一株家蚕异型微孢子虫感染特征和分类地位

【背景】家蚕微粒子病是一种对蚕业生产危害巨大的蚕病,该病病原是蚕种生产唯一检疫对象,而家蚕病原性微孢子虫种类多、来源复杂,给蚕种生产微粒子病的防控增加了难度。【目的】研究一株从蚕种检疫样品中分离的微孢子虫(命名为GXM15)的致病性和分类地位,鉴定并分析其来源,完善家蚕病原性微孢子虫分类和数据库,为蚕种生产控制家蚕微粒子病提供参考依据。【方法】采用生物试验方法测定GXM15微孢子虫对家蚕的半数感染浓度(IC50)和胚种传染率;显微镜观察GXM15微孢子虫孢子形态,利用透射电子显微镜观察GXM15微孢子虫的超微结构;采用PCR扩增、T克隆和测序获得GXM15微孢子虫的SSU rRNA基因和ITS片段DNA序列,并利用MEGA 5.0和DNAstar软件构建GXM15微孢子虫的系统发育树和遗传距离分析。【结果】GXM15微孢子虫对家蚕的IC50为8.29×104个/mL,是家蚕微孢子虫(Nosema bombycis,Nb)的2.28倍;GXM15微孢子虫对家蚕的胚种传染率为3.6%,明显低于Nb;GXM15微孢子虫形态呈短卵圆形,大小为(2.05±0.20)× (3.25±0.30) μm,GXM15微孢子虫体积是Nb微孢子虫的2.19倍;GXM15微孢子虫超微结构具双核,极丝13圈,极丝倾斜角约45°,符合Nosema属的特征;GXM15微孢子虫SSU rRNA基因在系统发育树中位于Nosema属分支中,遗传距离分析表明GXM15微孢子虫与Nb同属异种,是一株新微孢子虫。【结论】GXM15微孢子虫是一株家蚕病原性微孢子虫,根据GXM15微孢子虫致病性和分类地位研究,可以为蚕种生产防控家蚕微粒子病提供参考依据。     

Calcofluor White M2R荧光染色法识别家蚕微孢子虫

本研究探讨应用荧光染色试剂Calcofluor White M2R染色鉴别家蚕微孢子虫Nosema bombycis。结果表明：在荧光显微镜下可见家蚕微孢子虫孢子被染上强烈的青蓝色荧光, 而寄主组织碎片、病毒、细菌等不被染色。该法是一种快速有效鉴别微孢子虫的方法。     

家蚕微孢子虫在家蚕胚胎细胞系BmE-SWU1中的极丝弹出情况

本文借助扫描电镜(Scanning Electron Microscope,SEM)对在家蚕胚胎细胞(Bombyx mori-SWU1Embryoniccell line,BmE-SWU1)中增殖的家蚕微孢子虫(Nosema bombycis)进行了观察;同时,利用间接免疫荧光试验(Indirect Immunofluorescence Test,IFAT)对家蚕微孢子虫极丝弹出情况进行了研究。结果发现:在家蚕微孢子虫体外培养体系中观察到了二型孢子、发芽后的孢子空壳以及未成熟孢子等;此外,在接种后10d的细胞爬片中,发现了大量孢子弹出极丝的情况,其极丝的长度在58μm-120μm之间,其中100μm以上的居多,且其极丝的扭曲程度、方向性、粗细也大都不同。本研究表明了微孢子虫在培养细胞体系中自动发芽可以以长极丝孢子的形式来完成。     

梨花迁粉蝶微孢子虫对家蚕的侵染力与胚传性

【目的】对梨花迁粉蝶Catopsilia pyranthe分离的微孢子虫进行形态与分子鉴定,探究其对非天然宿主家蚕Bombyx mori的侵染力与胚传性。【方法】从田间采集的梨花迁粉蝶中分离得到梨花迁粉蝶微孢子虫液,测定其孢子的形态、大小、体积、长短轴比,同时对该孢子虫的16S r DNA进行PCR克隆测序与分析。将梨花迁粉蝶微孢子虫Nosema sp.CP与家蚕微孢子虫N.bombycis分别对2龄起蚕、4龄起蚕进行添食感染比对,测定家蚕食下两种微孢子虫的感染率和胚种传染能力。【结果】本研究分离的梨花迁粉蝶微孢子虫形态为长椭圆形,具双核;其16S r DNA序列与已报道的梨花迁粉蝶微孢子虫的序列一致性大于99%,为梨花迁粉蝶微孢子虫。梨花迁粉蝶微孢子虫和家蚕微孢子虫对家蚕综合感染率分别是68.8%和98.3%;在继代蚁蚕中,感染梨花迁粉蝶微孢子虫和家蚕微孢子虫的雌蛾所产蚕卵次代蚁蚕检出有孢子虫的检出率分别为100%和100%,卵壳的孢子虫的检出率分别为92.9%和100%;梨花迁粉蝶微孢子虫和家蚕微孢子虫对家蚕的胚种传染力分别为9.6%和23.2%。【结论】本研究分离得到的微孢子虫为梨花迁粉蝶微孢子虫,具有微孢子虫Nosema属的典型特征。梨花迁粉蝶微孢子虫能感染危害家蚕,也具有家蚕胚种传染性,但感染率和胚传率均明显低于家蚕微孢子虫,是蚕业生产中必须防控的对象。     

自桑兰叶甲分离出的一种微孢子虫(Mic-Ⅰ)的研究

从广州郊区罗岗的桑园及菜地捕捉到的桑兰叶甲Mimastra cyanura Hope成虫体中分离到一种卵圆形的微孢子虫（简称Mic-Ⅰ）。孢子大小为(3.35±0.46)×(1.96±0.17) μm;孢子具单核、双核两种类型;极丝10～11圈;孢子表面抗原的血清学类型与家蚕微粒子虫Nosema bombycis孢子不同;在家蚕Bombyx mori体内以两种不同的生活史发育,发育过程符合变态孢虫属Vairimorpha微孢子虫发育特征。Mic-Ⅰ微孢子虫对家蚕具强病原性,对斜纹夜蛾Prodenia litura和小菜蛾Plutella xylostella也有感染能力。     

自桑兰叶甲分离出的一种微孢子虫(Mic-Ⅰ)的研究

从广州郊区罗岗的桑园及菜地捕捉到的桑兰叶甲Mimastra cyanura Hope成虫体中分离到一种卵圆形的微孢子虫（简称Mic-Ⅰ）。孢子大小为(3.35±0.46)×(1.96±0.17) μm;孢子具单核、双核两种类型;极丝10～11圈;孢子表面抗原的血清学类型与家蚕微粒子虫Nosema bombycis孢子不同;在家蚕Bombyx mori体内以两种不同的生活史发育,发育过程符合变态孢虫属Vairimorpha微孢子虫发育特征。Mic-Ⅰ微孢子虫对家蚕具强病原性,对斜纹夜蛾Prodenia litura和小菜蛾Plutella xylostella也有感染能力。     

甜菜夜蛾微孢子虫研究:Ⅳ.孢子挤出器的超微结构

从甜菜夜蛾幼虫体内首次分离到一种侵染寄主脂肪体、马氏管和中肠 ,对甜菜夜蛾有很强致病力的微孢子虫 ,该微孢子虫新鲜孢子呈椭圆形 ,大小为 ( 3 98± 0 4 3 ) μm× ( 1 65± 0 3 3 ) μm (n =5 0 )。扫描电镜观察发现 ,孢子表面光滑 ,大小基本均匀一致。用透射电镜观察该微孢子虫孢子挤出器的超微结构 ,结果表明 :孢子的挤出器由极体、极丝及相关细胞器和后液泡三部分组成。极体位于孢子前端 ,约占据孢子 2 5 %～ 3 0 %的空间 ,由明暗相间、互相堆叠的片层结构所组成。纵切面上 ,极丝 11～ 13圈 ,单层螺旋状盘绕于孢子后端、孢原质的外层 ,如此典型的极丝切面在同类研究中尚未见报导。横切面上 ,可见极丝为同心管状结构 ,管壁由 6层明暗相间的同心层组成 ,极丝直径约 88 2～ 94 1nm ,极丝倾角在前端约为 5 5°～ 60° ,后端约 65°～ 70°。后液泡位于孢子后端 ,呈椭圆形 ,被一层膜结构包围 ,内含不规则颗粒状的内含体。此外 ,含有 1个极丝圈的初期孢子被发现。研究结果提示 ,在微粒子属 (Nosema)中 ,极丝的圈数、排列方式、极丝倾角的大小及微孢子虫在宿主细胞中的寄生部位等在微孢子虫种间存在着较大的差异 ,而在种内则相对稳定 ,这些超微结构水平上的形态学属性对微孢子虫种的鉴定具有重要价     

不同DNA抽提方法对普通PCR和实时荧光定量PCR方法检测家蚕微孢子虫的影响

为了优选快速、 灵敏、 特异的家蚕微孢子虫Nosema bombycis分子检测方法和DNA抽提方法, 本文通过对家蚕微孢子虫TaqMan探针荧光定量PCR检测方法和SYBR Green荧光定量PCR检测方法的建立以及反应体系优化, 并与普通PCR方法进行比较; 再采用4种不同DNA抽提方法分别对PCR和实时荧光定量PCR方法检测家蚕微孢子虫悬浮液的效果评价。结果显示: 不经过DNA抽提, 直接将家蚕微孢子虫发芽液进行PCR反应的效果优于其他方法, 检测灵敏度由高到低依次为直接法、 酚/氯仿抽提法、 动物组织DNA试剂盒抽提法和植物组织DNA试剂盒抽提法; TaqMan探针法检测家蚕微孢子虫发芽液的灵敏度和SYBR Green法相近, 达到微孢子10²个/mL, 两者均优于普通PCR方法。实验表明, 直接采用发芽液结合荧光定量PCR方法检测家蚕微孢子虫最为简便、 快速、 灵敏。该研究结果将有助于提高家蚕微粒子病监控技术和检疫能力, 对家蚕微粒子病的检疫和防治具有积极意义。     

一种短杆状耐辐射菌的分离与鉴定

从北京地区公园湖岸土壤中分离到一株橙红色杆状耐辐射菌,细胞壁革兰氏染色为阴性,电镜显示菌体大小为06μm～16μm,略大于日本学者报道的Deinobacter grandis菌,过氧化氢酶的含量和分子量不同于D.radiodurans R1菌,分离菌的(G+C)mol%含量为707%, 16S rDNA序列分析表明,分离到的杆状耐辐射菌(RR5332)16S rRNA基因序列与Deinobacter grandis菌高度同源,提示RR5332归于Deinobacter菌属,并可能是该菌属中的一个新种。     

抗真菌肽LP-1的分离纯化及特性分析

拮抗菌枯草芽孢杆菌(\%Bacillus subtilis)\%TG\|26分泌产生的小肽经两次盐酸沉淀、丙酮分级沉淀和Hi\|pore反相柱两次纯化,分离得到一种新的抗真菌的小肽,命名为LP\|1。经MALDI\|TOF质谱鉴定,分子 量为10573,等电聚焦测得其pI为475。LP\|1对温度有较高的稳定性,100℃保温30min,仍能保持75%的活性。抑菌谱表明,该抗菌肽对瓜果腐霉\%(Pythium aphanidermatum)\,玉蜀黍赤霉病菌(Gibberella zeae)\,长柄链格孢(Alternaria longipe)和番茄蔫萎座镰孢霉(Fusarium oxysporum \%f.\%lycopersici)等植物病原真菌有很强的抑制作用。LP1可造成绿色木霉(Trichoderma viride)\%菌丝生长形态异常：菌丝端部膨大,菌丝扭曲,分支加剧,菌丝内细胞质分布不均匀,发生凝聚。茚三酮反应以及测序结果均证实其为环肽。     

Fine structure and sporogonic development of a Vavraia sp. (Microsporida: Pleistophoridae) in the biting midge Culicoides edeni (Diptera: Ceratopogonidae)

A microsporidium with ultrastructural characteristics of the genus Vavraia was found in the fat body of an adult specimen of Culicoides edeni (Diptera: Ceratopogonidae) collected in northern Florida. The sporogonial stages developed within sporophorous vesicles, which contained variable numbers of oval spores at maturity. The wall of the sporophorous vesicle was composed of two electron-dense outer layers and an electron-lucent intermediate layer. Sporonts contained haplokaryotic nuclei and divided by rosette formation. Mature spores had anisofilar polar filaments and measured 3.8 +/- 0.28 microns in length and 2.2 +/- 0.16 microns in width in thick sections of resin-embedded material. This is the first report of a Vavraia sp. from a species of Culicodes.     

Heat curing Metaseiulus occidentalis (Nesbitt) (Acari,Phytoseiidae) of a fitness-reducing microsporidium

Laboratory colonies of the predatory mite Metaseiulus occidentalis in Gainesville, FL were found to be infected with an undescribed microsporidium. Experiments were performed to quantify the effect of infection on the fitness of M. occidentalis and to determine if heat treatment can cure mites of the microsporidium. The colonies tested were derived from an isofemale line so that differences in performance could be attributed to the presence of microsporidia. A subcolony of an uninfected isofemale line was infected with the microsporidium by feeding females infected eggs from another colony of M. occidentalis. Infected mites had a shorter mean (+/-SD) female life span (7.4 +/- 2.9 vs. 10.0 +/- 2.8 days), lower mean oviposition (1.6 +/- 0.7 vs. 2.2 +/- 0.4 eggs/day), and a male-biased sex ratio (43 +/- 16% vs. 57 +/- 15% female progeny). The infection was reduced temporarily in colonies initiated from mites that were reared in a growth chamber at 33 degrees C from egg to adult, but healthy colonies only were established from the progeny of the heat-treated adults. These colonies remained free of infection for 10 weeks.     

Light and scanning electron microscopy of fallow deer (Dama dama) spermatozoa

No basic differences in size (mean +/- s.d. for at least 300 spermatozoa), shape and ultrastructure of the spermatozoa of fallow deer were detected (1) in comparison to other artiodactyls, (2) between different fallow bucks, and (3) between different months of the fertile season. The total length of the normal spermatozoon was 67.2 +/- 1.2 microns. The flat, paddle-shaped head was 8.2 +/- 0.3 microns long, 4.4 +/- 0.2 microns for the greatest width, 1.9 +/- 0.2 microns for basal width and, approximately 0.7 microns in thickness. The tail measurements were 13.7 +/- 0.3 microns for the midpiece, 0.5 +/- 0.1 microns for the diameter of the midpiece, 42.6 +/- 0.9 microns for the principal piece, and 2.7 +/- 0.6 microns for the endpiece. Spermatozoa with abnormalities such as cytoplasmic remnants and droplets, bent and coiled tails, as well as microcephalic forms were observed.     

An intranuclear microsporidium associated with acute anemia in the chinook salmon, Oncorhynchus tshawytscha

An intranuclear microsporidium is described from hemoblastic cells of the chinook salmon, Oncorhynchus tshawytscha. The infection is associated with an acute anemia in the fish. Up to 47% of the hemoblast nuclei were infected in anemic fish. The organisms, found only in spleen and kidney tissues, were 1-2 microns in diameter and consisted of vegetative and early sporulation forms. This microsporidium differs from known species which parasitize fish in its tissue location; however, the absence of mature spores and other life cycle stages precludes determination of its precise taxonomic identity.     

Living high-training low increases hypoxic ventilatory response of well-trained endurance athletes.

This study determined whether "living high-training low" (LHTL)-simulated altitude exposure increased the hypoxic ventilatory response (HVR) in well-trained endurance athletes. Thirty-three cyclists/triathletes were divided into three groups: 20 consecutive nights of hypoxic exposure (LHTLc, n = 12), 20 nights of intermittent hypoxic exposure (four 5-night blocks of hypoxia, each interspersed with 2 nights of normoxia, LHTLi, n = 10), or control (Con, n = 11). LHTLc and LHTLi slept 8-10 h/day overnight in normobaric hypoxia (approximately 2,650 m); Con slept under ambient conditions (600 m). Resting, isocapnic HVR (DeltaVE/DeltaSp(O(2)), where VE is minute ventilation and Sp(O(2)) is blood O(2) saturation) was measured in normoxia before hypoxia (Pre), after 1, 3, 10, and 15 nights of exposure (N1, N3, N10, and N15, respectively), and 2 nights after the exposure night 20 (Post). Before each HVR test, end-tidal PCO(2) (PET(CO(2))) and VE were measured during room air breathing at rest. HVR (l. min(-1). %(-1)) was higher (P < 0.05) in LHTLc than in Con at N1 (0.56 +/- 0.32 vs. 0.28 +/- 0.16), N3 (0.69 +/- 0.30 vs. 0.36 +/- 0.24), N10 (0.79 +/- 0.36 vs. 0.34 +/- 0.14), N15 (1.00 +/- 0.38 vs. 0.36 +/- 0.23), and Post (0.79 +/- 0.37 vs. 0.36 +/- 0.26). HVR at N15 was higher (P < 0.05) in LHTLi (0.67 +/- 0.33) than in Con and in LHTLc than in LHTLi. PET(CO(2)) was depressed in LHTLc and LHTLi compared with Con at all points after hypoxia (P < 0.05). No significant differences were observed for VE at any point. We conclude that LHTL increases HVR in endurance athletes in a time-dependent manner and decreases PET(CO(2)) in normoxia, without change in VE. Thus endurance athletes sleeping in mild hypoxia may experience changes to the respiratory control system.     

Effects of in vitro culture methods on morphological development and infectivity of Strongyloides venezuelensis filariform larvae

The effects of in vitro culture methods on morphological development and infectivity of Strongyloides venezuelensis filariform larvae (L3) to rats were investigated. A significantly higher body length was observed in L3 from filter paper culture (597.3 +/- 32.2 microns) than those in fecal (509.9 +/- 35.0 microns) and nutrient broth culture (503.3 +/- 31.0 microns) (P < 0.05). Larval infectivity was assessed by exposing rats to 1,000 L3 from each culture and worms were recovered from the lungs and small intestines. Recovery rate of these worms did not show any significant difference. A significantly greater body length of adults was recorded in those corresponding to the L3 harvested from filter paper (2,777.5 +/- 204.4 microns) and nutrient broth culture (2,732.5 +/- 169.8 microns) than those corresponding to the L3 obtained from fecal culture (2,600.5 +/- 172.4 microns) (P < 0.05). Although worm fecundity and EPG counts differed among culture methods but worm burdens and course of infection did not. These findings suggest that the methods of cultures have a significant effect on the morphological development of the larvae to the L3 stage, but do not influence the infectivity to rats.     

Is ceruloplasmin an important catalyst for S-nitrosothiol generation in hypercholesterolemia?

Nitric oxide (NO) reacts with thiol-containing biomolecules to form S-nitrosothiols (RSNOs). RSNOs are considered as NO reservoirs as they generate NO by homolytic cleavage. Ceruloplasmin has recently been suggested to have a potent catalytic activity towards RSNO production. Considering that NO activity is impaired in hypercholesterolemia and that RSNOs may act as important NO donors, we investigated the relation between concentrations of ceruloplasmin and RSNOs in plasma of hypercholesterolemic (HC) patients compared to normolipidemic (N) controls. Concentrations of ceruloplasmin (0.36 +/- 0.07 x 0.49 +/- 0.11 mg/dl, N x HC), nitrate (19.10 +/- 12.03 x 40.19 +/- 18.70 microM, N x HC), RSNOs (0.25 +/- 0.20 x 0.54 +/- 0.26 microM, N x HC), nitrated LDL (19.51 +/- 6.98 x 35.29 +/- 17.57 nM nitro-BSA equivalents, N x HC), and cholesteryl ester-derived hydroxy/hydroperoxides (CEOOH, 0.19 +/- 0.06 x 1.46 +/- 0.97 microM) were increased in plasma of HC as compared to N. No difference was found for nitrite levels between the two groups (1.01 +/- 0.53 x 1.02 +/- 0.33 microM, N x HC). The concentrations of RSNOs, nitrate, and nitrated LDL were positively correlated to those of total cholesterol, LDL cholesterol, and apoB. Ceruloplasmin levels were directly correlated to apoB and apoE concentrations. Data suggest that: (i) ceruloplasmin may have a role in the enhancement of RSNOs found in hypercholesterolemia; (ii) the lower NO bioactivity associated with hypercholesterolemia is not related to a RSNOs paucity or a defective NO release from RSNOs; and (iii) the increased nitrotyrosine levels found in hypercholesterolemia indicate that superoxide radicals contribute to inactivation of NO, directly generated by NO synthase or originated by RSNO decomposition.     

Manual and automated AgNOR count in differentiating reactive mesothelial from metastatic malignant cells in serous effusions

OBJECTIVE: To distinguish reactive mesothelial cells from malignant cells in serous effusions using manual and automated methods of enumeration of argyrophilic nucleolar organizer regions (AgNORs). STUDY DESIGN: In this prospective study, 38 samples of benign (19 cases) and malignant (19 cases) serous effusions were included. AgNOR stain was used in each case along with routine Papanicolaou stain. The smears were examined under an oil immersion objective, and AgNOR dots were counted by direct observation independently by 2 observers. Automated AgNOR counting and morphometry were performed with a Quantimet 600 image cytometer (Leica, Cambridge, England). At least 100 cells were counted in each case. The number of AgNOR dots in individual cells, AgNOR area, nuclear area, AgNOR vs. nuclear area and nuclear perimeter were measured. Data on benign and malignant cells were compared. RESULTS: The AgNOR dots were discrete and smaller in benign effusion cases as compared to coarse and aggregated in malignant effusion cases. In benign reactive effusion cases the mean number of AgNOR dots per nucleus was 2.33 +/- 0.71 and 2.83 +/- 1.15 by the manual and automated method, respectively, whereas that for malignant effusion cases was 7.48 +/- 2.51 and 8.09 +/- 1.69 by the manual and automated method, respectively. Mean total AgNOR areas in benign and malignant groups were 4.77 +/- 2.66 microns 2 and 38.22 +/- 13.71 microns 2, respectively. Mean nuclear area, nuclear perimeter and ratio of AgNORs vs. nuclear area were 48.72 +/- 19.30 microns 2, 24.68 +/- 10.25 microns and .098 in benign effusion cases as compared to 174.25 +/- 82.36 microns 2, 69.03 +/- 27.23 microns and 0.22 in malignant effusion samples. All these values were significantly higher (P < .001, Student's t test) in malignant cells as compared to benign reactive cells. CONCLUSION: AgNOR dot enumeration, AgNOR area and ratio of AgNORs to nuclear area are valuable adjuncts to cytomorphology in differentiating reactive mesothelial cells from malignant cells in serous effusions. Automated AgNOR counting is rapid and less cumbersome.