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1.
《中国奶牛》2020,(6)
为了解新疆和静县绵羊细粒棘球蚴病的感染情况及流行株基因分型,对和静县屠宰场的1 115只1岁以上绵羊的细粒棘球蚴病感染情况进行调查和统计,并利用PCR技术对包囊病灶进行了基因分型鉴定。结果表明:有88只绵羊脏器表面发现棘球蚴包囊,感染率为7.89%(88/1115),其中来自农区的感染率为3%(10/331),来自牧区的感染率为9.9%(78/784)。通过线粒体细胞色素氧化酶基因1(mtCO1)特异性引物对剖检的感染病料进行PCR扩增、克隆、测序和NCBI中的Blast比对,发现新疆和静县绵羊细粒棘球蚴流行株基因型均为G1型。本研究为和静县绵羊细粒棘球蚴病的防控提供了科学依据。 相似文献
2.
本研究旨在分析青海地区细粒棘球蚴的种群基因多态性,为细粒棘球蚴病的防控提供基础资料。对采自青海地区的42株细粒棘球蚴(33株采自绵羊肝脏,9株采自绵羊肺脏)进行了线粒体12S基因的全序列测序并构建了NJ系统发生树。结果显示:在本研究样品的线粒体12S基因序列中共检测出5种单倍型(即H1～H5),其中以单倍型H5为主(占32株),并且系统发生树分析支持这一结果。单倍型多样性(H)和核苷酸多样性(Pi)分别为0.418、0.000 66,与E.granulosus G1(AF297617)的12S基因序列的核苷酸相似性达到99.86%以上。采自青海地区的42株细粒棘球蚴均鉴定为E.granulosus sensu stricto(基因型G1-G3),在检测出的5种单倍型中,单倍型H1～H4是本地区特有的单倍型。 相似文献
3.
为建立一种快速、高效的诊断家畜细粒棘球蚴病方法,提取绵羊细粒棘球蚴包囊新鲜囊液,盐析囊液抗原,点样于硝酸纤维膜,以胶体金-驴抗羊IgG和胶体金-兔抗鼠IgG为检测标记物,采用垂直流渗滤装置检测绵羊与人工感染细粒棘球蚴小鼠血清和全血特异性抗体。患病绵羊阳性血清及全血检出率在90.91%~94.4%,细粒棘球蚴感染小鼠血清及全血检出率均为100%;细粒棘球蚴阴性羊血清和全血假阳性率为4.00%~4.59%;与脑多头蚴病血清交叉反应率为28.57%(2/7)。研究结果表明细粒棘球蚴全血金标渗滤法(DIGFA)可应用于绵羊棘球蚴病的诊断与检疫。 相似文献
4.
我国三省区细粒棘球绦虫基因的变异分析 总被引：1，自引：0，他引：1
对从青海省、甘肃省和新疆维吾尔族自治区采集的24株细粒棘球蚴，用线粒体DNA的CO1基因和ND1基因结合rDNA的ITS2测序，调查了不同地区分离株基因型的变异情况。结果显示，所有分离株均为普通羊株(G1基因型)；CO1基因序列的变异率为0～0．8％，ND1基因的变异率为0．1％～0．7％；青海分离株ITS2序列的变异率为0．4％～3．1％；2株青海省西宁市和1株甘肃省武威市分离株分剐在C01基因和ND1基因序列不同位点发生的1处核苷酸非同义替换，导致1个编码的氨基酸替代。研究表明，我国上述3省、区部分区域存在的细粒棘球蚴属于同一株(G1基因型)。 相似文献
5.
《中国畜牧兽医》2020,(4)
本研究旨在对西藏自治区那曲地区和拉萨市牦牛、绵羊体内棘球蚴病原进行分子生物学鉴定并分析其遗传变异规律。对2016年11月底采自西藏拉萨市当雄县和那曲地区嘉黎县的5只绵羊体内的5个棘球蚴包囊、15头牦牛体内的18个棘球蚴包囊分别分离棘球蚴原头蚴或生发层组织,提取基因组DNA,应用PCR方法扩增nad1基因,通过测序获得nad1全基因序列。运用DNAStar MegAlign软件对序列进行同源性分析。以GenBank中已公布的棘球属的nad1全基因序列为比对对象,采用最大似然法(ML)构建系统发育树。结果显示,所测定的牦牛和绵羊的23个棘球蚴病原nad1基因序列与GenBank登录的细粒棘球蚴狭义种(G1基因型)nad1基因序列高度同源,同源性为99.6%～99.8%,23条nad1基因的遗传距离为0～0.0022447。同源基因的碱基变异率为0.2%～0.4%;与棘球属其他棘球绦虫同源基因的碱基变异率为14.9%～19.8%。有5个样本的nad1基因在不同位点发生碱基突变,变异位点发生序列转换。以上结果表明,本研究所采集牦牛和绵羊的棘球蚴为细粒棘球绦虫G1基因型,其nad1基因变异小,序列一致性高。 相似文献
6.
[目的]了解新疆维吾尔自治区部分规模化屠宰场羊的细粒棘球蚴感染情况及其分子遗传特征。[方法]收集屠宰场羊肝脏组织内经肉眼观察鉴定为细粒棘球蚴的包囊样本96份,全部提取基因组DNA后,基于细粒棘球绦虫cox1基因位点和nad1基因位点进行PCR扩增和测序。通过序列比对数据鉴定其基因型,构建遗传进化树解析其分子遗传特征。[结果]在cox1基因位点上,有81份样本呈PCR扩增阳性（84.37%,81/96）,经序列分析鉴定,所有阳性样本均为细粒棘球蚴G1基因型（n=81）,存在15个单倍型（Hap＿1～15）;在nad1基因位点上,有67份样本呈PCR扩增阳性69.79%(67/96),经序列分析鉴定出2种基因型,分别为细粒棘球蚴G1基因型（n=66）和G3基因型（n=1）,存在19个单倍型（Hap＿1～19）。种系发育分析显示,获得的细粒棘球蚴G1基因型和G3基因型序列,与国内外已报道的多种宿主源的G1基因型和G3基因型序列均处于同一个进化支。[结论]新疆部分地区羊源细粒棘球蚴呈现遗传多样性分布特征,研究结果为该地区羊包虫病的防治提供了基础数据。 相似文献
7.
本研究旨在对西藏自治区那曲地区和拉萨市牦牛、绵羊体内棘球蚴病原进行分子生物学鉴定并分析其遗传变异规律。对2016年11月底采自西藏拉萨市当雄县和那曲地区嘉黎县的5只绵羊体内的5个棘球蚴包囊、15头牦牛体内的18个棘球蚴包囊分别分离棘球蚴原头蚴或生发层组织，提取基因组DNA，应用PCR方法扩增nad1基因，通过测序获得nad1全基因序列。运用DNAStar MegAlign软件对序列进行同源性分析。以GenBank中已公布的棘球属的nad1全基因序列为比对对象，采用最大似然法（ML）构建系统发育树。结果显示，所测定的牦牛和绵羊的23个棘球蚴病原nad1基因序列与GenBank登录的细粒棘球蚴狭义种（G1基因型）nad1基因序列高度同源，同源性为99.6%~99.8%，23条nad1基因的遗传距离为0~0.0022447。同源基因的碱基变异率为0.2%~0.4%；与棘球属其他棘球绦虫同源基因的碱基变异率为14.9%~19.8%。有5个样本的nad1基因在不同位点发生碱基突变，变异位点发生序列转换。以上结果表明，本研究所采集牦牛和绵羊的棘球蚴为细粒棘球绦虫G1基因型，其nad1基因变异小，序列一致性高。 相似文献
8.
9.
探究细粒棘球绦虫半胱氨酸蛋白酶抑制剂的基本特性并初步评价其诊断价值,旨在为细粒棘球绦虫的防控提供依据。本研究原核表达出Eg-cystatin重组蛋白并对其进行生物信息学、免疫印迹分析,用荧光免疫定位的方法检测该蛋白质的分布情况,并以绵羊细粒棘球蚴阳性血清评价Eg-cystatin重组蛋白的诊断价值。结果如下:Eg-cystatin生物信息学分析结果显示,该蛋白质含有一个N端信号肽以及cystatin结构域,是典型的2型半胱氨酸蛋白酶抑制剂,进化树分析显示Eg-cystatin属于绦虫cystatinⅡ型,并且与其他绦虫的cystatin相似性为72.20%~80.66%,免疫荧光定位发现该蛋白质主要分布在原头蚴的表皮和顶突钩,生发层,成虫虫体内部和虫卵。基于Eg-cystatin建立的间接ELISA方法的特异性为79.1%,敏感性为83.3%,与剖检符合率为81.25%。Eg-cystatin在成虫和幼虫时期均有表达,提示该基因与虫体生命活动息息相关,但Eg-cystatin蛋白不适合作为绵羊细粒棘球蚴病的诊断抗原候选。 相似文献
10.
羊细粒棘球蚴病抗体间接ELISA检测方法的建立 总被引：1，自引：0，他引：1
本研究旨在通过建立检测羊细粒棘球蚴病抗体的间接酶联免疫吸附试验(ELISA)方法,为羊细粒棘球蚴病的检测提供快速、简便的手段.作者利用DNAStar软件对GenBank上发表的细粒棘球蚴EG95蛋白的氨基酸序列进行分析,筛选出高度亲水的优势表位区EG95s,对该区域编码基因进行克隆并表达.以纯化的重组融合蛋白为包被抗原,按常规方法建立检测羊细粒棘球蚴病抗体的间接ELISA,并对各种条件进行优化.SDS-PAGE结果表明成功获得了可溶性好、表达效率高、纯化简便的重组融合蛋白GST-1EG95s和HIS-1EG95s,Western blot检测结果表明表达产物具有较好的反应原性.间接ELISA方法优化结果显示:HIS-1EG95s作为包被抗原效果优于GST-1EG95s.经统计学分析,确定间接ELISA方法的判定标准:OD450nm值≥0.235时判定为阳性,OD450nm值≤0.191时判定为阴性,介于二者之间则为可疑.分别对采自新疆的70份羊包虫阳性血清和70份阴性血清进行检测,结果表明该方法与新西兰Wallaceville动物研究中心提供的间接ELISA方法符合率为100%,阻断试验结果显示与其他蛋白无交叉反应,批内变异系数介于3.8%～5.6%,批间变异系数介于5.7%～8.5%,结果表明该方法特异性强、敏感性高、重复性好.作者所建立的羊细粒棘球蚴病抗体的间接ELISA检测方法有望为羊细粒棘球蚴病的检测提供快速、简便的手段. 相似文献
11.
羊泰勒虫PCR检测方法的建立和初步应用 总被引：1，自引：0，他引：1
利用羊泰勒虫18SrRNA基因的序列特点,设计合成种特异性引物,建立羊泰勒虫PCR检测方法,该方法能特异性扩增398bp的羊泰勒虫18SrRNA基因片段,而对羊巴贝斯虫、羊无浆体、牛环形泰勒虫和牛伊氏锥虫的基因组DNA没有扩增带出现。对羊泰勒虫基因组DNA的最小检测量为0.12fgDNA。通过检测124份临床样品,24份为羊泰勒虫感染阳性,其余为阴性。结果表明,建立的PCR检测方法具有极高的敏感性和特异性,可用于羊泰勒虫病和临床健康带虫羊的诊断。 相似文献
12.
In this study, a pair of oligonucleotide primers were designed according to the nucleotide sequence of the small subunit ribosomal RNA (ssu rRNA) gene of Babesia ovis isolated from sheep in eastern Turkey. The primers were used to detect parasite DNA from blood samples of B. ovis-infected sheep and goats by polymerase chain reaction (PCR). A 549-bp DNA fragment was specifically amplified from blood samples from sheep and goats, naturally infected with B. ovis. No PCR products resulted from Babesia motasi, T. ovis, Theileria sp. OT1, Theileria sp. OT3, T. lestoquardi, B. canis, B. microti,T. annulata or normal sheep leucocytes DNA using these specific primers. B. ovis-infected erythrocytes with 1% parasitemia were subjected to 10-fold serial dilutions (from 10(-1) to 10(-9)) using an uninfected sheep erythrocytes, and DNA was extracted from each diluted sample for testing the sensitivity of the PCR. The PCR was sensitive enough to detect parasite DNA from the dilution of 10(-5) with 0.00001% parasitemia. This is more sensitive than examining 200 fields under light microscopy. In addition, 98 field samples collected from small ruminanats in eastern Turkey were tested for B. ovis infection. Four samples were positive Babesia spp. in blood smears, 21 samples were positive for B. ovis DNA by PCR. These results indicate that the PCR provides a useful diagnostic tool for the detection of B. ovis infection in sheep and goats. 相似文献
13.
Suzuki J Sasaoka F Fujihara M Watanabe Y Tasaki T Oda S Kobayashi S Sato R Nagai K Harasawa R 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2011,73(8):1113-1115
We examined the presence of hemoplasmas, hemotropic mycoplasmas, among 11 sheep (Ovis aries) with regenerative and hemolytic anemia and found six of them were positive by real-time PCR. The positive samples were then subjected to conventional PCR for direct sequencing of the 16S rRNA gene. Nucleotide sequences of all the positive samples were identified as the 16S rRNA gene of `Candidatus Mycoplasma haemovis' by phylogenetic analysis, demonstrating the infections with this particular hemoplasma species in Japan. 相似文献
14.
J M Miller A L Jenny J L Ellingson 《Journal of veterinary diagnostic investigation》1999,11(5):436-440
A PCR procedure previously developed for identification of Mycobacterium bovis in formalin-fixed tissues was used to identify mycobacteria of the M. avium complex. Tissues were examined from 100 culture-positive cases of M. avium complex infection, including 86 in which the subspecies was not identified and 14 that had been identified as M. avium subsp. paratuberculosis. Each sample was tested with 5 primer sets, 16S ribosomal RNA (rRNA), IS900, IS901, IS1245, and a heat shock protein (hspX), that detect 1 or both M. avium subspecies. The success rate of PCR detection varied with the primers used and the animal species tested. Among the 86 cases with no M. avium subspecies designation, primers for the 16S rRNA gene were clearly the most efficient because they produced amplicons from all samples that reacted with any other primer set. The overall detection rate in this group of samples was 71%: highest in avian tissues (89%) followed by swine (72%) and ruminants (57%) None of the avian or swine tissues reacted with primers for IS900 or hspX, which identify M. a. paratuberculosis. In contrast, 7 of the 12 ruminant samples that were 16S rRNA positive reacted with 1 or both of these primers. All of the 14 cases shown by culture to be M. a. paratuberculosis infections were positive with IS900 primers, whereas only 11 were positive for 16S rRNA. These results indicate that 16S rRNA primers are the most useful for PCR identification of M. avium in formalin-fixed tissues of nonruminant species. However, IS900 primers should also be used when ruminant tissues are examined because these primers provide the greatest sensitivity for detection of M. a. paratuberculosis infections. 相似文献
15.
The Xinjiang plateau of western China has been shown to have a high prevalence for human cystic echinococcosis (CE) caused by Echinococcus granulosus, and human alveolar echinococcosis (AE) caused by Echinococcus multilocularis. The domestic dog is suspected to be the primary definitive host for the transmission of both E. granulosus and E. multilocularis to humans in this locality. Seventeen of 30 stray dogs from Hejing County of Xinjiang were found positive for E. granulosus post mortem, and one double infection was suspected. Worm samples were collected, dyed by carmine, and observed microscopically. Carmine staining examination clearly revealed the differences in number of proglottids and appearance of uterine branches and lateral genital pore for those two species of Echinococcus. Furthermore, gene target DNA fragments were amplified for formal identification of the two parasite species, based on 12s rRNA mitochondrial gene. The PCR products were purified and sequenced. Compared with NCBI GenBank, the DNA sequences demonstrated 100% identity with E. granulosus (sheep strain, G1 genotype) and E. multilocularis. 相似文献
16.
Cystic echinococcosis (CE), caused by hydatid cysts, is a widespread and hazardous disease in humans and animals worldwide. The aim of the current study was to investigate the genetic characteristics of sheep and cattle isolates of Echinococcus granulosus obtained from eastern Turkey using Single Stranded Conformation Polymorphism (SSCP) analysis and conventional PCR method. A total of 54 isolates collected from Erzurum and Elazig provinces of east-Turkey were examined. The 31 of these were obtained from liver of sheep while 23 cattle isolates (12 of liver and 11 of lung) were tested. After the total genomic DNA isolation 12S rRNA gene of all isolates were examined by PCR for the aim of genetic characterization by conventional PCR and mitochondrial CO1 gene for SSCP analysis. The 12S rRNA-PCR yielded 254 bp of amplification product with all samples analyzed. Thus, these samples were identified as G1-G3 cluster (E. granulosus sensu stricto). At least two major single stranded bands were resolved for G1-G3 cluster and G5 in SSCP analysis. While the resolution of more than two additional single stranded bands in SSCP indicated the existence of G7 genotype. The SSCP analysis was identified the G5 and G7 while failed to G1 and G3. The present SSCP analysis classified all 54 cyst isolates from sheep and cattle as E. granulosus sensu stricto (G1-G3 cluster). However, some sequenced samples for G1 and G3 showed the same band patterns by SSCP. 相似文献
17.
A nested polymerase chain reaction (PCR) for the detection of Theileria ovis in sheep using oligonucleotide primers designed from the small subunit ribosomal RNA (SSU rRNA) gene sequence of T. ovis from sheep in eastern Turkey is described. A 398-bp DNA fragment was specifically amplified from blood samples from sheep, naturally infected with T. ovis. No PCR products resulted from T. lestoquardi, T. annulata, T. parva, T. buffeli and Babesia spp. DNA using these specific primers. The sensitivity of the nested PCR for T. ovis, which was assessed showed that one infected cell in 10(7) sheep erythrocytes, equivalent to a blood parasitemia of 0.00001%, could be detected. This is more sensitive than examining 200 fields under light microscopy. In addition, of the 124 field samples obtained from sheep in eastern Turkey tested, 19.35% (24/124) were positive for the presence of Theileria spp. by microscopic examination compared to 54.03% (67/124) positive for T. ovis by nested PCR. The primer pairs described in this study will be useful for epidemiological studies on ovine theileriosis and for discrimination between T. lestoquardi and T. ovis infections in sheep. 相似文献
18.
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. 相似文献
19.
Criado-Fornelio A Rey-Valeiron C Buling A Barba-Carretero JC Jefferies R Irwin P 《Veterinary parasitology》2007,144(3-4):261-269
The prevalence of hematozoan infections (Hepatozoon canis and Babesia sp., particularly Babesia canis vogeli) in canids from Venezuela, Thailand and Spain was studied by amplification and sequencing of the 18S rRNA gene. H. canis infections caused simultaneously by two different isolates were confirmed by RFLP analysis in samples from all the geographic regions studied. In Venezuela, blood samples from 134 dogs were surveyed. Babesia infections were found in 2.24% of the dogs. Comparison of sequences of the 18S rRNA gene indicated that protozoan isolates were genetically identical to B. canis vogeli from Japan and Brazil. H. canis infected 44.77 per cent of the dogs. A representative sample of Venezuelan H. canis isolates (21.6% of PCR-positives) was sequenced. Many of them showed 18S rRNA gene sequences identical to H. canis Spain 2, albeit two less frequent genotypes were found in the sample studied. In Thailand, 20 dogs were analyzed. No infections caused by Babesia were diagnosed, whereas 30 per cent of the dogs were positive to hematozoan infection. Two protozoa isolates showing 99.7-100% identity to H. canis Spain 2 were found. In Spain, 250 dogs were studied. B. canis vogeli infected 0.01% of the animals. The sequence of the 18S rRNA gene in Spanish isolates of this protozoa was closely related to those previously deposited in GenBank (> 99% identity). Finally, 20 red foxes were screened for hematozoans employing semi-nested PCR and primers designed to detect Babesia/Theileria. Fifty percent of the foxes were positive to Theileria annae. In addition, it was found that the PCR assay was able as well to detect Hepatozoon infections. Thirty five percent of the foxes were infected with two different H. canis isolates showing 99.8-100% identity to Curupira 1 from Brazil. 相似文献