山羊脑多头蚴线粒体nad1基因的克隆及序列分析

本研究旨在阐明脑多头蚴湖南分离株线粒体烟酰胺腺嘌呤二核苷酸(NADH)脱氢酶亚单位1基因(nad1)部分序列(pnad1)的遗传变异情况,并用pnad1序列重构脑多头蚴与其他带科绦虫的种群遗传关系。利用聚合酶链反应(PCR)扩增脑多头蚴的pnad1,应用ClustalX 1.81程序对序列进行比对,再用Phylip3.67程序MP法和Mage4.0程序NJ法绘制种系发育树,并用Puzzle5.2程序构建最大似然树,同时利用DNAStar 5.0中的Megalign程序进行同源性分析。结果显示,所获得的pnad1序列长度分别均为430 bp,湖南分离株与已知多头带绦虫位于同一分枝。由于脑多头蚴pnad1序列种内相对保守,种间差异较大,故均可作为种间遗传变异研究的标记,从而为脑多头蚴的分子流行病学和其相关疾病的诊断奠定基础。     

湖南省山羊脑多头蚴的线粒体nad1和nad4基因的序列测定及种系发育分析

本研究旨在阐明脑多头蚴湖南分离株线粒体烟酰胺腺嘌呤二核苷酸( NADH)脱氢酶亚单位1基因(nad1)部分序列(pnad1)和烟酰胺腺嘌呤二核苷酸(NADH)脱氢酶亚单位4基因(nad4)部分序列(pnad4)的遗传变异情况,并用pnad1和pnad4序列重构脑多头蚴与其它带科绦虫的种群遗传关系.利用聚合酶链反应(PCR)扩增脑多头蚴的pnad1和pnad4,应用ClustalX 1.81程序对序列进行比对,再用Phylip3.67程序MP法和Mage4.0程序NJ法绘制种系发育树,并用Puzzle5.2程序构建最大似然树,同时利用DNAstar5.0中的Megalign程序进行同源性分析.结果显示所获得的pnad1和pnad4序列长度分别均为666和887 bp,湖南分离株与已知多头带绦虫位于同一分枝.由于脑多头蚴pnad1和pnad4序列种内相对保守,种间差异较大,故均可作为种间遗传变异研究的标记,从而为脑多头蚴的分子流行病学和其相关疾病的诊断奠定基础.     

羊场暴发脑包虫病的诊治与防控

绵羊脑包虫病是由多头带绦虫（Taenia multiceps）的中绦期幼虫——脑多头蚴（Coenurus cerebralis）引起的一种严重危害羔羊的寄生虫病。脑多头蚴主要寄生于牛羊等反刍动物的脑和脊髓中,病畜出现一系列神经症状,最终导致死亡。介绍绵羊脑包虫病的病例情况、临床症状、病理剖解变化、诊断、手术治疗,分析发病原因,提出了针对脑包虫病的综合防控措施。     

脑多头蚴病研究进展

脑多头蚴病是由多头带绦虫的中绦期幼虫脑多头蚴寄生于绵羊、山羊、黄牛、牦牛等有蹄动物的脑及脊髓中引起的一种人兽共患寄生虫病,人可因误食虫卵而感染此病。该病在宿主出现典型临床症状后如果得不到治疗,多以死亡为转归,给畜牧业生产造成了巨大的经济损失。论文就脑多头蚴病的病原学、生活史、流行病学、临床症状、诊断、治疗及免疫预防等方面的研究进展进行了综述。     

多头带绦虫Tm16与Tm18抗原基因的克隆及原核表达

为原核表达Tml6和Tm18重组蛋白,本研究以自然感染羊源脑多头坳原头节基因组DNA为模板分别扩增Tml6和Tm 18杭原基因全基因片段,测序鉴定后合成其开放阅读框架DNA片段,将基因组序列中的内含子去除并对其稀有密码子进行改造优化,构建重组表达质粒pGEX-Tm 16和pGEX-Tml8.转化大肠杆菌BL21后以IPTG诱导表达,SDS-PAGE分析表达产物并进行纯化.结果显示:在大肠杆菌中表达出带有GST标签的大小约为39.6 ku和39.4 ku的重组蛋白,经谷胱甘肤琼脂糖树脂纯化得到高纯度的可溶性的GST-Tm 16及GST-Tml8重组蛋白,western blot分析表明重组蛋白均能够被兔抗GST单克隆杭体特异性识别.     

多头绦虫抗原特性的研究──应用IHA观察绵羊免疫及感染后的抗体消长规律

为了观察绵羊用多头蚴抗原免疫及感染后的抗体消长规律,为羊脑多头蚴病的免疫预防和免疫诊断提供依据,本试验应用多头蚴原头节可溶性抗原、囊壁可溶性抗原、囊液粗抗原致敏绵羊红细胞对绵羊免疫3次及虫卵攻击感染后的血清抗体进行间接血凝试验（IHA）检测。结果表明,原头节抗原免疫组、囊壁抗原免疫组、囊液抗原免疫组及原头节ES抗原免疫组在首次免疫后1周,抗体滴度迅速升高,第3次免疫后1周达到峰值,虫卵感染后开始下降,到感染后30周接近正常水平。多头蚴3种抗原对同种抗原免疫组血清检测敏感性、特异性优于其它抗原,原头节免疫组、囊壁免疫组、囊液免疫组抗体水平明显高于原头节ES抗原免疫组。     

奥芬哒唑对人工感染脑多头蚴绵羊的早期治疗试验

为了寻找一种治疗脑多头蚴早期感染的新药物，应用奥芬哒唑（Oxfendazole)于感染后第8d和第15d以30mg/kg的剂量，对人工感染脑多头蚴的绵羊进行了单剂量和三剂量早期治疗试验。结果表明：无论是第8d还是第15d，三剂量组对人工接种早期感染脑多头蚴的绵羊具有完全的治疗作用。奥芬哒唑是治疗羊脑多头病的理想药物。     

Variability in the Echinococcus granulosus cytochrome C oxidase 1 mitochondrial gene sequence from livestock in Turkey and a re-appraisal of the G1-3 genotype cluster

Investigations into the genetic strains of Echinococcus granulosus parasites occurring in sheep and cattle in Turkey were undertaken. A total of 112 hydatid cysts were investigated from sheep (100 isolates) derived from widely distributed sites within Turkey as well as from cattle (12 isolates) from the Turkish province of Kars. The parasite genotypes in these isolates were determined by DNA sequencing of part of the mitochondrial Cytochrome C oxidase 1 (cox1) gene. Haplotypes were identified which corresponded clearly to the previously described strain G1 in a total of 107 isolates, including 98 isolates from sheep and 9 isolates from cattle. Five isolates, including 2 sheep and 3 cattle, were determined to belong to the G3 genotype. Parasites of the G3 genotype were identified only in isolates derived from animals in the eastern regions of Turkey. While the majority of the isolates described here had haplotypes corresponding to the G1 genotype, none matched exactly the G1 sequence that was defined in previous studies. Analysis of all GenBank entries for E. granulosus cox1 sequences representing G1, G2 and G3 genotypes identified substantial microsequence variability. G1 and G3 could be distinguished as separate strains, however, the existence of G2 as a separate strain could not be supported. Rather, this can be regarded as a microsequence variation of G3.     

A survey of Taenia multiceps coenurosis in Sardinian sheep

A survey was carried out to assess the occurrence of Coenurus cerebralis infection in Sardinian sheep. A prevalence of 0.35% was observed when 566 regularly slaughtered sheep were examined. However, in 120 sheep with suspected symptoms of coenurosis examined from November 2001 to October 2002, a total of 299 cerebral coenurosis lesions were observed with an incidence of 1% per year. Lesions were classified as migratory, cystic and secondary. Most migratory lesions were found in sheep aged 3-6 months. Cavitary lesions containing cysts in different developing stages were found with high incidence per year in sheep aged 7-12 months. Secondary lesions due to the development of Coenurus were most frequent in sheep aged 19-36 months. Most sheep were found infected in spring and in early summer, between March and June. Most lesions were located in the cortex. The mean number of protoscolices per cyst was 149 (range 10-370).     

Clinicopathologic observations on Coenurus cerebralis in naturally infected sheep

Twelve sheep from 7 different flocks consisting of approximately 150-250 animals each were diagnosed with coenurosis caused by the larval stage of Taenia multiceps. Ataxia, incoordination, drowsiness, hind leg paralysis and coma were the most prominent clinical symptoms. Monocytosis and lymphocytosis were observed upon hematological examination. Creatin kinase (CKBB) levels of the animals varied between 421 and 495 U/l. Cysts were commonly localized in the parietal and frontal lobes of the brain and in the cerebellum. In two cases, cysts were found on the lumbar aspect of the medulla spinalis. Symptoms were related to cyst localization. Depression, tilting of the head either to the right or left and head pressing were seen when cysts were located in the cerebrum. Incoordination and hyperexcitability were noted if the cysts were involved with the cerebellum and when located in the spinal cord, hind leg paralysis was the typical clinical sign. On microscopic examination, atrophy was observed in the central nervous system (CNS) organs due to pression by the bladderworms. Nonpurulent meningoencephalitis with perivascular cuffings were the most common histopathological findings. In periodic acid Schiff staining (PAS), positive reaction was observed in protoscoleces. Neurons were the most affected cell type when stained by the Klüver Barrera method.This method also showed that in the CNS, Coenurus cerebralis caused a prominent glial reaction. When parasites were localized in the nervous system treatment was impossible. Animals without neurologic sings were treated with praziquantel (Tenikur tablet-Topkim A.S.) 50-100 mg/kg/day for three days.     

Diagnosis and treatment of coenurosis in sheep

Coenurosis is a disease of the central nervous system in sheep, caused by Coenurus cerebralis, the larval stage of Taenia multiceps, a tapeworm, which infests the small intestine of carnivores. In 80-90% of cases, the cyst is located in one cerebral hemisphere, whilst in 5-10% of cases, it is localised in the cerebellum; rarely it involves two sites in the brain of the affected animal. Listeriosis, louping-ill, sarcocystosis and polioencephalomalacia and brain abscessation should be considered when formulating a diagnosis of acute coenurosis. In all cases, it is essential to carefully examine the animal and not simply rely on results of ancillary tests (mainly of cerebrospinal fluid examination), as disorders other than coenurosis can be responsible for changes in the results of these tests. Treatment is based on surgical removal of the coenurus cyst after general anaesthesia of the animal; the approach has a very good success rate, especially after accurate localisation of the lesion. Despite that, many farmers may choose to slaughter those sheep fit for marketing for economic reasons and euthanise those in poor condition.     

Molecular discrimination of sheep and cattle isolates of Echinococcus granulosus by SSCP and conventional PCR in Turkey

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.     

Two rare clinical manifestations of coenurosis in sheep

Two rare clinical manifestations of coenurosis in sheep are reported. (i) A case of partial seizure disorder in a ram of 11 months old. During seizure episodes the animal lay down in lateral recumbency displaying initially a stuporous condition and subsequently began to revolve its head from the base of the cervix. At the necropsy of the case, Coenurus cerebralis cyst (young bladder worm) was found dorsally inside the brainstem, in the site of the tectum mesencephaly. (ii) The second-reported manifestation was a bacterial meningoengephalitis that was witnessed in two lambs of 6-7 weeks old. The lambs displayed lateral recumbency with seizure activity. At necropsy, meningoencephalitis with congestion and abscesses were observed in both of them. Interestingly, C. cerebralis cysts were also found in both brains. Streptococcus dysgalactiae was isolated from the abscesses. Possibly, S. dysgalactiae translocation of the blood-brain barrier was facilitated by the migration of the immature stages of C. cerebralis to and through the brain.     

Molecular epidemiology of Echinococcosis from food producing animals in north India

Echinococcosis is an important medical, veterinary and economic concern in India. Ten cysts were randomly selected from each intermediate host species (cattle, buffalo, sheep, goat and pigs). Either the germinal layer (sterile cysts) or protoscoleces (fertile cysts) were collected for molecular characterization. A 434 base pair fragment of the mitochondrial cytochrome oxidase-1 gene was amplified using PCR from each isolate. Ten representative samples (2 from each intermediate host species) were sequenced in both the directions from which readable sequences were obtained from nine for phylogenetic analysis (NCBI, Blast). Phylogenetic analysis of cytochrome oxidase I gene revealed that seven (77.7%) isolates, from cattle (2), pigs (2), buffaloes (1) and goat (2) were clustered with the Indian Buffalo (G3) strain of Echinococcus granulosus, while two (22.2%) isolates from sheep were clustered with the sheep strain (G1) of E. granulosus. Phylogenetic analysis of the cytochrome oxidase-1 gene revealed that the buffalo strain (G3) and common sheep strain (G1) are cycling among livestock in north India and that these strains are highly adapted to cattle, buffalo, sheep, goats and pigs.     

羊毛尾线虫线粒体nad4基因的序列变异分析

本研究旨在阐明中国羊毛尾线虫(Trichuris ovis)广东分离株线粒体NADH脱氢酶亚单位4(nad4)基因部分序列(pnad4)的遗传变异情况, 并用pnad4序列构建其与其他鞭虫的进化关系。应用PCR扩增羊鞭虫虫株的pnad4,将所获得的序列应用ClustalX 1.81程序进行比对,然后用Mega 5.0程序NJ法绘制种系发育树。本试验扩增所获得的pnad4序列长度一致,均为827 bp,种内变异在0~1.5%之间。种系发育分析结果表明,12个羊鞭虫分离株位于同一分支。由于羊鞭虫nad4序列种内相对保守,种间差异较大(37.8%~45.6%), 故可作为种间鉴定检测研究的遗传标记, 本研究结果为进一步研究羊鞭虫的群体遗传结构奠定了基础。     

Four p67 alleles identified in South African Theileria parva field samples

Previous studies characterizing the Theileria parva p67 gene in East Africa revealed two alleles. Cattle-derived isolates associated with East Coast fever (ECF) have a 129 bp deletion in the central region of the p67 gene (allele 1), compared to buffalo-derived isolates with no deletion (allele 2). In South Africa, Corridor disease outbreaks occur if there is contact between infected buffalo and susceptible cattle in the presence of vector ticks. Although ECF was introduced into South Africa in the early 20th century, it has been eradicated and it is thought that there has been no cattle to cattle transmission of T. parva since. The variable region of the p67 gene was amplified and the gene sequences analyzed to characterize South African T. parva parasites that occur in buffalo, in cattle from farms where Corridor disease outbreaks were diagnosed and in experimentally infected cattle. Four p67 alleles were identified, including alleles 1 and 2 previously detected in East African cattle and buffalo, respectively, as well as two novel alleles, one with a different 174 bp deletion (allele 3), the other with a similar sequence to allele 3 but with no deletion (allele 4). Sequence variants of allele 1 were obtained from field samples originating from both cattle and buffalo. Allele 1 was also obtained from a bovine that tested T. parva positive from a farm near Ladysmith in the KwaZulu-Natal Province. East Coast fever was not diagnosed on this farm, but the p67 sequence was identical to that of T. parva Muguga, an isolate that causes ECF in Kenya. Variants of allele 2 were obtained from all T. parva samples from both buffalo and cattle, except Lad 10 and Zam 5. Phylogenetic analysis revealed that alleles 3 and 4 are monophyletic and diverged early from the other alleles. These novel alleles were not identified from South African field samples collected from cattle; however allele 3, with a p67 sequence identical to those obtained in South African field samples from buffalo, was obtained from a Zambian field isolate of a naturally infected bovine diagnosed with ECF. The p67 genetic profiles appear to be more complex than previously thought and cannot be used to distinguish between cattle- and buffalo-derived T. parva isolates in South Africa. The significance of the different p67 alleles, particularly the novel variants, in the epidemiology of theileriosis in South Africa still needs to be determined.     

基于pcox1基因分析黑斑蛙小吻对盲囊线虫种系发育关系

以湖南省不同地区黑斑蛙体内采集的16条小吻对盲囊线虫样品为研究对象,利用PCR对其线粒体DNA中的细胞色素c氧化酶第1亚基(cox 1)基因部分序列(pcox 1)进行扩增,分析其遗传变异情况。并利用软件Clustal X 2.0对其序列进行分析,探讨小吻对盲囊线虫与其他线虫的种群遗传关系。测序结果显示,所获得的pcox 1序列长度一致,均为400 bp,湖南省各分离株之间相应序列的相似性在97%以上,与GenBank中登录的其他小吻对盲囊线虫pcox 1序列的相似度均低于89%。结果表明,小吻对盲囊线虫pcox 1序列种内相对保守,种间差异较大,可以作为种间遗传变异研究的分子标记,从而为小吻对盲囊线虫的分子流行病学调查研究等奠定基础。     

Molecular evidence of ovine (G1) and camel (G6) strains of Echinococcus granulosus in Tunisia and putative role of cattle in human contamination

Three hundred and seventy-two cysts coming from 50 humans, 166 cattle, 153 sheep and 3 camels were collected in order to establish some epidemiological molecular information in Tunisia for the first time. The analysis by PCR-RFLP of ITS1 sequence showed that all the human, ovine and bovine cysts were due to the common sheep strain of Echinococcus granulosus. The sequencing of the CO1 gene of 37 isolates confirm the G1 genotype of this strain. For seven of these isolates, we found the mutation C56T which is present in the three principal intermediate hosts: human (three cysts), cattle (three cysts) and sheep (one cyst). With regard to the G1 genotype, we identified three other point mutations. The camel strain G6 is uniquely found in the three camels isolates and not in the other intermediate hosts analysed. The fertility of the bovine cyst represents 48% that means that this host is involved in a bovine-dog cycle and consequently represents a reservoir of sheep strain in Tunisia. Our results confirm the importance of the prophylaxis measures in order to disrupt the cycle of transmission sheep-dog in Tunisia. Nevertheless, the supervision of bovine infection should be reinforced because this intermediate host may constitute an important link with the human contamination.     

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.     

Isolation of two lipoprotein antigens from the metacestodes of Taenia hydatigena (Pallas, 1766) and Taenia multiceps (Leske, 1780) and their evaluation in sero-diagnosis

Two identical host contaminant-free lipoprotein antigens were isolated from the metacestodes of T. hydatigena and T. multiceps, of which antigen 1 was more reactive than antigen 2. These antigens appear to be identical with antigens B and A, respectively, and antigen 2 with arc 5 of Echinococcus granulosus described by other workers. Attempts to use these antigens in serodiagnosis showed that only in the indirect haemagglutination (IHA) technique was there any increased sensitivity and specificity compared with the unpurified cyst fluid antigens. No correlation was found between the size of the infecting dose, the number of cysts found at necropsy, the IHA titres, or number of precipitation lines seen in immunodiffusion tests.