关键词： HBV   树鼩   动物模型   TLR-4   IL-6  

摘要： 目的通过对新生树鼩接种中、高滴度的人HBV血清,建立树鼩乙肝动物模型;观察树鼩感染HBV后体内相关免疫因子TLR-4和IL-6的动态变化,初步探讨HBV感染的免疫机制。方法第一部分用中、高滴度的人HBV血清接种新生树鼩,建立新生树鼩乙肝动物模型。将新生树鼩随机分组,中滴度组接种含人HBV血清（104-106IU/ml）,高滴度组接种含人HBV血清（107IU/ml）,对照组不接种。从接种后第8周开始,每隔2周经股静脉采血1次,接种后12周开始,每3个月行肝活检术取肝组织。ELISA法检测血清中HBs Ag的表达,荧光定量PCR（探针法）检测肝组织与血清中HBVDNA含量,免疫组化观察肝组织中HBs Ag表达情况。第二部分观察成功感染HBV的动物体内TLR-4与IL-6的动态变化。将第一部分实验中HBs Ag和（或）HBVDNA阳性树鼩确定为感染组,6只未接种且经检测HBs Ag和HBVDNA均为阴性的树鼩为对照组。ELISA检测两组动物血清中TLR-4与IL-6的表达情况,荧光定量PCR检测两组动物肝组织中TLR-4与IL-6的m RNA水平表达情况,免疫组化法观察两组动物肝组织中TLR-4与IL-6的表达情况。结果第一部分共有34只新生树鼩接种人HBV血清,高滴度组（107IU/ml）与中滴度组（104、105、106IU/ml）各17只动物。结果如下:（1）血清HBs Ag结果:在观察期内（8周24周）高滴度组共检测到8只动物出现HBs Ag阳性（8/17,47.05%）,中滴度组和对照组的HBs Ag均为阴性。在8只阳性的动物中,有7只动物在第8周即可检测出HBs Ag阳性,1只动物在22周第一次检测到HBs Ag阳性,且其中5只动物在整个观察期内（8周24周）HBs Ag持续阳性,最长可持续到第48周。（2）血清HBVDNA结果:按照临床检验标准,我们将HBVDNA≥500IU/ml定为阳性标准。在观察期内（8周24周）,高滴度组有11只动物血清检测出HBVDNA阳性（11/17,64.7%）,中滴度组有5只可检测出HBVDNA,但其拷贝数均低于阳性标准,对照组血清HBVDNA均为阴性。大部分阳性动物在第8周即可检测出,持续时间最短14周最长可达72周。11只阳性动物HBVDNA水平多在103IU/ml左右,少数可达到104IU/ml,随时间的延长,HBVDNA载量逐渐下降。（3）肝组织HBVDNA结果:高滴度组12周与24周肝组织检测都有12只动物出现HBVDNA阳性（12/17,70.5%）;中滴度组在第12周有5只检测出HBVDNA阳性（5/17,29.4%）,24周有3只检测出HBVDNA阳性（3/17,17.6%）,对照组肝组织未检测出HBVDNA阳性。高滴度组阳性动物HBVDNA水平多在103～104IU/ml左右,少数可达到105IU/ml,中滴度组阳性动物HBVDNA水平多在103IU/ml左右。（4）免疫组化结果显示,高滴度组与中滴度组大部分动物肝组织HBs Ag呈阳性表达,高滴度组肝组织HBs Ag阳性动物为12只（12/17,70.5%）,中滴度组肝组织HBs Ag阳性动物为5只（5/17,29.4%）,对照组染色均为阴性。阳性染色物为棕黄色,主要表达于肝细胞浆和细胞膜。第二部分将第一部分实验中12只HBs Ag和（或）HBVDNA阳性树鼩确定为感染组,6只未接种且经检测HBs Ag和HBVDNA均为阴性的树鼩为对照组。两组动物体内TLR-4与IL-6检测结果如下:（1）ELISA检测感染组与对照组血清中TLR-4结果显示,感染组动物血清中TLR-4的表达在第8w、12w、16w、20w、24w均比对照组高,差异均有统计学意义（p<0.05）,随时间的延长,血清TLR-4的表达呈降低趋势。感染组动物血清中IL-6的表达在8w、12w、16w、20w、24w均比对照组高,在第8w、12w、16w有显著性差异（p<0.05）,随时间的延长,血清中IL-6表达稍有下降。（2）q RT-PCR检测树鼩肝组织中TLR-4、IL-6m RNA的表达结果显示,感染组TLR-4 m RNA在12周、24周肝组织中的表达水平均比对照组高,差异均具有统计学意义（p<0.05）;感染组IL-6m RNA在12周、24周肝组织中的表达水平均比对照组高,第12周有显著性差异（p<0.05）。（3）免疫组化观察TLR-4在感染组中阳性表达率为（10/12,83.3%）,在对照组中阳性表达率为（1/6,16.6%）,感染组与对照组间差异具有统计学意义（p<0.05）。IL-6在感染组中阳性表达率为（9/12,75%）,在对照组中阳性表达率为（1/6,16.6%）,感染组与对照组间有显著性差异（p<0.05）。结论1.高滴度（1

关键词： animal welfare   animal welfare assessment   aquatic mammals   zoo animals   zoo inspection   Decision Tree   bottlenose dolphin   Antillean manatee  

摘要： During the last few decades, zoos and aquaria have made great improvements in their exhibit designs, feeding routines, social housing conditions, mixed species presentations, and environmental enrichment, as well as in the prevention of infectious and parasitic diseases, to enhance animal welfare. To monitor the effectiveness of all these changes, animal welfare science is needed. It is important to evaluate animal response by applying welfare metrics that include behaviour and/or physiology. To get a state-of-the-art overview of animal welfare metrics, Zoo Nuremberg organized a workshop in May 2016, inviting scientists from different disciplines. The workshop dealt with the challenges we face in developing and applying animal welfare indicators for zoo and aquarium animal species and clearly emphasized the need to assess the welfare of these animals. It was shown that animal welfare is science, and many scientific methods are available to assess welfare objectively at the species level, at least for some vertebrate species. However, it remains a challenge to apply different scientific methods for assessment of the broad species collection(s) of zoological parks and the huge number of individuals. The discussion also revealed that the assessment of animal welfare is a topic of much debate due to the complexity and practical implications of the evaluation. As a result, a written report was produced, Assessment of Welfare of Marine Mammal Species in Zoological Parks (Zoo Nuremburg, 2016), and a proposal for an animal welfare Decision Tree was created. The Decision Tree includes four different steps, involving keepers, veterinarians, biologists, and animal welfare inspectors: 1st Step: Survey - including life history, health protocol and nutrition plan, physical environment, animal management, and behavioural support system 2nd Step: Theoretical Analysis - including data analysis, data correlation, data evaluation, and preliminary report 3rd Step: In Situ Inspection -

关键词： 鼻咽癌   人源性EBV   树鼩   动物模型   感染阶段   基因测序   环磷酰胺  

摘要： 作为一种致瘤病毒,EB病毒(Epstein-Barr virus,EBV)与多种肿瘤的发生密切相关,如:伯基特淋巴瘤、鼻咽癌、肺癌、胃癌等。然而,EBV参与鼻咽癌发生发展的机制研究尚未明确,其因在于缺乏有效的细胞和动物模型。虽然目前已利用新世界猴、SCID小鼠、转基因动物和兔子、豚鼠等动物构建EBV相关动物模型,但因受限于感染率或动物机体的非自然免疫状态,仍未探明EBV参与NPC发生发展的机制。因此,急需寻找一种高感染率的具有正常机体免疫力的动物构建EBV感染的动物模型。在本课题组前期研究中,已证实机体免疫正常的树鼩可用于构建B95-8细胞系来源的EBV感染的动物模型,感染率可达80%,证实树鼩具有构建EBV感染的动物模型的潜力。因此,本实验希望能利用树鼩构建鼻咽癌患者来源的EBV感染的动物模型。首先,利用鼻咽癌患者来源EBV感染树鼩,拟构建EBV感染的树鼩动物模型,旨在探究人源性EBV感染树鼩的可能性。然后,通过摸索接种剂量,构建长期感染的动物模型,探讨EBV在树鼩内形成感染的时间及感染阶段分类等。再者,通过对BARF1基因片段进行测序和分析,以期阐明EBV分株与亚型。最后,应用环磷酰胺叠加EBV感染因素探索是否可诱导树鼩体内形成肿瘤。总之,本课题希望能通过一系列基础研究为后续开发抗EBV感染的药物和疫苗、研究EBV感染和致瘤机制、深入研究EBV与鼻咽癌发生发展的关系等提供依据。方法:1、鼻咽癌患者来源的EBV状况探索:收集10例血清EBVCA-IgA和EBNA-IgA为阳性的鼻咽癌患者的鼻咽部组织、抗凝外周血和不抗凝外周血,分别用于提取组织悬液、单个核细胞(PBMC)和血清。提取DNA后,利用qPCR方法检测各样品中的EBV DNA拷贝数。同时,使用透射电镜观察3类生物样本中是否有EBV病毒颗粒,再用免疫电镜技术验证。2、树鼩感染EBV需要的EBV DNA拷贝数探究:根据方法1的结果,将获取的鼻咽癌组织悬液按照EBV DNA拷贝数稀释成2×10～6 copies/μl、2×10～4 copies/μl、2×10～2 copies/μl、2×10～0 copies/μl。设置A-D四个实验组,每组5只树鼩,E组为对照组,含2只树鼩。实验组树鼩通过喷鼻的方式各接种100μl EBV混悬液,对照组给予同等剂量生理盐水喷鼻。每周为各组树鼩抽血,检测其感染程度。在连续观察2个月后,使用免疫抑制剂—环磷酰胺,按照20mg/Kg进行腹腔注射,连续观察3周评估感染效果。3、长期EBV感染树鼩模型的构建和观察:设置实验组树鼩10只,对照组3只。将鼻咽癌患者来源的含EBV病毒混悬液通过喷鼻方式感染实验组树鼩,对照组喷鼻同等体积的生理盐水,定期用qPCR、RT-PCR、ELISA的方法监测所有树鼩外周血PBMC中EBV DNA拷贝数变化、EBV相关基因片段的表达、血清EBVCA-IgG水平。通过对死亡树鼩的器官组织行免疫组织、Western blot和原位杂交检测,了解EBV相关蛋白和EBERs的表达情况。4、BARF1基因测序:采用RT-PCR和cDNA扩增产物基因测序等方法,对BARF1基因进行检测,并参照已发表的文献中人类疱疹病毒4型(HHV-4)、鼻咽癌EBV病毒株(HNNPC1-9)、淋巴瘤病毒株(B95-8)、胃癌病毒株(Akata-GC1)等序列,对所得序列结果进行比对分析,以寻找树鼩被感染的病毒基因型。5、应用环磷酰胺叠加EBV感染探索体内成瘤方法:通过对实验组树鼩连续应用环磷酰胺1周后,饲养3月处死动物。经石蜡包埋和病理切片后,HE染色观察实验组和对照组树鼩的鼻咽部和器官是否有肿瘤形成;免疫组化染色和原位杂交等方法,检测EBV相关蛋白和EBERs在树鼩组织中的表达。结果:1、NPC组织中EBV DNA拷贝数含量最高,可观察到EBV颗粒。通过鼻咽癌组织、外周血单个核细胞和血清三类生物样本的PCR检测,三者的EBV DNA拷贝数分别是(14.3±5.41)×10～7、(1.58±1.04)×10～4、(11.6±2.96)×10～2 copies/DNA,可知鼻咽癌组织中EBV DNA拷贝数的含量最高;透射电镜可观察到疱疹病毒样颗粒存在在7份NPC患者的生物样本中,免疫电镜可观察到8例患者的生物样本中EBV颗粒存在。2、本次实验中获得的树鼩感染EBV需要的EBV DNA拷贝数临界值为2×10～2 copies。通过对A-D四组树鼩外周血中BamHI-W、BZLF1、BARF1、BHRF1、LMP1、EBNA1等EBV相关基因片段的RT-PCR技术检测和分析可知,A组和B组中的10只树鼩均获得EBV感染,且出现感染的时间早,表达的目的指标强。虽然C组中2只树鼩被EBV感染,但是它们外周血中早期EBV相关指标表达不明显,仅在第2个月末

关键词： 轮状病毒   树鼩   细胞   感染   动物模型  

摘要： 轮状病毒(Rotavirus,RV)感染是引起婴幼儿及其他幼龄哺乳动物非细菌性腹泻最重要的原因,全世界因急性腹泻入院的儿童中,约60%左右为RV感染引起,因轮状病毒每年导致的死亡病例高达80万。我国儿童RV感染率和住院率分别为28%-65%和30-50%,造成相当大的的社会负担和经济损失。目前针对轮状病毒的治疗没有有效的药物,市场上现有的疫苗在不同地区作用效果差距较大,价格昂贵且存在发生潜在感染的安全隐患,因此研发安全性可靠、高效廉价的疫苗的成为当前比较重要的项目。而动物模型不仅可以研究病毒的感染进程和致病机理,同时也可以为疫苗的研发和药物的筛选提供基础性保障。目前轮状病毒的感染动物模型主要包括乳鼠、无菌猪、猴和大鼠等,由于这些模型在亲缘关系、实验周期、解剖学特性等方面的局限性,并没有动物模型的验证和清晰阐明病毒的致病机制。近年来,树鼩由于体型小、繁殖快、饲养成本低以及解剖学特性与人相近而受到重视,并且已经初步证实可以感染轮状病毒,因此成为研究轮状病毒的比较优势的动物。首先选择G1P[8]型的WA株和从临床病人粪便中分离培养的G9P[8]的JM-103作为研究对象,将病毒接种到MA-104细胞和Vero细胞中培养,通过形态学观察、测定病毒滴度等方法,筛选WA株作为优势病毒,确定MA-104细胞为宿主细胞。再将病毒进行浓缩,并且将测定病毒滴度的TCID技术条件进行优化,与此同时,建立了轮状病毒基因检测的PCR技术,为后续的实验做好准备。接着获取树鼩的原代细胞,将树鼩原代肾细胞、小肠上皮细胞进行培养、纯化,获得处于生长期的细胞再感染病毒,通过测定细胞悬液中病毒的滴度来鉴定病毒感染体外细胞的增殖特性。最后用高滴度的轮状病毒WA株通过灌胃的方式感染树鼩,记录树鼩的体重、体温、以及粪便等级和活动情况,采集树鼩不同时间段的血液、粪便、唾液等样本,同时将严重腹泻的树鼩处死获取体内组织,再用PCR技术和WB方法检测轮状病毒的表达。通过上述方法的建立全文结果如下:1.确定了高毒力和高感染力的WA株病毒和感染之后CPE更为明显的MA-104细胞用于后续实验。同时优化PEG-6000浓缩病毒的方法,获得高毒力、高滴度的轮状病毒WA株作为后续感染实验的材料。使用TCID50测定病毒滴度,通过对比不同条件的病毒滴度,建立了病毒激活的最优条件是胰酶终浓度20ug/ml,孵育时间60min。同时根据轮状病毒WA株感染增殖特性和细胞病变效应,建立轮状病毒RT-PCR检测和验证体系。2.使用0.25%的胰蛋白酶-EDTA消化法和机械振荡联合作用,成功分离获得树鼩原代肾细胞;用胶原酶Ⅺ型和中性蛋白酶I型混合消化液成功获取小肠上皮细胞。所分离出来的树鼩原代肾细胞、小肠上皮细胞贴壁速度快,生长状态好。通过利用树鼩原代细胞与成纤维细胞对胰蛋白酶敏感度的差异及贴壁时间的不同这两种特点在传代培养过程中对细胞进行纯化,得到了不含杂质细胞的树鼩原代细胞。同时对分离的树鼩原代细胞利用CCK法测定细胞增殖曲线,检测细胞的生长曲线。轮状病毒感染树鼩原代细胞,观察不同时间的细胞病变效应,感染72h原代细胞出现脱落、变圆现象。轮状病毒感染树鼩原代细胞之后,收取细胞悬液,检测细胞悬液中病毒的滴度,验证了轮状病毒WA株可以感染树鼩原代细胞。3.轮状病毒感染树鼩的最佳方式是灌胃,灌胃不同年龄的树鼩选择不同的剂量和毒力的病毒,我们选用每只树鼩灌胃2m L的病毒滴度为1?10(TCID)轮状病毒细胞悬液;感染病毒之后的树鼩,通过生命体征、活动情况、体温、体重、腹泻以及粪便等级等现象初步断定树鼩可以感染轮状病毒WA株,并且各个指标相互佐证,呈现相近规律;通过对感染树鼩的粪便、唾液、血液中轮状病毒的检测,证实轮状病毒在树鼩体内发生复制与增殖,并不简单的只是病毒的排出。我们初步建立了轮状病毒感染树鼩的动物模型,该模型的初步研究对于轮状病毒致病机制和疫苗的研发具有重要的参考意义。

关键词： Hollow-dependent wildlife   Mechanically created hollows   Hollow-bearing trees   Artificial hollows   Nest boxes   Bat boxes  

摘要： Tree hollow scarcity is a threat to cavity-dependent vertebrate wildlife world-wide across many landscapes. Currently, only nest boxes are commonly used to mitigate or offset lost cavity-bearing trees, with a number of shortfalls reported using this technique. There is a need to trial alternative techniques to improve artificial cavity provisions. This preliminary study investigated the use of carving hollows directly into tree trunks using chainsaws. Sixteen hollows of two simple cavity types were created in a timber production forest in southeastern Australia. One cavity type comprised a basal entrance (38 mm in diameter) which provided a space above the entrance, intended for bats, and the other cavity type provided a space below a 38 mm or 76 mm entrance, intended for marsupials and birds. Five species used the hollows over a 15-month cavity monitoring period;feathertail glider (Acrobates pygmaeus) (in 75% of the hollows), brown antechinus (Antechinus stuartii) (75%), sugar glider (Petaurus breviceps) (63%), long-eared bat (Nyctophilus sp.) (50%) and white-throated tree creeper (Cormobates leucophaea) (25%). Camera monitoring revealed hollow inspection after only one day post hollow creation by white-throated tree-creepers and feathertail gliders, and nest building by white-throated treecreepers after three days. No hollow host tree failure occurred for either of the two hollow wall widths trialled over two years of monitoring tree stability. Wound-wood formed and partly enclosed the hollows' faceplates over time, improving the sturdiness of the hollow and likely resulted in hollows closely resembling natural cavities. Mechanically created tree hollows have a broad application potential across many landscapes with the prospect to overcome some of the reported drawbacks of nest boxes. More research is required to document long-term performance and effectiveness of this technique.

摘要： Internally transcribed spacer (ITS) rRNA profiling is a novel tool for detailed analysis of microbial populations at low taxonomic ranks. Here we exploited this approach to explore species-level biogeography of the Bifidobacterium genus across 291 adult mammals. These include humans and 13 other primates, domesticated animals, such as dogs, cats, cows, sheep, goats, horses and pigs, and 46 additional species. The collected profiles revealed the presence of 89 putative novel bifidobacterial taxa in addition to 45 previously described species. Remarkably, in contrast to what is currently known for many gut commensals, we did not observe host-specialization among bifidobacterial species but rather their widespread distribution across mammals. Moreover, ITS rRNA profiling of wild relatives of domesticated dogs, rabbits and pigs clearly indicates that domestication and close contact with humans have impacted on the composition of the fecal bifidobacterial population. These data were complemented by analysis of bifidobacterial communities in milk of eight mammalian families, showing that bifidobacteria represent prototypical early gut microbiota members which are inherited by newborns from their lactating mother. Thus this study highlights the role of bifidobacteria as pioneering gut colonizers of a wide range of mammals.

关键词： Biodiversity patterns   Community assembly   Environmental change   Plant-herbivore interactions   Species-energy relationship   Species turnover  

摘要： In forests, local site conditions can affect both trees and herbivores and hence site-related factors act indirectly on herbivores mediated by tree growth rates. Here, tree foliage represents a fundamental prerequisite for insect herbivore development providing energy in the form of plant tissue quality. Resource-based theories, on the other hand, assume that the synthesis of defensive compounds is a trade-off with growth and peaks at low resource availability. However, the extent to which plant tissue quality in response to site productivity is relevant in the species-energy relationship is unknown. Therefore, we aimed at a better understanding of the form and structure of the species-energy relationship in forest insects. We used census data of foliage-feeding insects along a productivity gradient of Scots pine forests defined by relative growth rates of trees (RGR). As a result, diversity monotonically increases with decreasing RGR (as a proxy for energy) during almost two decades of sampling. Herbivore assemblages become more similar with available energy as species turnover linearly decreases and proportions of sites occupied by individual species rise. The results suggest that tree growth rate influences herbivore dynamics in this system by altering the chemical composition of needles, without necessarily affecting the form in the relationship. The site-specific resource availability requires trees to adjust their allocation to synthesis of carbon-based secondary metabolites or growth, which then results in fundamental differences in herbivore dynamics at low vs. lowest RGR (regular cycles (dominance) vs. dampened cycles (evenness)). However, these differences inevitably demonstrate that species richness is not necessarily a result of more individuals and implicate that different mechanisms are involved (facilitation vs. competition/temporal heterogeneity). The resulting pattern and structure of foliage-feeding insects advance our understanding of herbivore dy

关键词： bird diversity   ecological facilitation   Macrotermes   savanna dynamics   ungulates  

摘要： Vast areas of the African savanna landscapes are characterized by tree-covered Macrotermes termite mounds embedded within a relatively open savanna matrix. In concert with termites, large herbivores are important determinants of savanna woody vegetation cover. The relative cover of woody species has considerable effects on savanna function. Despite the potentially important ecological relationships between termite mounds, woody plants, large herbivores, and birds, these associations have previously received surprisingly little attention. We experimentally studied the effects of termites and large herbivores on the avian community in Lake Mburo National Park, Uganda, where woody vegetation is essentially limited to termite mounds. Our experiment comprised of four treatments in nine replicates;unfenced termite mounds, fenced mounds (excluding large mammals), unfenced adjacent savanna, and fenced savanna. We recorded species identity, abundance, and behavior of all birds observed on these plots over a two-month period, from late dry until wet season. Birds used termite mounds almost exclusively, with only 3.5% of observations occurring in the treeless intermound savanna matrix. Mean abundance and species richness of birds doubled on fenced (large herbivores excluded) compared to unfenced mounds. Feeding behavior increased when large mammals were excluded from mounds, both in absolute number of observed individuals, and relative to other behaviors. This study documents the fundamental positive impact of Macrotermes termites on bird abundance and diversity in an African savanna. Birds play crucial functional roles in savanna ecosystems, for example, by dispersing fruits or regulating herbivorous insect populations. Thus, the role of birds in savanna dynamics depends on the distribution and abundance of termite mounds.

关键词： 野生动物   管护责任   国有林场   护林员   海峰   城口县  

摘要： 【我的敝赘】2017年10月13日，城口县国有林场护林员陶海峰按例巡视其管护责任片区。茂密林间，陶海峰缓步穿梭，不时四处张望，仔细查看林中各个角落，以防疏漏可能存在的火睛。二十余年如一日，陶海峰在巡山护林过程中始终保持高度警惕。

关键词： Pan troglodytes   Meat eating   Early hominins   Ugalla   Predation   Faunivory  

摘要： There is broad consensus among paleoanthropologists that meat-eating played a key role in the evolution of Homo, but the details of where, when, and why are hotly debated. It has been argued that increased faunivory was causally connected with hominin adaptation to open, savanna habitats. If savanna-dwelling chimpanzees eat meat more frequently than do forest chimpanzees, it would support the notion that open, dry, seasonal habitats promote hunting or scavenging by hominoids. Here we present observational and fecal analysis data on vertebrate consumption from several localities within the dry, open Ugalla region of Tanzania. Combining these with published fecal analyses, we summarize chimpanzee vertebrate consumption rates, showing quantitatively that savanna chimpanzee populations do not differ significantly from forest populations. Compared with forest populations, savanna chimpanzees consume smaller vertebrates that are less likely to be shared, and they do so more seasonally. Analyses of chimpanzee hunting that focus exclusively on capture of forest monkeys are thus difficult to apply to chimpanzee faunivory in open-country habitats and may be misleading when used to model early hominin behavior. These findings bear on discussions of why chimpanzees hunt and suggest that increases in hominin faunivory were related to differences between hominins and chimpanzees and/or differences between modern and Pliocene savanna woodland environments. (C) 2017 Elsevier Ltd. All rights reserved.