以大蜡螟幼虫为模型研究伏立康唑抗茄病镰刀菌效果

目的构建大蜡螟幼虫动物茄病镰刀菌感染模型并观察伏立康唑治疗效果。方法选用临床分离获得茄病镰刀菌1株,实验所用孢子悬液浓度梯度为1×10⁵~1×10⁸CFU/mL,根据死亡率筛选出最佳感染浓度,并以此浓度感染大蜡螟幼虫,用伏立康唑(1.5 mg/kg)治疗;同时设置未处理组和生理盐水对照组,实验过程中收集大蜡螟幼虫尸体做病理检测并记录5 d内死亡情况,每24 h记录1次。通过感染后病理组织切片和大蜡螟幼虫生存率两个方面进行评价。结果大蜡螟幼虫在1×10⁷CFU/mL茄病镰刀菌感染后,5 d死亡率达100%,1×10⁷CFU/mL为本次实验组最佳感染浓度;大蜡螟幼虫感染第2 d死亡虫体病理检测发现大量茄病镰刀菌菌丝和孢子,经伏立康唑治疗后,单个幼虫体内菌体减少,菌体内菌丝减少;大蜡螟幼虫生存曲线显示伏立康唑治疗组较单茄病镰刀菌感染组生存率显著增高(P<0.05)。结论大蜡螟幼虫能够作为茄病镰刀菌体内感染的动物模型,并可用于观察药物治疗效果。     

基于ITS和ndhF-rpl32序列的荞麦种间亲缘关系分析

荞麦起源于我国, 演化形成了丰富的物种和遗传多样性。为了有效研究和利用荞麦及其野生种资源, 以从四川、甘肃、贵州等地采集的荞麦属(Fagopyrum)10个种(含变种、亚种和复合体种)共71份材料为对象, 通过ITS和叶绿体ndhF-rpl32序列分析, 利用MEGA5.0构建系统进化树, 探讨了荞麦种内及种间亲缘关系。结果表明, 在ITS序列矩阵中, 序列长度为725 bp, 信息位点为150个, 占序列总长度的20.7%; 在ndhF-rpl32序列矩阵中, 序列长度为940 bp, 信息位点为158个, 占序列总长度的16.8%。由ITS序列和ndhF-rpl32序列构建的两个进化树都可以将71份材料分为大粒荞麦种组和小粒荞麦种组; 其中, 大粒荞麦种组包括栽培苦荞和米苦荞(F. tataricum)、金荞复合体(F. cymosum complex)、栽培甜荞(F. esculentum)和野生甜荞(F. esculentum ssp. ancestralis); 小粒荞麦种组包括齿翅野荞(F. gracilipes var. odontopterum)、疏穗小野荞(F. leptopodum var. grossii)、小野荞(F. leptopodum)、密毛野荞(F. densovillosum)、细柄野荞(F. gracilipes)和硬枝万年荞(F. urophyllum)。而ndhF-rpl32序列构建的系统发育树还能区分栽培甜荞和野生甜荞, 具有更好的聚类效果。另外, 与栽培甜荞相比, 金荞复合体与栽培苦荞的亲缘关系更近。该研究为荞麦属种的分类和条形码研究提供了一定的科学依据。     

利用大蜡螟模型研究白念珠菌SUMO编码基因SMT3在致病性调控中的作用CSCD

目的探讨白念珠菌SMT3基因在致病性调控中的作用。方法利用瞬时CRISPR/Cas9系统构建SMT3纯合子缺失菌株,并检测其侵入生长和生物被膜形成能力;利用大蜡螟感染模型检测SMT3缺失株致病能力,通过糖原染色观察组织内白念珠菌菌体形态,并进一步检测SMT3缺失株对大蜡螟幼虫血细胞的破坏作用。结果SMT3缺失促进了侵入生长和生物被膜形成,其感染的大蜡螟幼虫平均存活期为3.5 d,致病性较野生型菌株(平均存活期为8.5 d)显著增强;SMT3缺失株感染的大蜡螟幼虫血细胞数为1.4×10^(7)/mL,较野生型组(2.9×10^(7)/mL)显著降低,仅为野生型组的48%。结论白念珠菌SMT3基因在致病性调控中发挥重要作用。     

影响大蜡螟幼虫体色的环境因素初探

大蜡螟幼虫体色各式各样 ,通过对比实验分析了温度、湿度、密度、光照和食物等环境因素对大蜡螟幼虫体色的影响。实验结果表明 :不同温度、湿度、密度、光照条件下大蜡螟幼虫的体色均未变化。在研究食物对大蜡螟幼虫体色的影响实验中食蜂巢的白黄色幼虫身体内部发黑 ,这可能是黑色的蜂巢透射出的颜色。实验为进一步研究大蜡螟幼虫体色的遗传机制打下了基础。     

三种鳞翅目幼虫肠道蛋白水解酶的活性

本工作以酪蛋白为底物,测定粘虫Mythimna separata Walker、棉铃虫Heliothis armigera Hubner和大蜡螟 Galleria mellonlla Linnaeus三种鳞翅目幼虫肠道蛋白水解酶的活性,并分别用BTEE和TAME为底物,测定了其中类胰凝乳蛋白酶和类胰蛋白酶的活性.结果表明:三种幼虫肠道都含有类胰凝乳蛋白酶和类胰蛋白酶.抑制剂TPCK可以部分地抑制类胰凝乳蛋白酶的活性,而胰酶抑制剂则显著地抑制类胰蛋白酶的活性.     

大蜡螟医学真菌感染模型的研究现况

在研究真菌感染中建立合适的真菌感染动物模型非常重要,大蜡螟幼虫作为昆虫动物模型之一,相比于其他的动物模型具有多种技术优势,目前已被广泛用于新型隐球菌、小孢子菌、红色毛癣菌、念珠菌属、暗色真菌、马尔尼菲青霉菌、黄曲霉和烟曲霉等多种致病菌的毒力、发病机制、免疫学改变、抗菌药物的开发以及系统性真菌感染的治疗等各个研究领域。研究表明大蜡螟感染模型研究结果与哺乳动物的结果相似,因此可以用大蜡螟来替代哺乳动物进行相关研究,从而减少了实验对哺乳动物的依赖性。     

分离自1例慢性阻塞性肺疾病患者的Aspergillus lentulus在蜡螟模型上的毒力初探

目的建立Aspergillus lentulus(A.lentulus或A.L)感染动物模型,借动物模型初步探究A.lentulus的毒力。方法将125只蜡螟随机分成5组,以Aspergillus lentulus临床株、Aspergillus lentulus标准株作为实验组,烟曲霉、白念珠菌为对照组,PBS为空白对照组。实验组及对照组菌株分别制成10~6 CFU/mL孢子悬液,感染各组蜡螟。记录72 h内蜡螟的生存情况并制作生存曲线,24 h后提取蜡螟肠道组织,HE染色组织切片观察肠道组织损伤情况,用组织匀浆法,测定蜡螟肠道内真菌载量及真菌逆培养阳性率,用真菌荧光染色法观察肠道培养真菌镜下形态。结果 A.lentulus临床株和A.lentulus标准株的蜡螟存活数与对照组之间差异有统计学意义(P0.05);结果显示A.lentulus临床株和A.lentulus标准株肠壁结构大致正常,局部可见水肿少量菌丝、孢子及炎症细胞浸润,对照组肠道结构破坏严重,可见菌丝、孢子及大量炎症细胞浸润;不同菌种感染蜡螟幼虫各组肠道载菌量及真菌逆培养阳性率比较,差异具有统计学意义(P0.05);真菌荧光显微镜观察,A.lentulus临床株和标准株菌丝多、孢子少,白念珠菌和烟曲霉组孢子多。结论与烟曲霉和白念珠菌相比,A.lentulus菌株对蜡螟毒力和肠道损伤能力较弱且致死率低。     

中国裂隙衣属地衣（文字衣科）的初步研究

在中国文衣科地衣系统研究中,对裂隙衣属Fissurina地衣进行了订正,报道了16种,其中中国新记录种3个,即连绵裂隙衣F. consentanea、球孢裂隙衣F. globulifica和皱体裂隙衣F. insidiosa。建议4个异名,即Fissurina isidiata Z.F. Jia 为Platythecium dimorphodes (Nyl.) Staiger的异名;Fissurina marginata Staiger为Fissurina elaocarpa (A.W. Archer) A.W. Archer的异名;Graphis canlaonensis Vain.为Fissurina consentanea Nyl.的异名;Graphis glauca Müll. Arg.为Fissurina dumastii Müll. Arg.的异名。本文提供了中国裂隙衣属每个种的描述和分布,并提供了鉴定检索表。     

异小杆线虫XJZL1409(Heterorhabditis bacteriophora)共生菌的分离及致病性研究

本研究分离了来自新疆和田地区间作绿豆的4年枣0-40 cm根际土壤中的昆虫病原线虫XJZL1409Heterorhabditis bacteriophora的共生菌,并进行致病性测定。通过侵染期线虫和线虫致死的大蜡螟幼虫血腔直接分离,采用传统形态学观察结合分子生物学方法对菌株进行鉴定。同时大蜡螟Galleria mellonella 5龄幼虫接种菌株细胞发酵液测定致病性。菌株形态学特性结合16S r DNA序列系统发育分析,线虫XJZL1409的共生菌被鉴定为发光杆菌属Photorhabdus luminescens subsp. laumondii。室内菌液注射毒性测定结果显示,侵染致死的幼虫体色呈灰绿色;接种48 h后,每头幼虫接种菌液量为500 CFU时校正死亡率就可以达到100%; 48 h时致死中浓度LC50为10. 74 CFU/m L。同时菌液口服杀虫毒性结果表明,第5天时待测菌株菌液对大蜡螟幼虫的校正死亡率为21. 21%,体重抑制率为20. 02%。因此,昆虫病原线虫XJZL1409的共生菌为P. luminescens subsp. laumondii对大蜡螟幼虫具有很强的注射毒性,为研究线虫的致病机制及发掘新抗性基因奠定基础。     

蜡螟抗白假丝酵母菌自噬机制的初步研究

目的探究白假丝酵母菌感染蜡螟时蜡螟的自噬相关通路蛋白的表达情况。方法用一定量的白假丝酵母菌的活化孢子感染蜡螟,经过12h,解剖蜡螟收集蜡螟细胞并裂解细胞,用真菌活性检测试剂盒检测孢子活性;解剖蜡螟取肠道组织并用PI染死细胞。取不同感染时间段的淋巴细胞,用裂解液裂解,离心取上清,用Western blot法检测上清液中的Dectin-1、ROS、LC3Ⅰ/Ⅱ的表达水平。结果活化的孢子注射至蜡螟体内后,其活性受到抑制;蜡螟的肠道细胞被定位在上面的菌丝损伤并且孢子活性受到抑制。随着蜡螟感染白假丝酵母菌时间的递增,其自身的Dectin-1、ROS、LC3Ⅰ/Ⅱ的表达水平在不断增高且在感染24h最高。结论白假丝酵母菌感染蜡螟后,蜡螟的淋巴细胞和肠道细胞通过升高Dectin-1、ROS、LC3Ⅰ/Ⅱ的表达水平发挥杀伤孢子的作用。     

蝙蝠蛾幼虫肠道中的冬虫夏草菌体研究

西藏林芝地区冬虫夏草天然产区野生虫草蝙蝠蛾幼虫的肠道液pH范围为9.69-10.99,体外在此pH下培养冬虫夏草菌长势较差。同时,在该pH的体外模拟中肠液中,虫菌体也会在极短的时间内消亡,而对自然侵染的蝙蝠蛾幼虫的血淋巴、消化道及其“反吐物”（肠液）进行切片和显微镜检却发现,蝙蝠蛾幼虫消化道、呕吐物中均存在与血淋巴中相同的冬虫夏草菌菌体形态,生存状态良好,正常增殖,表明蝙蝠蛾幼虫肠道的生理状态可以维系并支撑冬虫夏草菌菌体某种形态的存在与生长,并证实冬虫夏草菌由口器及消化道入侵蝙蝠蛾幼虫的可能性;肠道虫菌体与血淋巴虫菌体不一定同时存在,表明冬虫夏草菌对蝙蝠蛾幼虫的侵染可能同时存在表皮侵染和肠道侵染两种途径。本文为冬虫夏草菌对蝙蝠蛾幼虫的侵染途径研究提供更加多样化的信息支持。     

Baculovirus-mediated Expression of a Gene for Trehalase of the Mealworm Beetle, Tenebrio molitor, in Insect Cells, SF-9, and Larvae of the Cabbage Armyworm, Mamestra brassicae

It is of interest to understand what kinds of physiological and biochemical changes occur in insects if the homeostasis of trehalose in the hemolymph is disrupted by the infection with a recombinant baculovirus containing a secretory-trehalase gene. For this purpose, two recombinant non-occluded Autographa california multicapsid nucleopolyhedroviruses (AcMNPVs), vTREVL and vERTVL, containing a trehalase cDNA of the mealworm beetle, Tenebrio molitor, were constructed. The trehalase cDNA was inserted in the sense orientation downstream of the polyhedrin promoter for vTREVL, and in the anitsense orientation for vERTVL. The active trehelase of T. molitor was found outside of cells when SF-9 cells or larvae of the cabbage armyworm, Mamestra brassicae, were infected with vTREVL. In the hemolymph of vTREVL-infected larvae, expression of the active trehelase was followed by disappearance of trehalose and appearance of glucose. However, the mortality time of virus-infected 5th instar larvae increased in the following order: AcMNPV C6 (wild-type virus) ≤ vERTVL < vTREVL. The symptoms (the browning and liquefying of the host body) of NPV infection were moderated considerably in vTREVL-infected larvae.     

Studies on the immune response of sheep to Chabertia ovina (Fabricius, 1788)

The immunological basis of host resistance to C. ovina was established by the demonstration of a marked anamnestic response by 6 sheep to a second infection of C. ovina after previous sensitization. Reaginic antibody was detected in the circulation of 20 sheep tested during both primary and secondary infection. It was first detected 5 weeks after infection with 2000 larvae, 8 weeks after infection with 1000 larvae and 12 weeks after infection with 400 larvae. In contrast, precipitating antibody was detected in only 10 per cent of sheep tested, while in vivo and in vitro tests for delayed hypersensitivity were negative. Studies on the effect of antilymphocytic serum on immunity to C. ovina suggested that the development of resistance to C. ovina requires the participation of the thymus-derived lymphocyte.     

Observations on the self-cure reaction and other forms of immunological responsiveness against Haemonchus contortus in sheep

Self-cure reactions and immunological responses preventing establishment of Haemonchus contortus in sheep may operate as separate entities. In one experiment, self-cure occurred when challenge infection with 5000 larvae was superimposed on an infection with 5000 larvae given to worm-free sheep 6 weeks previously. Resident worms were rejected and establishment of infection by incoming larvae was impeded. The latter effect was not observed in sheep treated similarly but with resident parasites removed by treatment with oxfendazole before challenge. In another experiment, younger worm-free sheep primed by three infections with 2000 larvae at intervals of 2 weeks or a single infection with 6000 larvae were challenged with 10,000 larvae 6 weeks after the first priming infection. Self-cure was not incited but establishment of infection was impeded in sheep primed with three divided doses of larvae whether or not priming infections had been removed by oxfendazole. Infection regimes used for priming did not influence numbers of arrested fourth-stage larvae derived from challenge infection. However, more arrested larvae were present when challenge was superimposed on extant infections, indicating that resident worms or a factor activated by their presence induced developmental arrest. In a third experiment, large burdens with H. contortus were established in sheep immunosuppressed with the corticosteroid, dexamethasone, at the time of infection. Self-cure was not triggered by a challenge infection given 32 days later either in these sheep, or in sheep with a smaller worm burden derived from infection given without immunosuppression. Faecal egg counts, however, indicated that development of the challenge infection was prevented in both groups of sheep.

Investigation of self-cure is restricted by lack of a predictable system for reproducing the phenomenon. Self-cure was induced by a single infection with 5000 larvae in mature sheep but not with 6000 larvae in immature sheep. Three infections with 3000 larvae given at intervals of 2 weeks to mature sheep did not prime for self-cure. Procedures aimed at heightening immediate hypersensitivity, i.e. treatment with pertussis vaccine or concurrent infections with Ostertagia circumcincta, did not promote self-cure reactivity in the latter situation.     

Plant-mediated alteration of the peritrophic matrix and baculovirus infection in lepidopteran larvae

The peritrophic matrix (PM) lines the midgut of most insects, providing protection to the midgut epithelial cells while permitting passage of nutrients and water. Herein, we provide evidence that plant-mediated alteration of the PM contributes to the well-documented inhibition of fatal infection by Autographa californica multiple nucleopolyhedrovirus (AcMNPV) of Heliothis virescens F. larvae fed cotton foliage. We examined the impact of the PM on pathogenesis using a viral construct expressing a reporter gene (AcMNPV-hsp70/lacZ) orally inoculated into larvae with either intact PMs or PMs disrupted by Trichoplusia ni granulovirus occlusion bodies containing enhancin, known to degrade insect intestinal mucin. Larvae possessing disrupted PMs displayed infection foci (lacZ signaling) earlier than those with intact PMs. We then examined PMs from larvae fed artificial diet or plant foliage using electron microscopy; foliage-fed larvae had significantly thicker PMs than diet-fed larvae. Moreover, mean PM width was inversely related to both the proportion of larvae with lacZ signaling at 18 h post-inoculation and the final percentage mortality from virus. Thus, feeding on foliage altered PM structure, and these foliage-mediated changes reduced baculoviral efficacy. These data indicate that the PM is an important factor determining the success of an ingested pathogen in foliage-fed lepidopteran larvae.     

Epidemiological studies on horses infected with nematodes of the family Trichonematidae (Witenberg, 1925)

Observations are reported on seasonal changes in the age structure of populations of nematodes of the family Trichonematidae recovered at regular intervals from the lumens of the large intestines of horses slaughtered in S.W. England. The results show that changes in the size of parasitic populations of Trichonema nassatum follow seasonal variations in the rate of infection, more individuals maturing during summer/autumn than during winter/spring because of proportional differences in the numbers of infective larvae ingested from the pasture. In contrast, larvae of T. longibursatum, T. catinatum and T. goldi ingested by grazing horses during summer accumulate in the gut wall rather than promptly returning to the lumen and developing to maturity. Their development is apparently inhibited until the following spring when 4th-stage larvae emerge en masse and quickly reach the adult stage. The relationship between fluctuations in the size of the adult populations of the 4 species studied and the characteristic seasonal variations in horse faecal egg counts is discussed.     

Experimental infection routes of Angiostrongylus cantonensis in mice.

Stomach intubation is the most common method used in the experimental infection of animals with Angiostrongylus cantonensis. In order to compare the effectiveness of other possible transmission methods, groups of BALB/c mice were given infective third-stage larvae of A. cantonensis by different routes including intraperitoneal or subcutaneous injections, and penetration of anal mucosa, vaginal mucosa, conjunctival mucosa, lacerated skin, unabraded skin, foot pad and tail skin, while stomach intubation was used as control. Recovery of fifth-stage larvae was higher in mice inoculated with third-stage larvae subcutaneously. Successful infections were established through all experimental transmission routes except tail skin penetration. This study suggests that oral infection may not be the only route for the transmission of human angiostrongyliasis, and subcutaneous infection may be a better method for experimental infection.     

Trypanosoma cruzi: skin-penetration kinetics of vector-derived metacyclic trypomastigotes

In order to investigate the natural route of infection of nude and normal BALB/c mice with Trypanosoma cruzi via the skin, a drop of vector faeces/urine containing metacyclic trypomastigotes was placed onto the puncture site of a bite from Triatoma infestans. The periods of exposure, i.e. until removal of flagellates from the skin, and the time elapsed until surgical removal of the skin around the puncture were varied. After 15 min of exposure, T. cruzi developed in all nude mice without surgery, and in four of 10 mice if the puncture region of the skin was removed directly after exposure. In a shaved puncture region, 5 min of exposure were sufficient to infect all normal BALB/c mice without surgery and one of four mice with direct removal of the puncture region. Longer periods of exposure or time until removal of the skin only sometimes resulted in higher infection rates. Prepatent periods and the development of parasitaemia varied irrespective of the period of exposure or the period until skin removal at the puncture site. The importance of these findings is that they clearly prove that T. cruzi can rapidly invade the host via the puncture site of the bite of the vector and that at least some parasites are immediately transported away from this site.     

A case of myiasis in a patient with psoriasis from Turkey

Myiasis infestations caused by the larvae of flies mostly belonging to Cyclorapha suborders are frequently encountered in Turkey, which is located in the subtropical zone. The skin is a common site for myiasis, and the infestations are likely to develop in infected tissues and poorly attended wounds of the skin.

The case, a 30-year-old male patient, was diagnosed with psoriasis 18 years ago. He had psoriatic scales on his right big toe and was receiving corticosteroid and immunosuppressive drugs.

A total of 11 fly larvae were removed from the infected right first toe of the patient. Structures of the stigmas seen in the cross-sections taken from the final segments of these larvae were examined and determined as Sarcophaga spp. larvae.

Patients with infected tissues should be extremely cautious about their wound hygiene and take required fly control measures especially during summer as flies can find a suitable environment for sustenance.