埃博拉病毒经气溶胶感染与传播的研究进展

埃博拉病毒是一种可在人类及非人灵长类间通过接触传播的烈性病原微生物,致死率高达90%。目前关于埃博拉病毒能否在人际间经气溶胶传播,还未有定论。但经过多年研究表明,在实验室内,埃博拉病毒可通过气溶胶感染非人灵长类,极低剂量即可引起致命性感染。并且埃博拉病毒在动物间的气溶胶传播在实验室内已被证实。除此之外,埃博拉病毒变异速度惊人,不排除可导致大面积暴发的可能性。     

鼠疫耶尔森菌caf1基因缺失株体外生物学特性和对小鼠致病性分析

目的 探索caf1基因缺失对鼠疫耶尔森菌体外生物学特性与对小鼠致病性的影响。方法 利用CRISPR/Cas12a介导的基因编辑技术构建鼠疫耶尔森菌caf1基因缺失株,并通过PCR技术对caf1基因缺失验证。比较鼠疫耶尔森菌caf1基因缺失株和野生株201在生长速率、液体培养状态、聚集情况等体外生物学特性和对小鼠致病性等方面的差异。结果 PCR扩增结果证实,caf1基因缺失株构建成功。体外生物学特征分析表明,与野生株相比,caf1基因缺失株的形态学和生长速率并未发生明显变化,自凝聚率升高。肺递送途径LD₅₀分析表明,caf1的缺失导致鼠疫耶尔森菌气溶胶感染小鼠半数致死剂量显著降低。结论 不同途径感染结果表明,caf1基因缺失株相较于野生株,在肺递送、滴鼻、皮下、尾静脉注射4种途径下对BALB/c小鼠的毒力均降低。caf1基因的缺失导致鼠疫耶尔森菌自凝聚率升高,对BALB/c小鼠致病性下降。     

西藏地区首例血培养分离布鲁氏菌

目的 研究患者血液中分离出的病原菌生物学性状及布鲁氏菌病在西藏地区的病原学诊断。方法 按布鲁氏菌标准鉴定方法进行细菌学实验,包括形态学、培养特性、生化特征及血清学试验,并对患者的流行病学、临床资料、实验室资料进行分析。结果 患者血液中分离的病原微生物的形态学、培养特性、生化特征及血清学诊断结果均符合布鲁氏菌。结论 西藏地区首次从患者血液中分离到布鲁氏菌,说明临床机构已有病原诊断能力,同时要加强实验室生物安全。     

小毛莨内酯对结核休眠菌感染病人GLS mRNA表达的影响

目的　研究小毛莨内酯抗人体结核休眠菌感染的分子免疫学机理。方法 采用PCR方法检测结核病人PBLs中结核菌16kDα-晶状体同源蛋白DNA阳性的56例患者为本研究对象;采用反转录聚合酶链反应半定量 (QC-RT-PCR)方法,测定不同剂量的Tern.对以上病人PBL颗粒裂解肽mRNA表达水平的影响。结果 当Tern.在100mg/L,200mg/L浓度时,能诱导结核休眠菌感染病人体外活化的PBLGLSmRNA的表达。结论 Tern.可能通过促进颗粒裂解肽mRNA的表达,增强机体CTL杀菌能力,达到抗结核休眠菌的作用。     

云南家鼠鼠疫疫源地人群和指示动物血清鼠疫F1抗体调查

目的 了解云南省家鼠鼠疫疫源地静息期人和指示动物血清鼠疫F1抗体情况,分析鼠疫发生和流行的风险。方法 2019年7——8月,课题组选择云南省家鼠鼠疫疫源地弥勒市、芒市和梁河县作为研究区域,对8个曾经报告2次及以上鼠疫的自然村和8个从未报告鼠疫的自然村的人群及指示动物进行血清采集,每个自然村采集人血清不少于20份,指示动物血清不少于10份。用间接血凝法检测鼠疫F1抗体,并用上转发光法对阳性样本进行再次检测确认。结果 368份人血清中仅有1份鼠疫F1抗体阳性,307份犬血清和12份猫血清鼠疫F1抗体均为阴性。结论 云南家鼠鼠疫疫源地人群鼠疫F1抗体阳性率非常低,但鼠疫F1抗体阳性的自然村需要加强鼠疫监测。指示动物未检测出鼠疫F1抗体,当地发生鼠疫的风险不高。     

金黄色葡萄球菌ATCC25923人工感染BALB/c 雌鼠肾脏组织PRNP和PRND的表达变化研究

目的 为了研究细胞型朊蛋白和Doppel蛋白在金黄色葡萄球菌感染中的作用。方法 本实验用金黄色葡萄球菌ATCC25923感染BALB/c雌鼠后,分别在1~7 d内解剖小鼠,进行组织抹片革兰氏染色和石蜡切片制作,最后采用荧光定量PCR检测PRNP和PRND在1~7 d中的相对表达量。结果 金黄色葡萄球菌最先出现在肾脏,且对肾脏组织有损伤作用;PRNP和PRND基因在每个时段的表达量都高于空白对照组,其中PRNP在第1 d的表达量最高,为空白对照组的6.1倍,PRND在第2 d的表达量最高,为空白对照组的13.1倍。结论 小鼠肾脏的PRNP和PRND的mRNA表达水平随着金黄色葡萄球菌感染的时间发生变化。     

北京市鼠形动物感染巴尔通体的调查

目的 调查北京市鼠形动物巴尔通体感染状况和遗传特征。方法 采用夹夜法在北京市16个区捕获鼠形动物,使用PCR方法检测巴尔通体gltA基因,测序后进行系统发育树分析。结果 共采集7属9种鼠形动物样本448份,检测到巴尔通体阳性样本46份,总阳性率为10.3%,且阳性率在鼠种间、地区间和生境间的差异均具有统计学意义(X²_鼠种=114.980,X²_地区=22.133,X²_生境=96.466,P均<0.001)。系统发育树分析显示,40份样本序列位于进化树上的8个不同分支,提示北京市鼠形动物巴尔通体感染存在遗传多样性。结论 巴尔通体在北京市鼠形动物中广泛存在,并具有遗传多样性,本地人群存在一定的感染风险。     

新型疫苗佐剂Ov-ASP-1佐剂活性功能区的设计、表达及生物学特性研究

目的 获得保留Ov-ASP-1佐剂活性的功能区。方法 用前期制备的Ov-ASP-1单克隆抗体对Ov-ASP-1的9个肽进行特异性结合,根据肽的结合情况设计功能区ASPPRM,并进行密码子优化、构建原核表达载体后在大肠杆菌中表达。表达产物经 Western印迹鉴定后的用镍柱纯化蛋白,复性完全后对获得的ASPPRM活性蛋白进行生物学特性分析。结果 ASPPRM以包涵体形式表达,分子量为17 kD。将其和OVA共同免疫小鼠后ASPPRM组抗OVA的IgG抗体显著高于OVA组。结论 本研究获得了保留佐剂活性的ASPPRM蛋白,为后续ASPPRM体内的免疫增效作用及Ov-ASP-1佐剂活性功能域的探索奠定基础。     

广州市鼠类动物钩端螺旋体的感染调查

目的 调查广州市鼠类动物中钩端螺旋体病的流行情况和遗传特征。方法 笼捕法捕捉鼠类动物,采集脾组织提取总DNA,通过PCR技术检测钩端螺旋体16S rRNA和LipL32基因,测序后进行系统进化分析。结果 采集褐家鼠170只、板齿鼠4只、黄胸鼠3只、黄毛鼠1只和臭鼩鼱22只,共200只鼠类动物,在褐家鼠中检测到钩端螺旋体阳性样本3个,总阳性率1.5%,系统发育分析显示,3份阳性样本序列与致病性问号钩端螺旋体位于进化树上的同一个分支。结论 广州地区鼠类动物中存在致病性问号钩端螺旋体,本地人群存在一定的感染风险。     

云南省师宗县牛羊蓝舌病病毒感染情况及活动规律监测

目的 研究蓝舌病病毒近几年在云南省师宗县的感染情况及活动规律。方法 2014-2016年连续3年在师宗县设置监控点,每年选择蓝舌病抗体阴性的10头牛和5只羊作为监控动物,5-10月每周采血1次,11月、12月,每月采血1次,每次每头动物采集常规全血、肝素抗凝血各1份,用于蓝舌病抗体检测和病毒分离。结果 2014-2016年均有动物感染蓝舌病病毒,平均感染率为35.56%,感染时间均在6-10月之间,这一结果表明蓝舌病病毒感染动物主要集中在每年的6-10月,这一时期应加强蓝舌病的防控。在蓝舌病监测中,45只监控动物中的16只感染了蓝舌病病毒,并从7只感染动物中分离出9株蓝舌病病毒,分别为BTV-2、-4、-9、-12、-16型,其中BTV-16型4株,为优势血清型。结论 病毒分离和鉴定结果表明,师宗县长期存在多种血清型蓝舌病病毒。     

Spatial-temporal imaging of bacterial infection and antibiotic response in intact animals

We describe imaging the luminance of green fluorescent protein (GFP)-expressing bacteria from outside intact infected animals. This simple, nonintrusive technique can show in great detail the spatial-temporal behavior of the infectious process. The bacteria, expressing the GFP, are sufficiently bright as to be clearly visible from outside the infected animal and recorded with simple equipment. Introduced bacteria were observed in several mouse organs including the peritoneal cavity, stomach, small intestine, and colon. Instantaneous real-time images of the infectious process were acquired by using a color charge-coupled device video camera by simply illuminating mice at 490 nm. Most techniques for imaging the interior of intact animals may require the administration of exogenous substrates, anesthesia, or contrasting substances and require very long data collection times. In contrast, the whole-body fluorescence imaging described here is fast and requires no extraneous agents. The progress of Escherichia coli-GFP through the mouse gastrointestinal tract after gavage was followed in real-time by whole-body imaging. Bacteria, seen first in the stomach, migrated into the small intestine and subsequently into the colon, an observation confirmed by intravital direct imaging. An i.p. infection was established by i.p. injection of E. coli-GFP. The development of infection over 6 h and its regression after kanamycin treatment were visualized by whole-body imaging. This imaging technology affords a powerful approach to visualizing the infection process, determining the tissue specificity of infection, and the spatial migration of the infectious agents.     

Metronidazole prevents reactivation of latent Mycobacterium tuberculosis infection in macaques

Targeting Mycobacterium tuberculosis bacilli in low-oxygen microenvironments, such as caseous granulomas, has been hypothesized to have the potential to shorten therapy for active tuberculosis (TB) and prevent reactivation of latent infection. We previously reported that upon low-dose M. tuberculosis infection, equal proportions of cynomolgus macaques develop active disease or latent infection and that latently infected animals reactivated upon neutralization of TNF. Using this model we now show that chemoprophylaxis of latently infected cynomolgus macaques with 6 mo of isoniazid (INH) effectively prevented anti-TNF antibody-induced reactivation. Similarly, 2-mo treatment of latent animals with a combination of INH and rifampicin (RIF) was highly effective at preventing reactivation disease in this model. Metronidazole (MTZ), which has activity only against anaerobic, nonreplicating bacteria, was as effective as either of these treatments in preventing reactivation of latent infection. Because hypoxic lesions also occur during active TB, we further showed that addition of MTZ to INH/RIF effectively treated animals with active TB within 2 mo. Healing lesions were associated with distinct changes in cellular pathology, with a shift toward increasingly fibrotic and calcified lesions. Our data in the nonhuman primate model of active and latent TB supports targeting bacteria in hypoxic environments for preventing reactivation of latent infection and possibly shortening the duration of therapy in active TB.     

Influenza virus aerosol exposure and analytical system for ferrets

Understanding the transmission ability of newly emerging influenza viruses is central to the development of public health preparedness and prevention strategies. Animals are used to model influenza virus infection and transmission, but the routinely used intranasal inoculation of a liquid virus suspension does not reflect natural infection. We report the development of an inoculation method that delivers an influenza virus aerosol inoculum to ferrets and the characterization of size distribution and viable virus present in aerosols shed from infected ferrets during normal breathing and sneezing. By comparing virus deposition, infectivity, virulence, and transmissibility among animals inoculated intranasally or by aerosols with a human (H3N2) or avian (H5N1) influenza virus, we demonstrate that aerosol inoculations more closely resemble a natural, airborne influenza virus infection and that viable virus is measurable in droplets and droplet nuclei exhaled by infected ferrets. These methods will provide improved risk assessment of emerging influenza viruses that pose a threat to public health.     

Leukocytic response to inhaled bacteria.

Using histologic techniques, we have quantified the amount of infiltration of bronchi and alveoli by polymorphonuclear leukocytes during the 4 hours after an aerosol inoculation of mice with bacteria. Although the lungs of animals challenged with Staphylococcus aureus differed little from those of animals exposed only to a water aerosol, the lungs of animals exposed to Klebsiella pneumoniae or to Escherichia coli demonstrated significantly greater polymorphonuclear leukocyte infiltrations in bronchi and alveoli 2 and 4 hours afer exposure. These results suggest that the polymorphonuclear leukocyte may contribute to the early defense of the lung against some bacteria.     

大肠杆菌O157∶H7对小鼠感染的实验研究

目的　采用肠出血性大肠杆菌埃希氏菌 (EHEC)国际代表株O15 7∶H7-EDL933株 ,实验感染小鼠 (ICR) ,观察其感染和带菌消长情况。方法　ICR小鼠经口感染 ,剂量为 0 1～ 0 9ml(菌悬液浓度为 7 0× 10 8～ 4 0× 10 9CFU/ml) ,并在SPF动物实验室中饲养。结果　不同实验动物微生物等级的ICR小鼠对EDL933株表现出不同感染类型 ,Ⅰ级小鼠感染未成功 ;Ⅱ级小鼠为一过性排菌 (M =4h) ;Ⅲ级小鼠粪排菌中位数为 2 4h ,是Ⅱ级小鼠的 6倍。Ⅲ级小鼠发现盲肠带菌 ,阳性率为31 5 8% (6 / 19)。结论　研究结果提示 ,鼠可成为EHECO15 7∶H7的贮存宿主 ,可能是潜在的人类感染的传染源。不同实验动物微生物等级的ICR小鼠对EDL933株所表现出的感染类型 ,提示预防O15 7∶H7感染 ,可经口服菌苗来实现的可能性     

Congenital Trypanosoma cruzi infection in a laboratory-born squirrel monkey, Saimiri sciureus

A Colombian Phenotype, laboratory-born squirrel monkey, Saimiri sciureus, was found to be congenitally infected with biologically proven Trypanosoma cruzi. The parasite was observed in blood smears and by xenodiagnoses of the mother and the offspring, and the isolates produced infection in mice and amastigotes in VERO tissue culture cells. The finding was accidental; both animals were healthy. Tissues of the mother did not show macro-microscopic evidence of T. cruzi infection and the electrocardiograph of the offspring was normal. This seems to be the first report of congenital T. cruzi transmission in an otherwise healthy non-human primate.     

Natural and experimental oral infection of nonhuman primates by bovine spongiform encephalopathy agents

Experimental lemurs either were infected orally with the agent of bovine spongiform encephalopathy (BSE) or were maintained as uninfected control animals. Immunohistochemical examination for proteinase-resistant protein (prion protein or PrP) was performed on tissues from two infected but still asymptomatic lemurs, killed 5 months after infection, and from three uninfected control lemurs. Control tissues showed no staining, whereas PrP was detected in the infected animals in tonsil, gastrointestinal tract and associated lymphatic tissues, and spleen. In addition, PrP was detected in ventral and dorsal roots of the cervical spinal cord, and within the spinal cord PrP could be traced in nerve tracts as far as the cerebral cortex. Similar patterns of PrP immunoreactivity were seen in two symptomatic and 18 apparently healthy lemurs in three different French primate centers, all of which had been fed diets supplemented with a beef protein product manufactured by a British company that has since ceased to include beef in its veterinary nutritional products. This study of BSE-infected lemurs early in their incubation period extends previous pathogenesis studies of the distribution of infectivity and PrP in natural and experimental scrapie. The similarity of neuropathology and PrP immunostaining patterns in experimentally infected animals to those observed in both symptomatic and asymptomatic animals in primate centers suggests that BSE contamination of zoo animals may have been more widespread than is generally appreciated.     

耐药突变选择窗与细菌耐药的动物体内研究

目的探讨耐药突变选择窗(MSW)与体内细菌耐药发生的相关性。方法建立兔组织笼金黄色葡萄球菌感染模型,给予不同剂量的左氧氟沙星灌胃治疗。抽取组织笼内组织液进行药物浓度测定,同时监测组织笼内细菌药物敏感性变化。结果当左氧氟沙星浓度位于MSW内时,导致金黄色葡萄球菌耐药突变体的选择富集,引起耐药。当药物浓度低于抑制菌群中99%细胞生长的最低药浓度或高于防耐药变异浓度时,无耐药出现。当左氧氟沙星浓度在MSW内的下部时,最容易选择出耐药突变体,且耐药出现最快。结论MSW在体内存在,保持抗菌药物浓度在MSW以上可以限制耐药突变体的选择富集。     

Single-Dose Intranasal Treatment with DEF201 (Adenovirus Vectored Consensus Interferon) Prevents Lethal Disease Due to Rift Valley Fever Virus Challenge

Rift Valley fever virus (RVFV) causes severe disease in humans and ungulates. The virus can be transmitted by mosquitoes, direct contact with infected tissues or fluids, or aerosol, making it a significant biological threat for which there is no approved vaccine or therapeutic. Herein we describe the evaluation of DEF201, an adenovirus-vectored interferon alpha which addresses the limitations of recombinant interferon alpha protein (cost, short half-life), as a pre- and post-exposure treatment in a lethal hamster RVFV challenge model. DEF201 was delivered intranasally to stimulate mucosal immunity and effectively bypass any pre-existing immunity to the vector. Complete protection against RVFV infection was observed from a single dose of DEF201 administered one or seven days prior to challenge while all control animals succumbed within three days of infection. Efficacy of treatment administered two weeks prior to challenge was limited. Post‑exposure, DEF201 was able to confer significant protection when dosed at 30 min or 6 h, but not at 24 h post-RVFV challenge. Protection was associated with reductions in serum and tissue viral loads. Our findings suggest that DEF201 may be a useful countermeasure against RVFV infection and further demonstrates its broad-spectrum capacity to stimulate single dose protective immunity.