烟粉虱卵黄原蛋白通过影响自噬反应调控共生细菌Rickettsia的丰度

【目的】探明烟粉虱Bemisia tabaci卵黄原蛋白(vitellogenin,BtVg)调控共生细菌Rickettsia丰度的分子机制。【方法】通过显微注射烟粉虱MEAM1隐种雌成虫dsRNA对BtVg进行RNAi,采用qRT-PCR检测烟粉虱MEAM1隐种雌成虫BtVg和自噬基因BtAtg8的表达量,统计雌成虫死亡率,采用qPCR检测雌成虫中Rickettsia的丰度,采用免疫荧光标记显微镜技术检测BtVg和BtAtg8在雌成虫卵巢管中的定位和表达以及Rickettsia在卵巢管和中肠中的丰度。饲喂烟粉虱MEAM1隐种雌成虫含雷帕霉素(10μmol/L)的人工饲料诱导自噬后,采用免疫荧光标记显微镜技术检测BtAtg8在卵巢管中的表达和定位,采用qPCR检测卵巢中Rickettsia丰度。【结果】与显微注射dsGFP的对照相比,显微注射dsBtVg后3 d时烟粉虱MEAM1隐种雌成虫BtVg的表达量显著降低,BtAtg8的表达量显著升高,死亡率显著升高,Rickettsia丰度显著降低,卵巢管中BtVg表达量明显降低,BtAtg8表达量明显升高,Rickettsia在卵巢管和中肠中的丰度明显减少。与饲喂含DMSO的人工饲料的对照相比,饲喂含雷帕霉素(10μmol/L)的人工饲料后5 d时烟粉虱MEAM1隐种雌成虫卵巢管中BtAtg8表达量显著升高;饲喂含雷帕霉素(10μmol/L)的人工饲料后3和5 d时,烟粉虱MEAM1隐种雌成虫卵巢中Rickettsia的丰度显著降低。【结论】结果提示,BtVg能够保护Rickettsia免受自噬的降解,明确了BtVg能够调控Rickettsia在烟粉虱体内的丰度。

Molecular evidence and phylogenetic delineation of spotted fever group Rickettsia species in Amblyomma ticks from cattle in Gauteng and Limpopo Provinces,South Africa

Objective:To determine the prevalence of tick-borne pathogens with a particular focus on Rickettsia spp.in ticks collected from cattle in Gauteng and Limpopo Provinces,South Africa.Methods:A total of 200 ticks were collected from cattle within the Madala livestock,Pretoria,Gauteng Province and in Mankweng Township,Polokwane,Limpopo Province in 2019.The ticks were morphologically identified and processed individually for a total genomic DNA extraction.Specific primers targetting ompA,ompB,and the 17KDa genes were used for a molecular screening and delineation of Rickettsia from the extracted genetic materials using polymerase chain reaction(PCR)technique.PCR amplicons of positive samples were sequenced bidirectionally using the Sanger sequencing method.Sequences generated were processed and analysed using appropriate bioinformatics software.Results:The ticks were morphologically identified as Amblyomma spp.PCR profiling of the genomic DNA samples revealed the presence of the Rickettsia pathogen in 42(21%)of the ticks collected from both Provinces.Out of the genes profiled,14(7%)were positive for 17KDa,42(21%)for ompA and 32(16%)were positive for ompB genes respectively.The nucleotide blast of the sequenced genomes showed high similarity,as high as 100% with other reference Rickettsia(R.)africae in the GenBank.The phylogenetic analysis of the sequences further validated them as R.africae with their characteristic clustering pattern with related reference sequences.Conclusions:There is an abundance of R.africae in Amblyomma ticks collected from cattle in the study areas.This has serious public health implications as individuals who accidentally get infested with the ticks could acquire R.africae.Hence,adequate precautions in terms of sensitization of farmers about the risk and mass mobilization drive to control the vectors in the areas are highly recommended to safeguard public health.

昆虫次生内共生菌Rickettsia研究进展

Rickettsia隶属于变形菌纲Proteobacteria的α亚群立克次体科Rickettsiaceae革兰氏阴性菌,是形态多样的次生真核细胞内共生菌。Rickettsia的功能是多样的,在一些宿主中为营养共生菌,在另一些宿主中为生殖调控因子,或以昆虫为载体的植物病原菌,此外,Rickettsia还能增强宿主抗药性,提高宿主抵御天敌、高温或者其它致死因素的能力。本综述主要从Rickettsia的起源、分类、在昆虫体内的分布、传播方式、与昆虫生殖调控的关系以及基因组进化等方面,简述Rickettsia的研究进展,重点提出了Rickettsia研究中一些尚未解决的问题,期望通过这些研究进一步明确Rickettsia与昆虫之间的互作关系。

新疆地区扇头蜱中Candidatus Rickettsia barbariae的检测与序列分析

为了解新疆地区血红扇头蜱和图兰扇头蜱中Candidatus Rickettsia barbariae立克次体感染状况,从新疆尉犁县采集蜱标本510只,其中血红扇头蜱90只,图兰扇头蜱420只。利用PCR方法对C.R.barbariae立克次体ompA、ompB、glt A和17k Da基因片段进行扩增。10只血红扇头蜱和53只图兰扇头蜱检测出C.R.barbariae阳性,阳性率分别为11.11%和12.62%。经序列分析,同一目的基因阳性序列相同,为同一序列。经比对分析,ompA基因序列与意大利撒丁岛的图兰扇头蜱中检测出的C.R.barbariae序列(EU272186.1)同源性为99.8%(583/584)。所测得ompB基因序列与意大利撒丁岛图兰扇头蜱中检测出的的C.R.barbariae Omp B基因序列(EU272187.1)同源性达100%(768/768)。所测得的glt A基因序列、17k Da基因序列与新疆花蠕形蚤Vermipsylla alakurt检测出的相应基因序列(KT284716.1、KT284715.1)同源性达100%(375/375、356/356)。结果表明,新疆尉犁县地区血红扇头蜱和图兰扇头蜱中存在C.R.barbariae自然感染,感染率较高。

昆虫共生细菌Rickettsia的研究进展

Rickettsia是传播和引起人类与其他脊椎动物疾病的胞内共生菌。引起脊椎动物疾病的这些Rickettsia,其部分生活史是在节肢动物体内完成的;而另外许多Rickettsia,其整个生活史都是在宿主节肢动物体内完成。为了叙述方便,把前者称为脊椎动物Rickettsia,后者称为节肢动物Rickettsia。过去的研究主要集中在医学上具有重大意义的脊椎动物Rickettsia,而关于节肢动物Rickettsia的生物学特性等研究则相对较少。近年来,研究者们加大了对昆虫Rickettsia的研究,发现昆虫Rickettsia广泛分布于昆虫中,且有两种存在形式。其可以通过垂直卵传的方式在世代间传递,也可以通过寄生蜂和寄主植物达到在昆虫之间传播的目的。昆虫Rickettsia可通过诱导孤雌生殖、诱导杀雄等方式影响宿主的生殖行为。其对不同宿主昆虫可产生对宿主有利或有害的作用;可增强宿主昆虫抵御高温和寄生蜂的能力,与宿主昆虫对药剂的敏感性相关。最后,昆虫Rickettsia具有一个简化的基因组,且存在进一步减小的可能性。

Identification of Orientia tsutsugamushi,spotted fever group and typhus group Rickettsia by duplex and nested PCR methods

Objective:To identify members of genera of rickettsia and O.tsutsugammhi simultaneously.Methods:Rapid and duplex and nested PCR methods have been established by designing primers based on the conserved regions of heat shock protein GroEL gene.345 mouse viscera samples including liver,spleen and kidney,96 Xenopsylla cheopis and 32 chiggers collected from Hongta areas of Yuxi city,Yunnan province were tested by the new PCR methods.Results:The result of the study showed that the new PCR methods could identify most members of genera -Rickettsia and Orientia simultaneously with 100%specificity and its sensitivity could test one copy per microliter.The results of detection prevalence of rickettsioses in mouse,flea and mites DNA samples showed that the total rickettsia infection rate in mouse was 34.78%(120/345).The total infection rates in R.typhi,O.t Karp and R.sibirica of mouse samples were 28.12%(97/345),19.71%(68/345) and O. 29%(1/345) respectively.Co-infection rates in R.typhi and 0.t Karp of mouse samples were 13.33%(46/ 345).O.t Karp type has been the main epidemic strain in these areas.Conclusion:We concluded that this PCR method could be used to detect multi-genera rickettsia simultaneously.Molecular evidences provided in this and previous studies strongly support that Hongta areas of Yuxi city are a natural focus for typhus and scrub typhus with the common occurrence of their confection.

法国野猪中Rickettsia raoultii的流行(英文)

目的调查和鉴定法国野猪身上Rickettsia raoultii的流行情况以及野猪作为贮存宿主所起的作用。方法收集法国2007年9月到11月的野猪身上的边缘革蜱163只。应用柠檬酸合成酶基因和外膜蛋白基因进行实时定量PCR、普通PCR技术及序列分析方法检测立克次体。结果 113只(69.3%)边缘革蜱立克次检测阳性。进一步鉴定为斑点热的两类病原,其中R.raoultii占57.5%(存在于65只蜱中)和R.slovaca占42.5%(存在于48只蜱中)。结论证明R.raoultii是野猪身上边缘革蜱的主要病原,边缘革蜱中感染率较高。应警惕野猪对立克次体的传播作用。

Studies on rickettsia-like organism (RLO) disease of tropical marine pearl oyster-Epidemiological investigation of RLO disease in juvenile populations of maricultured Pinctada maxima

The death which occurred in juvenile population was a severe problem in the course ofPinctada maxima artificial culture. With the methods of field investigation, histological study and statistic analyses, the epidemiological study was carried out on the disease and death in juvenile populations of Pinctada maxima in the Xinying Pearl Oyster Mariculture Farm of Lingao County (for the A, B and C batches of cultured juveniles hanged in the sea) and the Xincun Pearl Oyster Mariculture Farm of Lingshui County (for the D batch of cultured juvenile hanged in the pond), Hainan Province from November 1993 to April 1995.The results show that the deaths which occurred in juvenile populations of Pinctada maxima presented an outbreak pattern. The peak of mortality rates, in general, occurred in 4-to 6-month old pearl oyster juveniles, and the mortality rates gradully declined with the extention of pearl oyster age after cul-turing 8 months. The correlation between the mortality rates of juvenile populations and mean body lengths of juvenile populations show that the mortality rate become higher under 4 cm of mean body lengths of juvenile population and become obvious declined over 5 cm of mean body lengths. The peak of mortality rate occurred in 1-3 cm of mean body lengths. The results of histological observation showed that rickettsia-like organism inclusions were common, histological widespread infective agent among every batches (A, B, C and D) of cultured juvenile populations. The mean severity indices (SI) of rickettsia-like organism (RLO) infection were positively correlated with mortality rates of juvenile populations. After or within every peaks of RLO infection were all accompanied with the peaks of mortality rates of host populations and the mortality rates declined with decreased RLO infection. So the evidences of histological observation and epidemiology in this study indicated that rickettsia-like organism (RLO) may be as an important pathogenic organism of disease and death of cultured Pinctada maxima. In addition, a few ciliates were discovered only in part of batch A of cultured juvenile population. The mortality rates in juvenile populations were not correlated with the weekly mean temperature and salinity of sea water.

Studies on rickettsia-like organism (RLO)disease of tropical marine pearl oyster——Epidemiological investigation of RLO disease in larvae populations of maricultured Pinctada maxima

The epidemiological investigations on the disease and death in mature eggs, embryonic de- velopmental periods and larvae populations [including oocytes, fertilized ovum, early embryonic phase larvae (6 h), D-shaped phase larvae (24 h), early umbo phase larvae, umbo phase larvae, post um- bo phase larvae and eyespot phase larvae] in the Luhuitou Pearl Oyster Mariculture Farm of Sanya City, Hainan Province in April 1995 showed that there were two peaks of mortality rates which occurred in early umbo phase or umbo phase and post umbo phase (Figs 3-5 and Table 1) respectively from pre- embryonic developmental periods to larvae phases. It indicated that the onset and death of Pinctada maxima larvae populations followed a pattem of outbreak. Between the prevalence, intensity of RLO infection and the mortality rates of larvae populations were of obvious positively correlations. Generally, every peak of RLO infection is always follwed by a peak of mortality rate of larvae hosts, and the mor- tality rates of larvae hosts declined with the decreased RLO infection (see Figs 3-5 ). Under the trans- mission electron microscopy (TEM), no rickettsia-like organisms were discovered in oocytes, fertilized ovum and early embryonic phase larvae (6 h). The RLO inclusions occurred first in the D-shaped phase larvae (24 h) under histological examination. Absences of RLO in transmission electron exami- nation of oocytes of RLO-infected adult females, ferilized ovum and early embryonic phase larvae (6 h) indicated that RLO may not be transmitted transovarially. But RLO for host infection may be trans- mitted by contact transmission since RLO inclusions were first identified regularly in D-shaped phase larvae (24 h), while these D-shaped phase larvae were still unable to take food during hatching 24 hours. In addition, the result of epidemiological investigation showed that no odservable death occurred in D-shaped larvae populations, but early obvious death occurred in larvae populations in the seventh day after fertilization, in a rate of about 21. 8%. It indicated that there was a incubation peried from RLO infection for host (D-shaped phase larvae) to host onset and death occurred clinically.

Molecular Study of Rickettsiae in Serum Cattle

A molecular survey of 230 serum samples from cattle was studied by PCR-amplification of the citrate synthase gene gltA, the gene coding for protein 190 kDa—ompA—and the gene ompB. The study was carried out in the Junta of Castilla y León (northern Spain). The results suggest that the molecular study of the serum cattle would not make a good method in epidemiological studies on rickettsiae in this region. But it is necessary to continue and expand the work with more sensitive molecular methods.

Tick-Borne Rickettsial Pathogens in Rodents from Mexico

Tick-Borne Rickettsial Diseases (TBRD) are emerging zoonotic diseases, and a problem of human health and veterinary medication. The distribution of these diseases is related to the distribution of vector. The presence of pathogens in the host is a risk indicator of population exposure to these areas. A total of 478 tissues samples from rodents, A. phagocytophilum 18 (3.7%), E. canis 47 (9.8%), Rickettsia rickettsii 18 (3.7%) and E. chaffeensis 19 (3.9%) were detected using species-specific PCR assay. It is the first report in Mexico the presence of rodents infected with A. phagocytophilum and E. chaffeensis. The rodent Peromyscus spp. were the most commonly prevalent host of infection for all the bacteria’s. We have to consider as host of TBRD transmitter and provide a useful contribution to understanding their epidemiology. The health sector should be considered all the fevers of unknown causes in humans and animals in Mexico as infections by these vector-borne rickettsial pathogens.

Rickettsia-like organism infection associated with mass mortalities of blood clam, Tegillarca granosa, in the Yueqing Bay in China

A series of mass mortalities of the cultured blood clam, Tegillarca granosa, occurred in the Yueqing Bay of China from 2005 to 2009. An obligate intracellular prokaryote, designated as rickettsia-like organism （RLO）, was frequently found in the moribund or dead blood clam sample during ultra- structural examination. These organisms were usually round, ellipsoid or occasionally dumbbell- shaped, ranged from approximately 0.28 to 0.71 #m in size and had a trilaminar cell wall. Two reproductive modes of organisms, transverse binary fission and budding, were observed. The or- ganisms were able to form eosinophilic inclusions. Most inclusions were found within epithelial and connective tissues of the mantle, gills and digestive tube. The biological and morphological char- acteristics indicate that these organisms may belong to the family Rickettsiaceae. RLOs exhibited significant pathogenicity. Cytopathological examinations revealed extensive necrosis and destruc- tion in the infected cell. The degree of tissue destruction was positively related to the number of RLO inclusions in the tissues, and the cytopathological effects were positively related to the number of intracellular RLO. RLOs and their inclusions were discovered throughout different disease areas and in different time periods. The infection intensity of the RLOs was positively correlated with the mortality rate of clams. Therefore, RLO infection might be associated with mass mortalities of cultured blood clams in the Yueqing Bay.

Use of eschar swab DNA to diagnose Rickettsia conorii subspecies conorii infection in Crimea:A case report

Mediterranean spotted fever(MSF)has been diagnosed clinically in the Crimean Peninsula since the 1930s.We describe the recent illness of an elderly patient from Crimea who had developed a classic triad of MSF symptoms consisting of fever,maculopapular rash,and eschar.Clinical diagnosis of rickettsiosis was confirmed using real-time PCR and sequencing of 4 Rickettsia protein genes.The strain causing clinical illness was characterized as Rickettsia conorii subspecies conorii Malish 7.This report corroborates the utility of eschar swab material as a source of DNA for PCR-based diagnostics that enables timely patient treatment and management.

The presence of foci of Rickettsia conorii infection in China

China is not considered as an endemic area of Rickettsia conorii,so there is no routine clinical way to diagnose this infection.This study aims to determine whether 2 febrile patients who had a tick bite in East China were indeed infected with R.conorii.The citrate synthase gene(gltA)was amplified with universal rickettsial primers by real-time fluorescent PCR from the patients’blood samples.Nested PCR was used to amplify the outer membrane protein A gene(ompA)for positive specimens.PCR products were further identified and analyzed through nucleic acid sequencing.Positive amplification of the gltA and ompA genes was found in both patients.The nucleotide sequences(303 bp)of the ompA gene of the 2 patients had high homology(99%)with the R.conorii Indian tick typhus strain in GenBank.A more than 4-fold increase in IgG against R.conorii provided supportive evidence of SFG Rickettsia infection.And the rapid recovery after doxycycline treatment also supported a rickettsial cause for the disease.Physicians in East China should be aware of human infections with R.conorii.PCR-based diagnostic methods offer a rapid and precise way to diagnose rickettsiosis,improving patient identification and management.

新疆地区Rickettsiaraoultii分子流行病学研究

Rickettsia（R．）raoultii是一种新发现的斑点热群立克次体（spottedfevergroupRickettsiae），为胞内革兰阴性菌，主要通过蜱叮咬传播。其分布广泛，目前已从13个国家和地区的革蜱（D．silvarum）等10种蜱中检出。近期研究表明，R．raoultii是蜱致淋巴结病（tick．

烟粉虱内共生菌Rickettsia在植物体内的分布及转移效率初探

【目的】检测Q型烟粉虱Bemisia tabaci(Gennadius)体内Rickettsia的感染情况,研究分析Rickettsia共生菌经烟粉虱传入豇豆植物后的分布、转移效率等。【方法】以Q型烟粉虱为实验材料,利用常规PCR及荧光原位杂交技术(FISH),检测了烟粉虱体内Rickettsia的感染率,以及Rickettsia传入豇豆植物体内后的存留情况。【结果】Q型烟粉虱可以通过取食将Rickettsia传至豇豆植株内;接虫数量与Rickettsia传入效率及其在取食部位相邻的下部叶片中检测到的起始时间呈负相关;Rickettsia经烟粉虱取食传入豇豆叶片后,集中分布在叶片的韧皮部筛管中;基于16S r RNA的系统发育分析结果表明,Q型烟粉虱体内的Rickettsia与经取食传入豇豆叶片的Rickettsia高度同源。【结论】Rickettsia可以通过烟粉虱的取食传入植物体内,并且可以在相邻叶片之间转移传播,Rickettsia在由寄主昆虫向植株传播过程中高度保守。

内共生菌Rickettsia对烟粉虱生物学特性的影响

【目的】阐明次生共生菌Rickettsia对烟粉虱生物学特性的影响。【方法】Rickettsia阳性(B^+)和阴性(B^–)的烟粉虱在Rickettsia阳性棉花(C^+)和阴性棉花(C^–)上取食15 d,调查不同处理组烟粉虱的单雌产卵量、发育历期、存活率、成虫寿命以及F1代雌雄比。【结果】(1)Rickettsia与烟粉虱共生可显著缩短烟粉虱的发育历期,B^+C^+及B^+C^-两处理组烟粉虱卵-成虫的世代发育历期均短于B^–C^+及B^–C^–两处理组。(2)Rickettsia可以提高烟粉虱各龄期的存活率,B^+C^+、B^+C^-、B^–C^+、B^–C^–各处理组烟粉虱世代存活率依次呈下降趋势。(3)Rickettsia对烟粉虱种群的雌雄比也有重要的影响,B^–C^+和B^–C^–处理组中烟粉虱种群雌性比显著小于B^+C^+和B^+C^-烟粉虱处理组。(4)Rickettsia可以影响烟粉虱成虫的寿命及繁殖力,Rickettsia阳性烟粉虱处理组成虫寿命及平均单雌产卵量显著高于阴性处理组。【结论】Rickettsia与烟粉虱共生以及Rickettsia在棉花植株中的存留对烟粉虱的发育、存活以及成虫雌性比、寿命和繁殖力都有有利影响,且Rickettsia与烟粉虱共生时对烟粉虱的影响力度要明显强于Rickettsia存留于棉花植株中时对烟粉虱产生的影响。

Sequencing and comparison of the Rickettsia genomes from the whitefly Bemisia tabaci Middle East Asia Minor I

The whitefly, Bemisia tabaci, harbors the primary symbiont ＇Candidatus Portiera aleyrodidarum＇ and a variety of secondary symbionts. Among these secondary symbionts, Rickettsia is the only one that can be detected both inside and outside the bacteriomes. Infection with Rickettsia has been reported to influence several aspects of the whitefly biology, such as fitness, sex ratio, virus transmission and resistance to pesticides. However, mechanisms underlying these differences remain unclear, largely due to the lack of genomic information of Rickettsia. In this study, we sequenced the genome of two Rickettsia strains isolated from the Middle East Asia Minor 1 （MEAM1） species of the B. tabaci complex in China and Israel. Both Rickettsia genomes were of high coding den- sity and AT-rich, containing more than 1000 coding sequences, much larger than that of the coexisted primary symbiont, Portiera. Moreover, the two Rickettsia strains isolated from China and Israel shared most of the genes with 100% identity and only nine genes showed sequence differences. The phylogenetic analysis using orthologs shared in the genus, inferred the proximity of Rickettsia in MEAM1 and Rickettsia bellii. Functional analysis revealed that Rickettsia was unable to synthesize amino acids required for complementing the whitefly nutrition. Besides, a type IV secretion system and a number of virulence- related genes were detected in the Rickettsia genome. The presence of virulence-related genes might benefit the symbiotic life of the bacteria, and hint on potential effects of Rickettsia on whiteflies. The genome sequences of Rickettsia provided a basis for further understanding the function of Rickettsia in whiteflies.

New insights into the transovarial transmission of the symbiont Rickettsia in whiteflies

Endosymbiont transmission via eggs to future host generations has been recognized as the main strategy for its persistence in insect hosts;however,the mechanisms for transmission have yet to be elucidated.Here,we describe the dynamic locations of Rickettsia in the ovarioles and eggs during oogenesis and embryogenesis in a globally significant pest whitefly Bemisia tabaci.Field populations of the whitefly have a high prevalence of Rickettsia,and in all Rickettsia-infected individuals,the bacterium distributes in the body cavity of the host,especially in the midgut,fat body,hemocytes,hemolymph,and near bacteriocytes.The distribution of Rickettsia was subjected to dynamic changes in the ovary during oogenesis,and our ultrastructural observations indicated that the bacteria infect host ovarioles during early developmental stages via two routes:(i)invasion of the tropharium by endocytosis and then transmission into vitellarium via nutritive cord and(ii)entry into vitellarium by hijacking bacteriocyte translocation.Most of the Rickettsia are degraded in the oocyte cytoplasm in late-stage oogenesis.However,a few reside beneath the vitelline envelope of mature eggs,spread into the embryo,and proliferate during embryogenesis to sustain high-fidelity transmission to the next generation.Our findings provide novel insights into the maternal transmission underpinning the persistence and spread of insect symbionts.

新疆昆玉市某牧场羊蜱蝇中携带立克次体的检测及分析

试验旨在了解新疆昆玉市某牧场绵羊体外寄生虫羊蜱蝇所携带立克次体的感染情况。从该牧场绵羊体表采集羊蜱蝇82只,经形态学鉴定后,提取羊蜱蝇DNA,对立克次体17 kDa基因和geneD基因运用分子生物学技术进行PCR扩增,测序结果采用序列分析软件MEGA7.0构建系统进化树并进行遗传进化分析。结果显示,采集物种为羊蜱蝇并检测到其携带立克次体,经鉴定为暂定巴布瑞立克次体(Candidatus Rickettsia barbariae)。研究表明,新疆昆玉市某牧场绵羊羊蜱蝇携带立克次体,结果可为新疆地区羊蜱蝇携带的立克次体病原提供一定的数据支撑。