湖南省部分地区中华竹鼠毕氏肠微孢子虫基因分型研究

为了明确湖南省部分地区中华竹鼠毕氏肠微孢子虫的感染情况,从湖南省4个市的6个养殖场共采集中华竹鼠新鲜粪便样品480份,利用小亚基核糖体RNA(SSU rRNA)基因的套式PCR对这些样品中毕氏肠微孢子虫的感染率和遗传多样性进行研究。结果发现,25份粪便样品存在毕氏肠微孢子虫感染,总感染率为5.2%,除吉首市的中华竹鼠未检出阳性外,其余各采样点及各年龄段的中华竹鼠均有毕氏肠微孢子虫感染,但感染率差异不显著(P0.05);进一步序列分析发现,所检测的中华竹鼠毕氏肠微孢子虫存在2种SSU rRNA基因型(Type 1和Type 2),其中Type 1为优势基因型;多位点序列分析发现,在MS1、MS3、MS7基因位点分别有7、1、14份样品成功扩增,呈现7、1、4个单倍型。本研究结果将为湖南省部分地区中华竹鼠微孢子虫病的防控提供基础数据。     

藏香猪毕氏肠微孢子虫感染情况和基因分型

采集了青海省和陕西省部分地区共计450份藏香猪新鲜粪便样品,基于小亚基核糖体RNA基因(small-subunit ribosomal RNA,SSU rRNA)的巢式PCR检测样品中毕氏肠微孢子虫(Enterocytozoon bieneusi)的感染情况,并分析其遗传多样性。结果显示,218份样品感染了毕氏肠微孢子虫,总感染率为48.4%(218/450);青海省所调查藏香猪的毕氏肠微孢子虫感染率为65.8%(154/234),显著高于陕西省的感染率29.6%(64/216)(P0.001);4个年龄段间藏香猪毕氏肠微孢子虫感染率差异显著(P0.001),其中哺乳仔猪感染率最高(92.7%),而成年猪感染率最低(9.2%)。序列分析发现,藏香猪存在5个SSU rRNA基因型(Type 1～Type 5),其中Type 1为优势基因型。对218份毕氏肠微孢子虫阳性分离株的多位点序列分型发现,分别有59,26,9和99份样品在MS1、MS3、MS4和MS7基因位点成功扩增,呈现18,4,4和4种单倍型,基于MS1、MS3和MS4等3个基因位点共形成6种多位点基因型(multi-locus genotypes,MLGs)。     

秦岭地区几种珍稀动物毕氏肠微孢子虫基因分型

为了评估秦岭地区珍稀野生动物的毕氏肠微孢子虫(Enterocytozoon bieneusi)的人兽共患风险,本研究采用PCR技术和多位点序列分型技术对秦岭地区5种珍稀动物毕氏肠微孢子虫的感染情况及基因型/亚型进行了分析。在采集的22份新鲜粪便中检测到7个阳性分离株,其中斑羚源5个,长颈鹿源1个,麂子源1个。基于ITS位点基因分型发现所有分离株均为人兽共患基因型,包括6个已报道的基因型D和1个尚未报道的D-new。多位点序列分型技术发现秦岭地区珍稀野生动物的毕氏肠微孢子虫存在遗传多样性,7个分离株在MS1、MS3、MS4和MS7位点分别有3、1、2、2个亚型。     

云南省怒江州部分地区独龙牛毕氏肠微孢子虫感染情况及基因分型研究

为了解云南省怒江州独龙牛毕氏肠微孢子虫感染情况,从怒江州的鸠门当、古泉村、亚左洛村、茨开镇、独龙江乡分四个季度(春、夏、秋、冬)采集独龙牛粪便样本1129份,采用分子生物学技术对其毕氏肠微孢子虫感染情况进行调查研究。结果发现,所调查地区独龙牛毕氏肠微孢子虫总感染率为1.68%(19/1129);各地点之间感染率差异显著(P<0.05),以独龙江乡感染率最高;4个季度中夏季感染率最高3.26%(9/276),春、秋和冬感染率分别为1.10%(3/272)、1.28%(4/312)和1.12%(3/269),但差异不显著(P>0.05)。序列分析发现,在19个阳性样本中成功鉴定出4种已知基因型(EbpC、BEB4、CHN3和CHN4)及2种新基因型(YNNJ1和YNNJ2),丰富了毕氏肠微孢子虫基因型数据。系统发育分析表明,所得6种基因型均为人兽共患基因型,提示怒江州独龙牛毕氏肠微孢子虫存在人兽互传的潜在风险。     

云南省部分地区荷斯坦牛毕氏肠微孢子虫感染情况调查及分析

为了解毕氏肠微孢子虫在云南部分地区荷斯坦牛中的感染以及基因型分布情况，本实验从云南大理、昆明、曲靖和楚雄等牛养殖集中地区总共9个养殖场采集荷斯坦牛粪便样品547份，采用套式PCR结合测序检测并分析毕氏肠微孢子虫感染情况。结果显示，毕氏肠微孢子虫总感染率为4.57%(25/547)；各地区的感染率为0～10.53%，其中昆明地区养殖场感染率最高，不同地区间感染率差异显著(P=0.025<0.05)。断奶前犊牛感染率为6.32%(23/364)，高于其他月龄牛的感染率，但各月龄牛感染率差异不显著(P=0.052>0.05)。公牛感染率为7.84%(8/102)高于母牛3.82%(17/445)，性别间感染率差异不显著(P=0.079>0.05)。进一步通过对样品扩增产物测序分析后鉴定其基因型，结果显示本研究共鉴定出5个基因型，I、J、BEB4为3个已知的基因型和YNY1、YNY2 2个新拟定的基因型，其中BEB4为优势基因型。在不同地区中，大理检出4种基因型(BEB4、I、YNN1、YNN2)，昆明仅检出基因型BEB4，曲靖仅检出基因型J，大理地区基因型分布较为多样；在0...     

陕西部分地区山羊毕氏肠微孢子虫多位点基因分型研究

为了阐明山羊毕氏肠微孢子虫的遗传多样性,笔者采用基于微卫星(MS1、MS3、MS7)和小卫星(MS4)位点的多位点序列分型技术首次对不同用途山羊的122个毕氏肠微孢子虫分离株进行了多位点序列分型研究。结果发现,在MS1、MS4和MS7位点的扩增效率依次为27.9%(34/122)、18.0%(22/122)、50.8%(62/122),而在MS3位点所有样品均未获得有效扩增。核苷酸序列分析表明,所有样品的MS1、MS4和MS7位点分别具有16、9和18个基因型,共形成了14个MLGs。其中,50份绒山羊阳性样品3个位点扩增效率分别为10.0%(5/50)、14.0%(7/50)和90.0%(45/50),分别具有3、3和10个基因型,共形成了5个MLGs;56份奶山羊阳性样品3个位点的扩增效率依次为28.6%(16/56)、19.6%(11/56)和8.9%(5/56),分别组成4、4、1个基因型,共组成7个不同的MLGs;16份黑山羊阳性样品在3个位点的扩增效率依次为81.3%(13/16)、25.0%(4/16)、75.0%(12/16),分别具有9、2、7个基因型,构成2个MLGs。     

动物园动物消化道寄生虫感染情况调查

为了解动物园动物肠道寄生虫感染情况,本试验采集了来自贵阳和北京动物园33种动物,共150份粪便样品。采用饱和盐水漂浮法和离心沉淀法富集虫卵和卵囊进行显微镜检查;基于隐孢子虫、芽囊原虫、毕氏肠微孢子虫和十二指肠贾第虫的特异性基因位点对人兽共患原虫进行PCR检测和序列比对分析。结果显示,动物园动物寄生虫总感染率为64.0%。显微镜检查出的寄生虫包括毛首线虫(20.0%)、细颈线虫(2.7%)、艾美耳球虫(14.7%)和其他线虫(25.3%)。PCR检测4种肠道原虫的感染率分别为：隐孢子虫1.3%、毕氏肠微孢子虫8.7%、十二指肠贾第虫12.0%和芽囊原虫32.0%。序列比对结果显示,存在2种隐孢子虫虫种(安氏隐孢子虫和泛在隐孢子虫);7种毕氏肠微孢子虫基因型(SC02、BEB6、Type IV、PigEBITS 7、Peru8、PtEb IX和D);2种十二指肠贾第虫基因型(B和E);8种芽囊原虫基因亚型(ST1、ST2、ST3、ST5、ST8、ST10、ST13和ST14)。结果表明,贵州和北京动物园动物肠道寄生虫感染非常普遍,并且存在多种人兽共患虫种和基因型。做好动物园寄生虫病的防控不...     

元江县山羊肠道寄生虫的感染状况调查与分析

为了解元江县部分山羊肠道寄生虫感染状况,采用饱和食盐水漂浮法结合麦克马斯特氏法,对该县2个采样点6个不同养殖场中采集的168份山羊粪样进行了寄生虫检测及阳性粪样虫卵计数,并采用巢氏PCR方法对粪样中毕氏肠微孢子虫、芽囊原虫和隐孢子虫进行检测.结果显示,该地区山羊肠道寄生虫总感染率为44.64％,以线虫和球虫感染为主,球...     

马属动物毕氏肠微孢子虫研究进展

毕氏肠微孢子虫是一种引起人和动物腹泻的常见病原体,可通过食物和水源传播,具有重要的公共卫生意义。根据系统进化分析,已鉴定出近300个毕氏肠微孢子虫基因型,分为9个组群,组群1中的基因型大多数具有潜在人兽共患性;组群2~8中的基因型多数具有宿主特异性,部分也可感染人。研究表明,马属动物可感染37个毕氏肠微孢子虫基因型,具有一定人兽共患风险。论文就近年来感染马属动物的毕氏肠微孢子虫基因型、人兽共患风险、流行情况和致病性等进行综述。     

新疆南疆某规模化绵羊养殖场腹泻羔羊隐孢子虫感染情况及其分子鉴定

[目的]了解新疆南疆某规模化绵羊养殖场腹泻羔羊隐孢子虫感染情况和基因亚型分布特点。[方法]采集新疆维吾尔自治区某规模化绵羊养殖场4个品种1月龄以内腹泻羔羊新鲜粪便样本60份,使用饱和蔗糖溶液漂浮法检查隐孢子虫卵囊,进行种类初步鉴定;全部粪便样本提取基因组DNA后,基于隐孢子虫SSU rRNA基因位点和微小隐孢子虫gp60基因位点,对其进行PCR扩增、测序和序列分析,鉴定隐孢子虫种属和基因亚型,构建遗传进化树解析其分子遗传特征。[结果]经显微镜观察,发现38份样本呈隐孢子虫卵囊阳性,形态学初步鉴定为微小隐孢子虫;基于SSU rRNA基因位点,采用PCR方法检测出52份样本呈隐孢子虫阳性,感染率为86.67%(52/60),经序列比对分析,均为微小隐孢子虫;基于微小隐孢子虫gp60基因位点,PCR扩增后成功获得49条序列,经比对分析均为ⅡdA19G1基因亚型。[结论]该养殖场腹泻羔羊普遍感染微小隐孢子虫,其基因亚型均为ⅡdA19G1。调查结果为新疆南疆绵羊隐孢子虫种属鉴定与遗传进化研究提供了基础数据。     

First report of Enterocytozoon bieneusi infection on a pig farm in the Czech Republic

Enterocytozoon bieneusi infects humans and animals and can cause life-threatening diarrhea in immunocompromised people. The routes of transmission and its zoonotic potential are not fully understood. Pigs have been frequently reported to have E. bieneusi; therefore, we surveyed farm-raised pigs in the Czech Republic to determine its presence and genetic diversity. Spores were detected by microscopy in the faeces of 65 out of 79 examined animals (82%). A species-specific polymerase chain reaction (PCR) identified E. bieneusi in 94% of samples. Genotyping based on the ITS regions of the SSU rRNA gene identified that most pigs were infected with the species-specific genotype F, while two animals had the zoonotic genotype D and two had genotype Peru 9. This is the first report of E. bieneusi in swine in the Czech Republic, and demonstrated that most infections were with pig-specific genotypes. Nonetheless, swine may still play a role in the transmission of E. bieneusi to humans.     

Cryptosporidium, Giardia and Enterocytozoon bieneusi in cats from Bogota (Colombia) and genotyping of isolates

The prevalence of Cryptosporidium, Giardia, and Enterocytozoon bieneusi in cats from Bogota (Colombia) was determined from fecal specimens and scrapings of duodenal and ileal mucosa screened by PCR. All PCR-positive specimens were sequenced to determine the genotype(s) present. Of 46 cats, 6 (13%) were positive for Cryptosporidium, 5 (11%) were infected with C. felis and one (2%) with C. muris. Three (6.5%) cats were infected with Giardia duodenalis Assemblage F. Eight (17%) cats were infected with four genotypes of E. bieneusi: genotype D-like (9%), K (4%), Peru 10 (2%), and Peru 5 (2%). This is the first report on the presence of zoonotic species/genotypes of Cryptosporidium and E. bieneusi in cats in Colombia.     

First report of Enterocytozoon bieneusi from dairy cattle in Argentina

Fecal specimens were obtained from a total of 70 dairy calves less than two months old on 11 municipalities in Buenos Aires, Argentina. After removal of fecal debris by sieving and sucrose flotation, specimens were subjected to PCR to detect the presence of Enterocytozoon bieneusi. PCR revealed a 14.3% of prevalence for E. bieneusi with 10 positive calves from 7 municipalities. Gene sequence analysis conducted in all samples positives by PCR revealed the presence of six genotypes; four previously reported in cattle as well as humans (D, I, J, and BEB4), one never reported in cattle before but previously reported in humans (EbpC), and one novel genotype (BEB10). These results constitute the first molecular characterization of E. bieneusi in Argentina, and suggest a potential risk of zoonotic transmission in this area.     

Detection of nucleolar organizer regions on chromosomes by flourescence in situ hybridization with human 28S rRNA gene and cloning of 28S rRNA gene in Sika deer

The number and loci of nucleolar organizer regions (NOR) on chromosomes in Sika deer (Cervus nippon centralis) were determined by fluorescence in situ hybridization with a human 28S ribosomal RNA (rRNA) gene as a probe. Sika deer that live in Nikko National Park and its neighboring areas (Asio and Seta) in Japan were used. All of the analyzed metaphases had three or four NOR at the end of the first and second longest telocentric autosomes. Nucleolar organizer region association, which is associated specifically on parts of NOR between chromosomes, was also observed clearly. A Sika deer 28S rRNA gene was produced by a polymerase chain reaction method. The nucleotide sequence of a Sika deer 28S rRNA gene determined by an automatic sequencer was 97 bp, and showed homogeneity of 88% for the human sequence.     

Longitudinal identification of Enterocytozoon bieneusi in dairy calves on a farm in Southern Xinjiang,China

Enterocytozoon bieneusi is the most common species responsible for human and animals microsporidiasis. A total of 250 samples were collected weekly from 25 newborn dairy calves of a farm in Southern Xinjiang, China at one to ten weeks of age. Enterocytozoon bieneusi was identified and genotyped by nested PCR amplification and sequencing of internal transcribed spacer (ITS) region.The cumulative prevalence of E. bieneusi infection was 100% (25/25), and the average infection was 52.0% (130/250). The highest infection rate was recorded at six weeks of age (92.0%, 23/25), and no infection was observed at one and two weeks of age. Sequencing analysis showed nine E. bieneusi genotypes (J, EbpC, PigEBITS5, CHV4, CHC3, CS-9, KIN-1, CH5, and CAM5) were identified. The highest genetic polymorphism was observed at ten weeks of age. Genotype J was the predominant E. bieneusi genotype. Phylogenetic analysis clustered genotype J into Group 2 and other eight genotypes (EbpC, PigEBITS5, CHV4, CHC3, CS-9, KIN-1, CH5, and CAM5), detected in 22 (16.9%, 22/130) samples, into Group 1. Among the genotypes, EbpC, KIN-1, and J have been identified in humans. The highest E. bieneusi infection rate (57.9%, 124/214) was observed in fecal samples with formed feces with no diarrhea (p < 0.01), and high genetic polymorphism was observed in class I fecal samples. The presence of zoonotic E. bieneusi genotypes in dairy calves suggests the possibility of transmitting zoonotic infections to humans. It provides the basic data on dynamic change of E. bieneusi in calves.     

全国梅花鹿养殖状况调查

中国野生动物保护协会于2005年6～12月,在全国31个省(区、市)开展了梅花鹿养殖情况调查.调查数据显示,截止到2004年底,全国31个省(区、市)共有9 465家养殖场,圈养繁殖梅花鹿452 355头.其中养殖梅花鹿数量最多的是吉林省,养殖场5 985家,目前存栏量278 000头.2004年全国生产梅花鹿鹿茸73 t,吉林省居全国首位,年产量约为21 t,占总数的28.8%.本文以调查数据为基础,提出我国梅花鹿养殖中存在的问题和改进建议.     

Analysis of mitochondrial DNA protein-coding region in the Yeso Sika deer (Cervus nippon yesoensis)

In the present study, mitochondrial DNA sequences of the Yeso Sika deer (Cervus nippon yesoensis) were studied. Specifically, protein‐coding genes as mitochondrial NADH dehydrogenase subunits (ND1, ND2, ND3, ND4L, ND4, ND5 and ND6), cytochrome c oxidase subunits (CO I and CO III), ATP synthase subunits (ATPase8 and ATPase6) and cytochrome b. Also, phylogenetic analyses on eight mammalian species were performed, including the Muntjac deer (Muntiacus reevesi). The rate of amino‐acid substitution was lowest (3.74%) between Yeso Sika deer and Muntjac deer, and the values between Yeso Sika deer and other species (sheep, cattle, horse, pig, mouse, human and chimpanzee) were 6.63%, 7.30%, 12.55%, 13.03%, 23.59%, 24.82% and 25.04%, respectively. Among them, the highest value of divergence was recognized in ATPase8, and the second structure of ATPase8 showed a difference between the Yeso Sika deer and Muntjac deer as a result of the substitution of 34His→Tyr and 49Thr→Ile. In addition, we identified a substitution of an amino‐acid sequence (19Thr→Ala) between the Yeso Sika deer and Yakushima Sika deer (C. n. yakushimae). From these results, ATPase8 was also a variable region in Cervidae.     

吉林省梅花鹿副结核病、结核病及布鲁菌病血清流行病学调查

采集吉林省具有代表性的4个地区的8个梅花鹿场（四平2个梅花鹿场、通化2个梅花鹿场、长春市2个梅花鹿场、辽源2个梅花鹿场）中630份血清样本作为研究对象,进行血清学（ELISA_方法）检测,了解吉林省梅花鹿副结核病、结核病及布鲁菌病感染现状。上述4个地区均存在梅花鹿副结核病,结核病及布鲁菌病发生和流行,其中副结核病的整体阳性率为18．73％、结核病的整体阳性率为19．21％、布鲁病的整体阳性率为28．42％。吉林省梅花鹿副结核病、结核病及布鲁菌病流行不容乐观,提示广大饲养者及相关部门应该高度重视梅花鹿副结核病的防治工作。     

马鹿特异性SNP分子标记的验证

试验旨在对基于基因分型测序（genotyping by sequencing,GBS）技术筛选出的马鹿特异性SNPs位点的准确性进行验证,为梅花鹿、马鹿及其杂交后代的鉴别提供可靠的分子遗传标记。随机选取30个马鹿特异性SNPs位点,根据SNPs位点前后各200 bp的序列,利用Primer Premier 6.0软件设计特异性引物,以随机选取的验证样本DNA作为模板进行PCR扩增,并进行Sanger测序,对测序结果利用BioEdit软件进行峰图的观察,利用Mega 6.0软件对测序得到的序列进行比对分析并观察每个特异性SNP位点在不同验证群体中的基因型,对每一个马鹿特异性位点的峰图和比对信息进行统计分析。结果表明,30个马鹿特异性位点中有28个和前期研究结果一致,其中1个SNP位点（SNP3）中G等位基因在梅花鹿中的基因频率为0.05,而G等位基因在马鹿中的基因频率为1,G等位基因在马鹿个体中的基因频率比在梅花鹿个体中高,同时,利用SPSS 22.0进行统计分析发现,该位点在马鹿和梅花鹿中基因型分布表现出显著性差异（P<0.05）;另外1个SNP位点（SNP5）中T等位基因在梅花鹿的基因频率为0.15,而在马鹿中的基因频率为1,且这个SNP位点在梅花鹿和马鹿中基因型分布差异显著（P<0.05）,所以这2个位点仍然可以作为马鹿的特异性SNPs位点。研究结果说明了GBS测序筛选出的马鹿特异性SNPs可以作为鉴定的分子标记,对梅花鹿、马鹿及其杂交后代的鉴别奠定了理论基础。