恶性疟原虫ApiAP2蛋白质家族研究进展

疟疾是由顶复门寄生虫——疟原虫引起的一种人畜共患病。疟原虫不仅可以感染人，还能够感染家禽、鼠、食蟹猴等多种动物，其中，鸡疟原虫病、鼠疟原虫病对畜牧业影响较大。为完成其复杂的生命周期，疟原虫需要严格的基因调控，而转录因子对基因表达的调控作用十分关键。截至目前，ApiAP2 蛋白质家族是在所有顶复门原虫中作为候选转录因子出现的唯一蛋白质家族。论文在总结已有研究的基础上，预测恶性疟原虫ApiAP2 蛋白质家族中未知功能蛋白质的调控机制，并开展对ApiAP2 蛋白质家族发生蛋白质翻译后修饰功能的展望，为畜牧业研发原虫病疫苗提供借鉴与参考。     

顶复门原虫钙依赖蛋白激酶的研究进展

钙依赖蛋白激酶(Calcium-dependent protein kinases,CDPKs)是一类大的蛋白激酶家族,属于丝氨酸/苏氨酸类蛋白激酶,广泛存在于各种植物和原生动物中,参与多种生命活动的调控.随着生物信息学、分子生物学及基因工程的迅速发展,对顶复门原虫CDPKs的研究日益增多.研究结果表明,这类蛋白家族成员参与调控寄生虫入侵、外出宿主细胞、配子形成、宿主体内移行等顶复门原虫生活史的多个重要时期,其特殊的分子结构或可成为研究抗寄生虫疫苗或药物的候选靶标.本文以疟原虫、弓形虫和艾美耳球虫CDPKs为重点,综述了顶复门原虫CDPKs的结构、功能及生物学意义,展望了顶复门原虫CDPKs的研究和应用前景,以期为研究顶复门原虫的致病机理及研发抗原虫生物制剂提供参考.     

顶复门原虫多胺合成研究进展

多胺是一类聚阳离子脂肪族化合物,广泛存在于生物体内,其在顶复门原虫的生长、发育和繁殖等多个阶段发挥着重要作用。顶复门原虫的多胺合成途径存在多样性,其与宿主细胞的经典路径不同,且顶复门原虫多胺生物合成限速酶(鸟氨酸脱羧酶与S-腺苷甲硫氨酸脱羧酶)的半衰期与宿主细胞限速酶的半衰期存在显著差异。因此,顶复门原虫多胺合成限速酶及转运载体成为抗原虫药物研发的候选靶标,以顶复门原虫多胺合成限速酶及多胺转运载体为靶标的抗寄生原虫抑制剂研究取得一定进展。论文综述了顶复门原虫多胺的生物学功能、生物合成途径及其抑制剂研究进展,以期为抗原虫药物靶标研究及新型抑制剂研发提供参考。     

顶复门原虫醛缩酶的生物学功能与应用进展

顶复门原虫是一类专一性细胞内寄生原虫,不仅危害畜禽的健康,有些种类还是人兽共患病原,威胁人类的健康。醛缩酶是一类可逆醇醛裂合酶,根据作用机理可分为Ⅰ型与Ⅱ型醛缩酶。研究发现,顶复门原虫存在Ⅰ型醛缩酶,它们主要参与顶复门原虫的能量代谢、虫体黏附及入侵宿主细胞等过程,或可成为抗原虫药物研发的候选靶标。论文主要对顶复门原虫醛缩酶的生物学功能与应用研究现状进行综述,以期为顶复门原虫感染的防控提供参考。     

应用CRISPR/Cas9敲除HeLa细胞DDX21基因对新城疫病毒复制的影响

解旋酶家族是一类对所有生物都至关重要的酶,主要功能是基因解旋,DEAD/H-box家族RNA解旋酶是一类重要的解旋酶。DEAD-box RNA解旋酶DDX21被证实可能调控宿主细胞先天性免疫。新城疫病毒(NDV)是一种对养禽业危害严重的病原,NDV能够通过多种手段调控宿主先天性免疫,为了探讨DDX21如何调控NDV复制,本研究应用CRISPR/Cas9系统建立DDX21基因敲除的HeLa细胞系,并验证其对NDV复制的影响。针对DDX21基因共设计6条sgRNA,构建敲除载体并电转染细胞,通过药物筛选、亚克隆筛选后,以PCR和Western blot进行敲除效率的双重鉴定,并比较野生型和DDX21敲除细胞中NDV的复制效率。结果显示,由于DDX21对细胞生长至关重要,因此仅获得一株DDX21杂合子敲除细胞,Western blot结果显示DDX21表达量被显著抑制,NDV对在敲除细胞上的复制滴度明显低于野生型细胞,说明DDX21发挥正调控NDV复制的作用,本试验为DDX21调控抗病毒先天性免疫研究奠定了基础。     

寄生虫半胱氨酸蛋白酶抑制剂的研究进展

半胱氨酸蛋白酶抑制剂是一类在进化相关的蛋白质,能够抑制半胱氨酸蛋白酶类的活性。根据氨基酸序列和一级结构特征,将半胱氨酸蛋白酶抑制剂分为stefins家族,cystatins家族,kininogens家族和fetuin家族四个亚家族。本文通过结合半胱氨酸蛋白酶抑制剂功能的研究进展,对吸虫、绦虫、线虫和原虫半胱氨酸蛋白酶抑制剂的结构、种类、免疫调节和免疫逃避功能进行综述,以期对寄生虫半胱氨酸蛋白酶抑制剂的应用和深入研究提供参考。     

顶复门原虫电子转移链代谢及Ⅱ型NADH脱氢酶研究进展

顶复门原虫是包括刚地弓形虫、疟原虫及球虫等在内的一大类寄生性原虫的总称,可引起重要的人畜寄生虫病.抗顶复门原虫药物的长期使用,甚至是滥用,使得这类寄生虫对现有药物产生了明显的抗药性,急需开发新型药物.Ⅱ型NAD(P)H脱氢酶是电子转移链途径中的关键酶,由于其仅存在于某些植物、细菌、真菌和寄生原虫等一些低等生物体内,而在高等动物体内缺失,是研发新型抗感染性药物的重要靶标.笔者主要针对顶复门原虫线粒体电子转移链代谢途径以及Ⅱ型NAD(P)H脱氢酶的研究概况进行综述.     

发酵乳中沙门氏菌依赖解旋酶恒温基因扩增快速检测方法的建立

建立依赖解旋酶恒温基因扩增（helicase-dependent isothermal DNA amplification,HDA）快速检测发酵乳中沙门氏菌方法。以沙门氏菌invA基因序列为目的基因,设计特异性引物,优化反应体系中UvrD解旋酶及T4 gp32添加量,建立最优反应体系。通过HDA方法直接检测发酵乳中沙门氏菌,扩增其产物后进行电泳检测,验证方法特异性。结果表明：采用HDA快速检测法检测发酵乳中沙门氏菌特异性良好,优化后反应体系体积50 μL时,UvrD解旋酶添加量为0.10 μg,T4 gp32添加量为5.0 μg,得到与设计序列长度（304 bp）一致的扩增产物,检出限为2.6×102 CFU/g;该方法用于快速检测发酵乳中沙门氏菌能够满足检测需求,具有较高的灵敏度、易操作,可作为一种基础且快速的方法检测发酵乳中沙门氏菌。     

顶复门原虫棒状体蛋白疫苗候选分子的研究进展

顶复门原虫是一类专一性的胞内寄生性原虫,其种类丰富、分布广泛,包括艾美耳球虫、巴贝斯虫、隐孢子虫、疟原虫和弓形虫等.此类原虫具有保守的棒状体、致密颗粒以及微线等顶端复合器结构,这些细胞器可分泌大量的入侵相关蛋白分子介导虫体入侵宿主细胞.而棒状体蛋白是这类原虫在入侵过程中由棒状体分泌的一类蛋白,其在虫体入侵宿主细胞、纳虫...     

自噬核心蛋白Beclin 1在动物生殖生理中的作用研究进展

自噬是一种进化上高度保守的自我消化过程，是维持细胞内稳态所必需的。Beclin 1(Becn1)是酵母自噬相关基因6(ATG6)的同源物，Beclin 1蛋白在自噬中起核心作用，与多种因子相互作用，介导自噬蛋白定位于自噬前体结构而推进自噬进程。Beclin 1的功能障碍会导致动物生育力下降等多种疾病，且Beclin 1与生殖系统肿瘤发生也存在多种联系。本文综述了Beclin 1在动物生殖生理以及生殖系统恶性疾病中的研究进展，讨论了Beclin 1对于维持生殖系统健康的重要性，为提高动物繁殖力及治疗人类繁殖障碍提供参考。     

酸乳中葡糖醋杆菌赖解旋酶恒温基因扩增检测方法的建立

建立酸乳中葡糖醋杆菌的赖解旋酶恒温基因扩增(helicase-dependent isothermal DNA amplification,HDA)检测方法。根据葡糖醋杆菌16S基因序列(基因号为HQ677466.1)设计特异性引物,并优化反应体系中UvrD解旋酶和T4 gp32的浓度。通过HDA法直接检测酸乳中的葡糖醋杆菌,并确定其检出限,在建立的HDA体系中对酸乳中多种菌株进行扩增和电泳检测,验证方法的特异性。结果表明:用HDA法检测酸乳中葡糖醋杆菌,得到了与设计序列长度(101 bp)一致的基因片段,检出限为102 CFU/g,反应体系中UvrD解旋酶和T4 gp32的适宜添加量分别为0.1 μg和5.0 μg。该方法用于检测酸乳中葡糖醋杆菌的灵敏度高、耗时短。     

Neospora-like protozoal infections associated with bovine abortions

Eighty bovine fetuses with presumed protozoal infections from a previous 2-year retrospective study were examined by immunohistochemistry using antisera against Neospora caninum. In 66 (83%) of the fetuses, protozoa were found that reacted positively with anti-N. caninum sera. In three (4%) additional fetuses, protozoa identified as Sarcocystis species did not react, and in two fetuses (3%) single protozoal clusters were found only in hematoxylin and eosin-stained slides. A group of 20 fetuses were chosen for further evaluation. They included 14 fetuses from the first group of 80 fetuses plus six additional fetuses that had large numbers of protozoa in the fetal brain. The 20 fetuses were examined immunohistochemically with antisera to N. caninum, Hammondia hammondi, and Toxoplasma gondii. Protozoa from 3/20 fetuses, identified as Sarcocystis species, failed to react with any antisera. In 16/20 fetuses protozoa reacted positively to antisera against N. caninum, and in most cases reacted to H. hammondi, and weakly to one or more of the antisera against T. gondii. Thick-walled protozoal tissue cysts were found in the brain of four of these 16 fetuses by transmission electron microscopy. The cyst wall morphology was comparable to N. caninum. The results suggested that a single protozoal parasite of unknown identity was responsible for most of the bovine abortions. By immunohistochemistry, the unknown protozoon reacted most strongly and consistently to N. caninum antisera, but was antigenically distinct from N. caninum. Ultrastructurally, tissue cysts found in four fetuses most closely resembled Neospora caninum.     

快速检测发酵乳中葡萄糖杆菌的赖解旋酶恒温基因扩增方法的建立

建立赖解旋酶恒温基因扩增（helicase-dependent isothermal DNA amplification，HDA）快速检测发酵乳中葡萄糖杆菌的方法。选择葡萄糖杆菌ITS基因序列（基因号为KF896260.1）为目的基因，设计特异性引物，并优化反应体系中UvrD解旋酶和T4 gp32的添加量，建立最优反应体系。采用建立的HDA体系对多种菌株进行扩增和电泳检测，验证方法特异性，通过HDA法直接检测发酵乳中的葡萄糖杆菌，并确定其检出限。结果表明：采用HDA法检测发酵乳中葡萄糖杆菌，反应体系中UvrD解旋酶和T4 gp32的适宜添加量分别为0.10 μg和5.0 μg，扩增产物与设计序列长度（309 bp）一致，特异性良好，检出限为4.3×101 CFU/g。该方法用于检测发酵乳中葡萄糖杆菌的灵敏度高、耗时短。     

Advances in diagnosis of protozoan diseases

Microscopy still remains the gold standard procedure for the diagnosis of many protozoan infections in animals, but the specific identification requires skilled and experienced personnel. Immunoassays, detecting antibodies or specific protozoan antigens, have been developed but often lack sensitivity and specificity due to close relationship between many protozoa. Recent research has focussed almost exclusively on molecular based techniques for the identification and quantification of parasite DNA in samples. Opinion differ on most appropriate targets to use and there are very few diagnostic kits available making comparison between laboratories difficult. Future research needs to focus on robust, cheap field diagnostic assays.     

Prevalence and molecular characterization of Giardia duodenalis and Cryptosporidium spp. in dairy cattle in Ontario, Canada

Giardia duodenalis and Cryptosporidium spp. are intestinal protozoan parasites that infect a wide range of host species, including humans. Molecular characterization of these parasites has demonstrated that a number of genotypes and species are common to both humans and animals, and that zoonotic transmission may occur. Numerous studies have reported a high prevalence of G. duodenalis and Cryptosporidium spp. in cattle, particularly calves, and these animals are frequently associated with zoonotic transmission. In the present study, a total of 143 faecal samples from adults, heifers and calves were collected from two dairy cattle farms in eastern Ontario, Canada. The prevalence and molecular characteristics of G. duodenalis and Cryptosporidium spp. in these animals were determined in order to investigate the potential for transmission between cattle and humans in this region. Following DNA extractions from faecal samples, nested-PCR protocols were used to amplify fragments of the 16S rRNA gene and the heat-shock protein 70 (HSP-70) gene for determining the prevalence of G. duodenalis and Cryptosporidium spp. infections, respectively. Genotypes of G. duodenalis, and species of Cryptosporidium, were determined by means of DNA sequencing of amplicons, and subsequent sequence alignment. Cattle on both farms showed a high prevalence of G. duodenalis (42.0%) and Cryptosporidium spp. (27.3%). G. duodenalis infections were more prevalent in calves and heifers than in adults, and Cryptosporidium spp. infections were only observed in calves and heifers. The zoonotic genotype, G. duodenalis Assemblage B was isolated from 24.5% of the cattle tested, while G. duodenalis Assemblage E was found in 17.5% of the cattle tested. The overall prevalence of the zoonotic species Cryptosporidium parvum in the animals tested was found to be 21.7%, while only 1.4% were infected with C. bovis. These findings suggest that there is a potential risk of zoonotic and/or zooanthroponotic transmission of G. duodenalis and C. parvum infections between cattle and humans in eastern Ontario, likely by means of contaminated water or food, or through direct faecal-oral transmission in the case of farmers and veterinary staff.     

Prevalence of agglutinating antibodies to Sarcocystis neurona in raccoons, Procyon lotor, from the United States.

Equine protozoal myeloencephalitis (EPM) is the most important protozoal disease of horses in North America and it is caused by Sarcocystis neurona. Natural cases of encephalitis due to S. neurona have been reported in raccoons, Procyon lotor. We examined 99 raccoons for agglutinating antibodies to S. neurona using the S. neurona agglutination test (SAT) employing formalin-fixed merozoites as antigen. Raccoons originated in Florida (N=24, collected in 1996), New Jersey (N=25, collected in 1993), Pennsylvania (N=25, collected in 1999), and Massachusetts (N=25, collected in 1993 and 1994). We found that 58 (58.6%) of the 99 raccoons were positive for antibodies to S. neurona using the SAT; 44 of 99 raccoons (44%) had titers of ≥1:500. This prevalence is similar to the reported seroprevalence of 33–60% for S. neurona antibodies in horses from the United States using the Western blot test.     

Technical note: Specific PCR amplification of protozoal 18S rDNA sequences from DNA extracted from ruminal samples of cows

A protozoa-specific primer (P-SSU-342f) was designed and paired with a eukarya-specific primer to amplify a 1,360-bp fragment of DNA encoding protozoal small subunit (SSU) ribosomal RNA from ruminal fluid of cows fed a mixed forage:grain diet or alfalfa hay. Sequencing of clones showed that P-SSU-342f is specific to ruminal protozoa and, with slight modifications, the primer will be useful for ecological studies of ruminal protozoa.     

水源性原虫病流行病学与分子检测研究进展

水源性原虫病是由经水传播的原虫所引起的一类寄生虫病。近年来,由水污染原虫造成大量疾病的爆发引起了对水源性原虫病的高度重视。本文就近年来国内外对隐孢子虫、贾第虫和环孢子虫等水源性原虫病流行病学与分子检测研究进展作 一概述。