牛卵形巴贝斯虫巢式PCR诊断方法的建立及初步应用

根据GenBank上发表的牛卵形巴贝斯虫CCTη基因序列设计合成2对巢式PCR引物,建立牛卵形巴贝斯虫巢式PCR诊断方法,对该方法的最佳反应条件进行了筛选,并进行了特异性、敏感性及临床样本检测试验。结果表明,建立的巢式PCR方法外引物扩增牛卵形巴贝斯虫基因组片段的长度为1 008bp,内引物为537bp;该方法扩增不出牛瑟氏泰勒虫、弓形虫、犬新孢子虫基因组DNA;最低检测DNA含量为16fg;通过对46份临床样本的检测,该巢式PCR较常规PCR阳性检出率高8.7%。本试验为牛卵形巴贝斯虫病的诊断提供了一种更为特异、敏感的检测技术。     

马梨形虫病双重PCR检测方法的建立

为了建立马梨形虫病双重PCR检测方法,试验根据Gen Bank发表的驽巴贝斯虫和马泰勒虫18S rRNA保守基因序列分别合成2对特异性引物,以核酸混合物为模板,优化反应条件建立马梨形虫病双重PCR检测方法,并检验该方法的特异性和敏感性。同时用该双重PCR检测方法对采自昭苏种马场的马疑似病例全血进行检测,并与镜检法和单重PCR法进行比较。结果表明:该双重PCR检测方法能对驽巴贝斯虫和马泰勒虫核酸扩增出大小为529 bp和789 bp特异性目的片段,而对双芽巴贝斯虫、环形泰勒虫、羊泰勒虫、牛巴贝斯虫核酸的扩增均为阴性;驽巴贝斯虫和马泰勒虫阳性DNA被稀释1×108倍时均能检出其相应目的片段;对采集的46份马疑似病例血样进行PCR诊断,其中驽巴贝斯虫感染率为30.4%(14/46),马泰勒虫感染率为41.3%(19/46);双重PCR检测方法与单重PCR方法的符合率为100%。说明建立的双重PCR检测方法具有一定的特异性和敏感性,可用于驽巴贝斯虫和马泰勒虫临床感染隐性病例的联合检测与鉴别诊断。     

套式PCR检测马巴贝斯虫

根据GenBank中马巴贝斯虫(Babesia equi)ema-1基因序列,设计合成内外2对引物,其中外引物扩增ema-1基因60～627nt间567 bp片段,内引物扩增ema-1基因259～488nt间229bp片段。从实验室感染马巴贝斯虫阳性马匹全血样本中提取DNA,采取2次扩增的方法,扩增到229bp特异性条带,建立了适合马巴贝斯虫快速检测的套式PCR方法。经重复性试验和特异性试验,结果显示,马巴贝斯虫阳性样本在229bp均出现条带,而驽巴贝斯虫、牛巴贝斯虫扩增结果为阴性。采用该方法对已知阴、阳性的16份马匹全血样品进行检测,有8份为阳性,与实际结果的符合率为100%。表明,所建立的方法具有较高的重复性和特异性,可用于马巴贝斯虫病的临床诊断、病料检测和分子流行病学调查。     

蜱传牛巴贝斯虫套式PCR检测方法的建立及应用

为建立一种快速、敏感检测牛巴贝斯虫的方法,本研究根据GenBank中登录的牛巴贝斯虫rap-1基因保守区设计2对特异性引物,经反应条件的优化,建立了牛巴贝斯虫套式PCR检测方法。结果显示,该方法可以特异地检测牛巴贝斯虫,而对双芽巴贝斯虫、牛环形泰勒虫和弓形虫的检测均为阴性;该方法的灵敏度可达1.3×10~1拷贝/μL,是牛巴贝斯虫荧光定量PCR检测试剂盒的10倍,是常规PCR的1 000倍。对50只扇头蜱和微小牛蜱DNA进行检测,套式PCR、牛巴贝斯虫荧光定量PCR检测试剂盒和常规PCR的阳性检出率分别为100.0%、72.0%和0。本实验建立的套式PCR检测方法适用于牛巴贝斯虫病的早期诊断和分子流行病学调查,为蜱传牛巴贝斯虫病的防控提供技术支持。     

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。     

牛卵形巴贝斯虫PCR检测方法的建立及初步应用

为建立牛卵形巴贝斯虫(B.ovata)快速检测方法,本研究根据GenBank中登录的B.ovata CCTη基因序列设计引物,建立了PCR检测方法。对该方法的最佳反应条件进行优化,并进行特异性、敏感性及临床样本检测试验。结果表明,建立的PCR方法扩增B.ovata CCTη基因片段大小为1 008 bp,与参考株序列同源性为100%。该方法对牛巴贝斯虫、牛双芽巴贝斯虫、牛瑟氏泰勒虫基因组DNA扩增结果均为阴性。最低可以检测样品中34个拷贝的DNA。通过对49份临床样本的检测,该方法比姬姆萨染色镜检阳性率高8.2%。本实验为B.ovata的诊断提供了一种特异、敏感的检测技术。     

牛巴贝斯虫TaqMan荧光定量PCR检测方法的建立

为建立一种灵敏、特异、快速的牛巴贝斯虫检测方法,针对牛巴贝斯虫Rap-1a基因设计引物进行PCR扩增,然后构建重组质粒制作标准品,经过优化反应体系、绘制标准曲线,建立了牛巴贝斯虫TaqMan荧光定量PCR方法,并进行灵敏度、特异性及稳定性检测,同时利用该方法对37份田间样品进行检测。结果显示：建立的牛巴贝斯虫TaqMan荧光定量PCR检测方法的标准曲线方程式为y=-3.362×Log（X）+43.32,相关系数R2=0.999,扩增效率为98.4%。该方法的灵敏度为1.0×102 copies/μL,是普通PCR（1.0×104 copies/μL）的100倍。该方法对牛双芽巴贝斯虫、大巴贝斯虫、卵形巴贝斯虫等7种常见的牛梨形虫病检测结果均为阴性,组内和组间重复试验的变异系数均小于2.5%,37份田间样品的阳性检出率为67.5%。结果表明,本试验建立的牛巴贝斯虫TaqMan荧光定量PCR灵敏度高,且特异、稳定,适用于牛巴贝斯虫的诊断,从而为其流行病学调查提供了快速有效的检测方法。     

卵形巴贝斯虫与中华泰勒虫双重PCR检测方法的建立

为建立一种能快速对卵形巴贝斯虫(Babesia ovata)和中华泰勒虫(Theileria sinensis)同时进行检测的双重PCR方法。根据GenBank已报道的卵形巴贝斯虫AMA1基因和中华泰勒虫MPSP基因设计合成了2对特异性引物,通过条件优化,建立了双重PCR检测方法。结果显示：双重PCR可特异扩增出卵形巴贝斯虫和中华泰勒虫目的条带,片段大小分别为500 bp和986 bp。该方法具有较好的特异性,对卵形巴贝斯虫和中华泰勒虫的最低检出浓度为16 fg/μL。对采集的90份牛血液样本进行双重PCR检测,卵形巴贝斯虫阳性率为30%(27/90),中华泰勒虫阳性率为16.67%(15/90),混合感染率为10%(9/90)。结果表明,双重PCR方法可用于卵形巴贝斯虫和中华泰勒虫的快速诊断和流行病学调查。     

蜱传反刍动物艾立希体巢式PCR检测方法的建立及应用

为建立一种快速、敏感检测反刍动物艾立希体的方法,本研究根据GenBank中登录的反刍动物艾立希体pCS20基因保守区设计2对特异性引物,经各反应条件的优化,建立了反刍动物艾立希体巢式PCR检测方法。结果显示,该方法可以特异性检测反刍动物艾立希体DNA,而对牛巴贝斯虫、双芽巴贝斯虫、牛环形泰勒虫和弓形虫的检测均为阴性,具有良好的特异性;该方法灵敏度可达1.04×101拷贝/μL,是反刍动物艾立希体实时荧光PCR检测试剂盒的10倍,是常规PCR的1 000倍。对50只血蜱、花蜱和微小牛蜱DNA进行检测,巢式PCR、反刍动物艾立希体实时荧光PCR检测试剂盒和常规PCR的阳性检出率分别为26.0%,14.0%和0.0%。本试验建立的巢式PCR检测方法适用于反刍动物艾立希体病的早期诊断和分子流行病学调查,为蜱传反刍动物艾立希体病的防控提供技术支持。     

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。     

Detection of Theileria and Babesia in brown brocket deer (Mazama gouazoubira) and marsh deer (Blastocerus dichotomus) in the State of Minas Gerais, Brazil

Intraerythrocytic protozoan species of the genera Theileria and Babesia are known to infect both wild and domestic animals, and both are transmitted by hard-ticks of the family Ixodidae. The prevalences of hemoprotozoa and ectoparasites in 15 free-living Mazama gouazoubira, two captive M. gouazoubira and four captive Blastocerus dichotomus from the State of Minas Gerais, Brazil, have been determined through the examination of blood smears and the use of nested polymerase chain reaction (nPCR). The cervid population was inspected for the presence of ticks and any specimens encountered were identified alive under the stereomicroscope. Blood samples were collected from all 21 animals, following which blood smears were prepared, subjected to quick Romanowsky staining and examined under the optical microscope. DNA was extracted with the aid of commercial kits from cervid blood samples and from tick salivary glands. The nPCR assay comprised two amplification reactions: the first was conducted using primers specific for a 1700 bp segment of the 18S rRNA gene of Babesia and Theileria species, whilst the second employed primers designed to amplify a common 420 bp Babesia 18S rRNA fragment identified by aligning sequences from Babesia spp. available at GenBank. The ticks Amblyomma cajennense, Rhipicephalus microplus and Dermacentor nitens were identified in various of the cervids examined. Of the animals investigated, 71.4% (15/21) were infected with hemoprotozoa, including Theileria cervi (47.6%), Theileria sp. (14.3%), Babesia bovis (4.8%) and Babesia bigemina (4.8%). However, only one of the infected wild cervids exhibited accentuated anaemia (PCV=17%). This is first report concerning the occurrence of Theileria spp. in Brazilian cervids.     

用重组的牛巴贝斯虫棒状体蛋白1作为ELISA诊断抗原进行牛群中牛巴贝斯虫病的血清流行病学调查

用重组的牛巴贝斯虫棒状体蛋白1（Bc—RAP-1）作为ELISA诊断抗原,对采自青海省湟中县的120份奶牛血清样品,进行抗Bc—RAP-1特异性抗体的检测。结果检出7份阳性,阳性率为5．83％,说明该地区牛群存在牛巴贝斯虫的感染。     

Phylogenetic relationships of Mongolian Babesia bovis isolates based on the merozoite surface antigen (MSA)-1, MSA-2b, and MSA-2c genes

We conducted a molecular epidemiological study on Babesia bovis in Mongolia. Three hundred blood samples collected from cattle grazed in seven different districts were initially screened using a previously established diagnostic polymerase chain reaction (PCR) assay for the detection of B. bovis-specific DNA. Positive samples were then used to amplify and sequence the hyper-variable regions of three B. bovis genes encoding the merozoite surface antigen (MSA)-1, MSA-2b, and MSA-2c. The diagnostic PCR assay detected B. bovis among cattle populations of all districts surveyed (4.4-26.0%). Sequences of each of the three genes were highly homologous among the Mongolian isolates, and found in a single phylogenetic cluster. In particular, a separate branch was formed only by the Mongolian isolates in the MSA-2b gene-based phylogenetic tree. Our findings indicate that effective preventative and control strategies are essential to control B. bovis infection in Mongolian cattle populations, and suggest that a careful approach must be adopted when using immunization techniques.     

Detection of bovine babesiosis in Mozambique by a novel seminested hot-start PCR method

Babesiosis is a tick borne disease (TBD) caused by parasites of the genus Babesia, with considerable worldwide economic, medical, and veterinary impact. Bovine babesiosis and other TBDs were considered responsible for 50% of the deaths of cattle that occurred in Mozambique in the first year after importation from neighbouring countries. Here, we present the detection of Babesia bigemina and Babesia bovis in cattle from Mozambique using two distinct PCR methods. For this study, blood samples were collected in one farm located near Maputo city. The DNA samples were analyzed using a previously described nested PCR and a novel hot-start PCR method. Primers were selected for the hot-start PCR based on the putative gene of an undescribed aspartic protease named babesipsin, present in both B. bovis and B. bigemina. The combination of hot-start polymerase and long primers (29-31 bp) were in this study determinant for the successful amplification and detection in only one PCR. With a seminested approach the sensitivity was further increased. The babesipsin seminested hot-start PCR was in this study more sensitive than the nested PCR. A total of 117 field samples were tested by seminested hot-start PCR, and 104 were positive for B. bigemina (90%), 97 were positive for B. bovis (82%), 86 were mixed infections (52%) and only 2 were negative for both Babesia species (1.7%). The results confirm that this area of Mozambique is endemic for babesiosis, and that this TBD should be regarded as a threat for imported cattle.     

Comparison of duplex PCR and microscopic techniques for the identification of Babesia bigemina and Babesia bovis in engorged female ticks of Boophilus microplus

A single-step duplex polymerase chain reaction (PCR) technique and traditional microscopic examination of haemolymph smears were used to detect Babesia bigemina and/or Babesia bovis infection in engorged female ticks of Boophilus microplus recovered from calves raised in an endemic area of the State of Minas Gerais, Brazil. In the PCR amplification of tick-derived DNA, pairs of oligonucleotide primers specific for a 278-bp sequence from B. bigemina and for a 350-bp sequence from B. bovis were used conjointly. The microscopic examination of haemolymph revealed that 16.7% of the engorged ticks were infected with Babesia spp., although no significant differences (rho > 0.05) were found in the infection rate of ticks collected from calves of different age groups. PCR analysis showed that 77.8% of the engorged ticks whose haemolymph contained sporokinetes were infected with B. bigemina, 7.8% with B. bovis and 14.4% with both protozoan species. However, the PCR assay further revealed that, amongst the engorged female ticks whose haemolymph was apparently negative for the presence of sporokinetes, 15.6% were infected with B. bigemina, 2.2% with B. bovis and 10.0% with both species. The duplex PCR method is thus more efficient and sensitive than the microscopic assay and also permits facile identification of the protozoa species present in engorged female ticks.     

Babesia spp. infection in Boophilus microplus engorged females and eggs in Sao Paulo State, Brazil

Babesia spp. infections were investigated in Bos taurus x Bos indicus dairy cows and calves and in Boophilus microplus engorged female ticks and eggs. Blood samples and engorged female ticks were collected from 25 cows and 27 calves. Babesia spp. was detected in ticks by microscopic examination of hemolymph of engorged female and by squashes of egg samples. Cattle infection was investigated in blood thin smears and by DNA amplification methods (PCR and nested PCR), using specific primers for Babesia bovis and Babesia bigemina. Merozoites of B. bovis (3 animals) and B. bigemina (12 animals) were detected exclusively in blood smears of calves. DNA amplification methods revealed that the frequency of B. bigemina infection in calves (92.6%) and in cows (84%) and of B. bovis in calves (85.2%) and in cows (100%) did not differ significantly (P > 0.05). Babesia spp. infection was more frequent in female ticks and eggs collected from calves (P < 0.01) than from cows, especially in those which had patent parasitemia. Hatching rates of B. microplus larvae were assessed according to the origin of engorged females, parasitemia of the vertebrate host, frequency and intensity of infection in engorged female tick, and frequency of egg infection. Hatching rate was lower in samples collected from calves (P < 0.01) than from cows, and in those in which Babesia spp. was detected in egg samples (P < 0.01).     

牛分枝杆菌特异性PCR检测方法的建立及初步应用

根据已发表的牛分枝杆菌的pncA的基因序列，设计和合成了一对可扩增294bp目的片段的引物，建立了特异性检测牛分枝杆菌的PCR方法。对牛分枝杆菌国际参考株和国内分离株成功扩增出294bp的特异性基因片段；对人结核分枝杆菌、副结核分枝杆菌、鸟胞内分枝杆菌和草分枝杆菌DNA的PCR扩增结果均为阴性。本PCR方法检测的敏感度可达到50pg。对10份牛分枝杆菌培养阳性和10份阴性样品的DNA分别进行了PCR检测，结果10份阳性样品中有9份样品为PCR扩增阳性，阳性符合率为90％（9／10）；而10份阴性样品则PCR扩增全部为阴性，阴性符合率为100％（10／10）。本方法可做为牛分枝杆菌的快速检测和流行病学调查的工具。     

Seroepidemiological studies of bovine babesiosis on Pemba Island, Tanzania.

Serological evidence of infection with Babesia bovis and Babesia bigemina at a number of sites in Pemba was obtained using an enzyme-linked immunosorbent assay (ELISA) capable of detecting the appropriate parasite-specific antibody. Overall, 96% of animals were found to be positive for B. bovis, 88% were positive for B. bigemina and 88% were positive for both Babesia species. Antibody to B. bovis and B. bigemina was detected early in life in a number of calves born on Pemba, and was considered to be of maternal origin. The amount of maternal antibody in the serum of individual animals fell throughout the first 3 months of life. Later in life, antibody levels increased, probably in response to Babesia infection from natural tick challenge. These results suggest that infection with both Babesia parasites is widespread throughout Pemba and that both parasites probably exist in an enzootically stable situation.