovis-derived antigen[J]. Veterinary Parasitology. 1991 39(3-4):225~231.
[102]. Reinhard T, Sundmacher R. Strunck-Kortenbusch B. Corneal puncture in recurrent corneal erosion[J]. Ophthalmologe, 1993, 90 (6): 694~697.
[103]. Molloy J B, Bowles P M, Bock R E, et al. Evaluation of an ELISA for detection of antibodies to Babesia bovis in cattle in Australia and Zimbabwe[J]. Prev Vet Med, 1998, 33: 59~67.
[104]. Gray M A, Lukins A G, Rae P F, et al. Evaluation of an enzyme immunoassay for serodiagnosis of infections with Theileria parva and T annulata[J]. Research in Veterinary Science, 1980, 29: 360~366.
[105]. Gubbels M J, d'Oliveira C, Jongejan F. Development of an indirect Tams1 enzyme-linked immunosorbent assay for diagnosis of Theileria annulata infection in cattle[J]. Clinical Diagnostic Laboratory Immunology, 2000, 7(3): 404~411.
[106] Araújo F R, Madruga C R, Leal C R, et al.Comparison between enzyme-linked immunosorbent assay, indirect fluorescent antibody and rapid conglutination tests in detecting antibodies against Babesia bovis[J]. Vet Parasitol. 1998 Jan 31;74(2-4):101~108.
[107]. Manuja A, Nichani AK, Kumar R, Rakha NK, Kumar B, Sharma RD. Comparison of cellular schizont, soluble schizont and soluble piroplasm antigens in ELISA for detecting antibodies against Theileria annulata[J]. Veterinary Parasitology, 2000, 87(2-3): 93~101.
[108]. Shuzhen Guo, Yongjuan Mu, Zhijie Liu, et al. Serological investigation of ovine theileriosis by ELISA in Gannan Tibet Region of Gansu Province in China[J]. Parasitol Research, 2007, 101:S197~S200.
[109]. Mclaughlin G L, Edlind T D, Ihler G M. Detection of Babesia bovis using DNA hybridization[J]. Journal of Protozoology, 1986, 33: 125~128.
[110]. Allsopp B, Carrington M, Baylis H, et al. Improved characterization of Theileria parva isolates using the polymerase chain reaction and oligonucleotide probes[J]. Mol Biochem Parasitol.1989, 15, 35(2):137~147.
[111]. Jasmer D P, Reduker D W, Goff W L, et al. DNA probes distinguish geographical isolates and identify a novel molecule of Babesia bovis[J]. Journal of Parasitology, 1990, 76: 834~841.
[112]. Kajiwara N, Kirisawa R, Onuma M, et al. Specific DNA probe for the detection of Theileria sergenti infection in cattle[J]. Japanese Veterinry Journal, 1990, 52(6): 199~204.
[113]. Matsuba T, Kawakami Y, Iwai H, Onuma M. Genomic analysis of Theileria sergenti stocks in Japan with DNA probes[J]. Vet Parasitol. 1992, 41(1-2):35~43.
[114]. Hodgson J L, Stiller D, Jasmer D P, et al. Babesia bigemina: Quantitation of infection in nymphal and adult Boophilus microplus using a DNA probe[J]. Experimental Parasitology, 1992, 74: 117~126.
[115]. Bishop R, Sohanpal B, Kariuki D P, et al. Detection of a carrier state in Theileria parva-infected cattle by the polymerase chain reaction[J]. Parasitology, 1992, 104(Pt2): 215~232.
[116]. Figueroa J.V, Chieves L.P, Johnson G.S, et al. Detection of Babesia bigemina-infected carriers by polymerase chain reaction amplification[J]. Journal of Clinical Microbiology, 1992, 30(10): 2576~2582.
[117]. D'Oliveira C, van der Weide M, Habela MA, Jacquiet P, Jongejan F. Detection of Theileria annulata in blood samples of carrier cattle by PCR[J]. Journal Clinical Microbiology, 1995, 33(10): 2665~2669.
[118]. Akta? M, Altay K, Dumanli N. Development of a polymerase chain reaction method for diagnosis of Babesia ovis infection in sheep and goats[J]. Veterinary Parasitology, 2005, 133(4):277~281.
[119]. Aktas M, Altay K, Dumanli N. Determination of prevalence and risk factors for infection with Babesia ovis in small ruminants from Turkey by polymerase chain reaction[J]. Parasitol Research. 2007,100(4):797~802.
[120]. 陈德明. 绵羊焦虫病调查报告[J]. 兽医科技杂志, 1982 (3): 31~32.
[121]. 赵锡荣,李繁如,闵友贵, 等. 绵羊巴贝斯焦虫病的调查[J]. 中国兽医科技, 1986 (1) : 26~27.
[122]. Leonhard Schnittger, Hong Yin, Marc J. Gubbels. Phylogeny of sheep and goat Theileria and Babesia parasites[J]. Parasitol Research, 2003, 91: 398~406.
[123]. HILLIS D M, DIXON M T. Ribosomal DNA: molecular evolution and phylogenetic inference[J]. Quart Rev Biol 1991, 66: 411~453.
[124]. María Haro, Carmen del A′ guila, Soledad Fenoy, et al. Intraspecies Genotype Variability of the Microsporidian Parasite Encephalitozoon hellem[J] .Journal of Clinical Microbiology 2003, p. 4166~4171.
[125].关贵全,殷宏,罗建勋等.羊的大型巴贝虫未定种的形态学和致病性初步研究[J].中国兽医科技,2001,(11)35~36.
[126]. Mehlhorn H, Schein E. The piroplasms: life cycle and sexual stages[J]. Adv Parasitol 1984, 23:37~103.
[127]. Mehlhorn H, Schein E, Ahmed J S. Theileria. In: Kreier JP (ed) Parasitic protozoa. vol 7. Academic Press San Diego, 1994, pp 217~304.
[128]. Yin H, Schnittger L, Luo J X et al.Ovine theileriosis in China: a new look at an old story[J]. Parasitol Research, 2007,101:S191~S195.
[129]. Preston P M. Theilerioses. In: Service, M.W., (Ed.), Encyclopedia of Arthropod-transmitted infections of man and domesticated animals. CABI Publishing Wallingford, 2001, pp 487~502.
[130]. Uilenberg G. Babesiosis. In: Service MW, (Ed.) Encyclopedia of Arthropod-transmitted infections of man and domesticated animals. CABI Publishing Wallingford, 2001, pp 53~60.
[131]. Kirvar E, Wilkie G, Katzer F, et al.Theileria lestoquardi--maturation and quantification in Hyalomma anatolicum anatolicum ticks[J]. Parasitology. 1998, 117 ( Pt 3): 255~263.
[132]. Bruning A. Equine piroplasmosis an update on diagnosis[J]. Br Vet J, 1996, 152: 139~151.
[133]. Papadopoulos B, Brossard M, Perie N M. Piroplasms of domestic animals in the Macedonia region of Greece.3[J]. Piroplasms of small ruminants. Veterinary Parasitology, 1996, 63:67~74.
[134]. Nagore D, Garc′?a-Sanmart′?n J, Garc′?a-Pérez A L, et al. Detection and identification of equine Theileria and Babesia species by reverse line blotting:epidemiological survey and phylogenetic analysis Veterinary assistant[J]. Veterinary Parasitology, 2004, 123:41~54.
[135]. Alhassan A, Pumidonming W, Okamura M, et al. Development of a single-round and multiplex PCR method for the simultaneous detection of Babesia caballi and Babesia equi in horse blood[J]. Veterinary Parasitology, 2005, 129: 43~49.
[136]. Altay K, Dumanli N, Holman PJ, Aktas M, Detection of Theileria ovis infected sheep by nested PCR[J]. Veterinary Parasitology, 2005, 127:99~104.
[137]. Birkenheuer A J, Levy M G, Breitschwerdt E B. Development and evaluation of a seminested PCR for detection and differentiation of Babesia gibsoni (Asian genotype) and B. canis DNA in canine blood samples[J]. J Clin Microbiol, 2003, 41:4172~4177.
[138]. Criado-Fornelio A, Martinez-Marcos A, Buling-Sarana A, et al. Molecular studies on Babesia, Theileria and Hepatozoon in southern Europe. Part I. Epizootiological aspects[J]. Veterinary Parasitology, 2003, 113(3-4): 189~201.
[139]. Quick R E, Herwaldt B L, Thomford J W, Babesiosis in Washington State: a new species of Babesia?[J]. Ann Intern Med. 1993, 119(4):284~290.
[140]. Thomford J W, Conrad P A, Telford S R . Cultivation and phylogenetic characterization of a newly recognized human pathogenic protozoan[J]. J Infect Dis. 1994, 169(5): 1050~1056.
[141]. Persing D H, Herwaldt B L, Glaser C, et al. Infection with a babesia-like organism in northern California. N Engl J Med, 1995, 332: 298~303.
[142]. Herwaldt B L, Kjemtrup A M, Conrad P A, et al. Transfusiontransmitted babesiosis in Washington State: first reported case caused by a WA1-type parasite[J]. J Infect Dis, 1997, 175(5): 1259~1262.
[143]. Katzer F, McKellar S, Kirvar E, et al. Phylogenetic analysis of Theileria and Babesia equi in relation to the establishment of parasite populations within novel host species and the development of diagnostic tests[J]. Mol Biochem Parasitol, 1998, 95(1): 33~44.
[144]. Schnittger L, Yin H, Jianxun L, et al. Ribosomal small-subunit RNA gene-sequence analysis of Theileria lestoquardi and a Theileria species highly pathogenic for small ruminants in China[J]. Parasitology Research, 2000, 86(5): 352~358.
[145]. Gubbels M J, Yin H, van der Weide M, et al. Molecular characterisation of the Theileria buffeli / orientalis group[J]. Int J Parasitol, 2000, 30(8): 943~952.
[146]. 殷宏. 羊的泰勒虫生物学及分子生物学特性研究.[博士学位论文],南京,南京农业大学,2002,112~124.
[147]. Centeno-Lima S, do Rosário V, Parreira R, et al. A fatal case of human babesiosis in Portugal: molecular and phylogenetic analysis[J]. Trop Med Int Health, 2003, 8(8): 760~764.
[148]. Nijhof A M, Penzhorn B L, Lynen G, et al. Babesia bicornis sp. nov. and Theileria bicornis sp. nov.: tick-borne parasites associated with mortality in the black rhinoceros (Diceros bicornis)[J]. J Clin Microbiol 2003, 41:2249~2254.
[149]. Oura C A, Bishop R P, Wampande E M, et al. Application of a reverse line blot assay to the study of haemoparasites in cattle in Uganda[J]. Int J Parasitology 2004a, 34(5): 603~613.
[150]. Oura C A, Bishop R P, Wampande E M, et al. The persistence of component Theileria parva stocks in cattle immunized with the Muguga cocktail live vaccine against East Coast Fever in Uganda[J]. Parasitology[J], 2004b, 129(Pt1):27~42.
[151]. Brígido C, da Fonseca I P, Parreira R, et al. Molecular and phylogenetic characterization of Theileria spp. parasites in autochthonous bovines (Mirandesa breed) in Portugal. Veterinary Parasitology, 2004, 123(1-2): 17~23.
[152]. Nagore D, Garc′?a-Sanmart′?n J, Garc′?a-Pérez A L, et al. Detection and identification of equine Theileria and Babesia species by reverse line blotting:epidemiological survey and phylogenetic analysis Veterinary assistant[J]. Veterinary Parasitology, 2004, 123(1-2): 41~54.
[153]. Nagore D, Garc?′a-Sanmart?′n J, Garc?′a-Pe′rez AL, et al. Identification, genetic diversity and prevalence of Theileria and Babesia species in a sheep population from Nortern Spain[J]. Int J Parasitology, 2004, 34(9):1059~1067.
[154]. Georges K, Lori G R, Riili S, et al. Detection of haemoparasites in cattle by reverse line blot hybridisation with a note on the distribution of ticks in Sicily[J].Veterinary Parasitology, 2001, 99(4): 273~286.
[155]. García-Sanmartín J, Nagore D, García-Pérez A L, et al. Molecular diagnosis of Theileria and Babesia species infecting cattle in Northern Spain using reverse line blot macroarrays[J]. BMC Veterinary Research, 2006, 16:1~7.