基于TaqMan qPCR检测凡纳滨对虾样品中虾肝肠胞虫并样检测方法的评价

对山东海阳和潍坊的2个养殖凡纳滨对虾群体取样，采用TaqMan qPCR逐尾检测肝胰腺中的虾肝肠胞虫(Enterocytozoon hepatopenaei, EHP)载量，再将提取的DNA样品按5并1(5∶1)、25并1(25∶1)、50并1(50∶1)、100并1(100∶1)和150并1(150∶1)进行并样，检测并样的EHP载量。设定不同临界循环数为假定灵敏度，定性判断各单尾检测阳性及并样组阳性，比较不同并样模式与检测阳性率、诊断灵敏度、诊断特异性等之间的关系以及定量的准确性。结果表明，检测灵敏度过低，会降低高并样率检测的准确性；阳性率在30%以上时，高并样率的检测结果与单样品检测相符性很好；高载量感染的阳性率不低于6.7%时，50∶1以内的并样能准确得出检测结果；低载量感染的阳性率不低于16%时，25∶1以内的并样能得出较好结果；高载量感染的1.3%阳性率和低载量感染的8%阳性率可能导致所有并样出现假阴性结果；各种并样模式均有很好的诊断特异性，50∶1并样的诊断灵敏度与OIE标准推荐的5∶1并样的接近；各并样检测的EHP载量与单样品检测平均值之比在0.27~2.83范围，二者存在极显著相关性，各种并样模式的定量检测结果在数量级水平能大致反映样品的平均EHP载量。本研究为水生动物疫病诊断和流行病学调查的样品检测提供了参考依据。     

长春地区梅花鹿毕氏肠微孢子虫分子流行病学调查

为了掌握长春地区梅花鹿毕氏肠微孢子虫(Enterocytozoon bieneusi)的感染率及基因型种类的分布情况,探究其在人与动物之间传播的源由,本研究于2018年12月-2019年12月期间采集长春地区8个养殖场共计538份梅花鹿粪便样品进行毕氏肠微孢子虫的流行病学调查及基因型分析,经PCR鉴定,测序分析、序列拼接比对以及系统进化分析。试验结果表明,长春地区梅花鹿毕氏肠微孢子虫的感染率为17.84%(96/538),不同养殖场的感染率为0~47.96%;研究共发现14种基因型(BEB6、EbpC、Ⅰ、JLD-Ⅲ、JLD-Ⅸ、JLD-ⅩⅤ、JLD-ⅩⅥ、JLD-ⅩⅦ、JLD-ⅩⅧ、JLD-ⅩⅨ、JLD-ⅩⅩ、JLD-ⅩⅪ、JLD-ⅩⅫ和JLD-ⅩⅩⅢ),其中BEB6为优势基因型。应用多位点序列分型(MLST)方法,选择MS1、MS3、MS4以及MS7位点对毕氏肠微孢子虫阳性样本进行多态性和群体遗传结构分析,结果显示,在96份阳性样品中,共有16份阳性分离株在至少2个位点同时扩增成功,形成10个多位点基因型(multilocus genotypes,MLGs),相同的基因型之间存在不同亚型,表明长春地区梅花鹿毕氏肠微孢子虫具有丰富的遗传多样性。本研究应用分子生物学方法对长春地区梅花鹿毕氏肠微孢子虫的流行病情况及基因型进行分析,研究结果不仅为梅花鹿毕氏肠微孢子虫防控提供科学依据,也为防控毕氏微孢子虫在人和动物间的传播提供基础数据,对保障公共卫生与健康意义重大。     

Microsporidiosis: Enterocytozoon bieneusi in domesticated and wild animals

Microsporidia are a ubiquitous group of obligate intracellular parasites that infect all major animal groups. Enterocytozoon bieneusi is the most commonly identified Microsporidia in humans and has also been reported worldwide in animals with importance in veterinary medicine (e.g. cats, dogs, horses, cattle and pigs). The identification of E. bieneusi in animals has raised the question of the importance of animal reservoirs in the epidemiology of this pathogen, and the implications of the infection with this pathogen in infected animals. Considerable genetic diversity within E. bieneusi has been found with over 90 genotypes identified based on the ITS nucleotide sequence of E. bieneusi spores recovered from the feces of infected humans and animals. Both host-adapted E. bieneusi genotypes with narrow host ranges and potentially zoonotic genotypes with wide host specificity have been identified. The information presented in this review should be useful in understanding the taxonomy, epidemiology, zoonotic potential, and importance in public health of E. bieneusi.     

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。     

Biochemical changes and tissue distribution of Enterocytozoon hepatopenaei (EHP) in naturally and experimentally EHP‐infected whiteleg shrimp,Litopenaeus vannamei (Boone, 1931), in India

Stunted growth in pond‐reared Litopenaeus vannamei was observed in different farms located in Tamil Nadu and Andhra Pradesh, India. No mortality was associated with stunted growth. PCR assay on these samples revealed the presence of Enterocytozoon hepatopenaei (EHP) in stunted shrimp. Tissue distribution of EHP in naturally and experimentally infected shrimp was studied by PCR and histology. Histological examination revealed the presence of EHP in hepatopancreas and gut, but not in other organs. The PCR assay revealed the presence of EHP in all the organs tested in both naturally and experimentally infected shrimp. Healthy shrimp were challenged with E. hepatopenaei by intramuscular injection and oral route, and no mortality was observed in both routes after 30 days post‐challenge. Different developmental stages of the microsporidian parasite were observed in the hepatopancreatic epithelial cells. Biochemical parameters such as total protein, albumin, aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase were measured in the haemolymph of naturally and experimentally EHP‐infected shrimp. All biochemical parameters mentioned were found to be significantly higher in EHP‐infected shrimp when compared to normal shrimp. This is the first report relating AST and ALT levels to EHP infection in naturally and experimentally infected shrimp.     

Cryptosporidium hominis Subtypes and Enterocytozoon bieneusi Genotypes in HIV‐Infected Persons in Ibadan,Nigeria

Cryptosporidium and Enterocytozoon are common opportunistic pathogens in HIV+ patients in developing countries, especially those do not have access to antiretroviral therapy. To determine the distribution of genotypes/subtypes of Cryptosporidium and Enterocytozoon bieneusi, faecal specimens were collected from 132 HIV+ persons attending a tertiary hospital in Ibadan, Nigeria. By polymerase chain reaction, eight and ten patients were identified as positive for Cryptosporidium spp. and E. bieneusi, respectively. Seven of the Cryptosporidium specimens were identified as C. hominis, while the remaining one as the new species C. viatorum recently identified in the United Kingdom. DNA sequencing of the 60‐kDa glycoprotein gene showed that the C. hominis belonged to three common subtype families: Ia (in three patients), Ib (in one patient) and Ie (in one patient). In contrast, DNA sequencing of the E. bieneusi internal transcribed spacer products showed the occurrence of genotypes associated with both humans (Peru 8 in one patient, Nig2 in two patients and a new genotype in one patient) and animals (D in one patient and Type IV in five patients). Low CD4+ cell count was identified as a risk factor for both cryptosporidiosis and microsporidiosis.     

凡纳滨对虾感染虾肝肠胞虫的群体及组织间差异性分析

对来自河北黄骅(HH)、山东平度(PD)、江苏吴江(WJ)和山东日照(RZ)的4个凡纳滨对虾(Litopenaeus vannamei)群体进行了对虾生长参数测量,用Taq Man q PCR检测了凡纳滨对虾各群体的肝胰腺组织中和RZ群体多种组织中的虾肝肠胞虫数量(Amount of Enterocytozoon hepatopenaei,EHP)。结果显示,在主要生长相关参数中,RZ群体最优,该群体EHP载量也最低。不同群体的样本数EHP对数直方图的模式存在差异,HH和PD群体的EHP对数呈双峰分布,而WJ和RZ群体的EHP对数呈单峰分布,代表EHP在不同群体中可能存在不同的传播模式。EHP对数呈单峰分布的群体或从多峰分布的群体中分离出的高EHP对数子群体的对虾体长或体重与EHP对数呈显著的负相关。RZ群体中,各个体不同组织中EHP从高到低的顺序依次是肝胰腺中肠血淋巴鳃肌肉。肝胰腺、中肠和鳃3个组织中EHP对数相互间的相关性为99.9%的极显著水平(P0.001);除了中肠与血淋巴和肝胰腺与血淋巴以外,其余组织间EHP对数的相关性也达到极显著(P0.01)或显著(P0.05)水平。用DIG标记的EHP探针对肝胰腺、肌肉、鳃、肠道组织的原位杂交显示,肝胰腺是主要的EHP感染组织,其他组织中杂交信号较弱,但各组织中有少数细胞的EHP易感。     

虾肝肠胞虫水通道蛋白基因 EHP00_492 的克隆与表达特征分析

【目的】克隆虾肝肠胞虫（Enterocytozoon hepatopenaei,EHP）水通道蛋白编码基因 EHP00_492,分析其在 EHP 成熟孢子中的表达特征,为研究 EHP 水通道蛋白的功能提供理论基础。【方法】以虾肝肠胞虫为材料,克隆获得完整的 EHP00_492 基因,对其进行序列特征分析,构建 pCold-TF-EHP00_492 重组表达载体,转化至大肠杆菌中异源表达重组蛋白,制备兔多克隆抗体。利用蛋白免疫印迹实验和间接免疫荧光实验分析 EHP00_492 在 EHP 成熟孢子中的表达定位特征。 【结果】 EHP00_492 的编码基因全长 729 bp,由 242 个氨基酸组成,富含亮氨酸,预测分子量为 25 kD,无信号肽,含有 6 个跨膜结构域,具有 MIP 蛋白家族结构域和 2 个水通道蛋白保守的 NPA/G 基序。此外,EHP00_492 与其他微孢子虫水通道蛋白序列同源性较高,系统进化树分析结果显示,EHP00_492 与毕氏肠胞虫 EBI_27080 蛋白亲缘关系最近。AlphaFold 分析发现,EHP00_492与已鉴定的家蚕微孢子虫水通道蛋白 NbAQP 和兔脑炎微孢子虫水通道蛋白 EcAQP 的蛋白质三维结构高度相似,推测 EHP00_492 是一个潜在的水通道蛋白。蛋白免疫印迹结果显示,EHP00_492 蛋白在 EHP 成熟孢子中有表达,分子量为 21 kD。亚细胞定位分析结果显示,EHP00_492 蛋白定位于 EHP 成熟孢子的孢壁上。【结论】初步明确了 EHP00_492 蛋白的序列特征、结构特征及其与其他微孢子虫水通道蛋白的亲缘关系,以及其在 EHP 成熟孢子中的表达定位特征,可为进一步研究 EHP 水通道蛋白的功能提供基础。     

EHP、SHIV双重TaqMan实时荧光定量PCR检测方法的构建及应用

本研究根据GenBank中已有的虾肝肠胞虫(EHP)和虾血细胞虹彩病毒(SHIV)基因的保守序列,设计特异的引物和探针.建立了快速诊断EHP和SHIV的双重TaqMan实时荧光定量PCR检测方法,并对其特异性、敏感性和稳定性进行检测.结果表明:该方法检测限可达10 copies/μL,其敏感性是SYBR Green r...     

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。