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.

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。

从厩真厉螨中检出与猫立克次体近缘的立克次体核酸片段

本研究用立克次体属特异的gltA和ompB基因扩增引物，从吉林长白县捕获鼠中分拣的534只厩真厉螨中扩得gltA和ompB基因片段。通过基因片段的序列测定、BLAST比对和系统发育分析，显示两个扩增基因与猫立克次体Rickettsia fells同源性最高（99％），证明该地区厩真厉螨携带与猫立克次体近缘的立克次体。

External quality assessment of the detection of rickettsioses in China

Objective:To report a training course on the laboratory diagnoses of rickettsioses that 10 provincial/city CDCs participated in laboratory external quality assurance(EQA) based on the serological specific antibodies detection and rapid PCR amplifying targeted genes of rickettsiae. Methods:An EQA program to evaluate the following laboratory procedures was developed to detect rickettsiae:(1) immunofluorescent assay(IFA) to detect specific antibodies of A. phagocytophilum,R.heilongjiangensis and 0.tsutsugamshi respectively.(2) Two sets of nested PCR were used amplifying groEL genes for most members of the family Rickettsiaceae and amplifying 16SrRNA genes for the most members of family anaplasmae,respectively.A scoring scheme based on the distribution of the median antibody titer values of the serologic assays was designed and a ranking list of the scores of the PCR results based on the detected minimal copy numbers of reference DNA was created.Results:Among nine laboratories who reported the results on time,eight laboratories gave acceptable serologic results,the other one provided an unacceptable antibody titer(1:2 vs median 1:64) results for 0.tsutsugamshi.The limits of detection(LOD) for the PCR amplifying for five references DNA ranged from 1copy/μL to 10~6 copy/μL.Conclusions:We successfully trained and popularized modern diagnostic methods of rickettsiae in 10 provincial CDCs in China and first conducted the EQA projects and evaluated the results.

桑粉虱4种次级内共生菌的检测分析

桑粉虱(Pealius mori)是对我国蚕区桑树造成严重危害的粉虱类害虫。应用Wolbachia次级内共生菌wsp基因和Rickettsia、Cardinium、Hamlitonella次级内共生菌16S rDNA特异引物对采自田间的桑粉虱样本基因组DNA进行PCR扩增、测序,通过分析测序得到的基因序列确定次级内共生菌种类,并对桑粉虱样本感菌情况进行检测分析。桑粉虱样本Rickettsia的扩增序列(963 bp)与烟粉虱(Bemisia tabaci)样本Rickettsia扩增序列(KY620205、GU563843、FJ915603)的碱基一致性为99.90%~100%;桑粉虱样本Cardinium的扩增序列(446 bp)与烟粉虱样本Cardinium扩增序列(FJ766341、GU563845、HG421084、KT827382)的碱基一致性为99.78%;桑粉虱样本Hamlitonella的扩增序列(694 bp)与烟粉虱样本Hamlitonella扩增序列(MH908669、KM197206、AB981348、AF400475)的碱基一致性为99.71%。次级内共生菌除Wolbachia外,Rickettsia、Cardinium、Hamlitonella均可感染桑粉虱。81份桑粉虱样本中,次级内共生菌Rickettsia、Cardinium、Hamlitonella的感染率分别为6.2%、11.1%、11.1%;Cardinium与Hamlitonella的共同感染率为11.1%,Rickettsia与Hamlitonella、Rickettsia与Cardinium、Rickettsia与Cardinium和Hamlitonella的共同感染率皆为6.2%;88.9%的样本没有检测到次级内共生菌感染。结果表明,桑粉虱体内检测到Rickettsia、Cardinium、Hamlitonella3种次级内共生菌感染,Hamlitonella、Cardinium的感染率相同并高于Rickettsia;桑粉虱体内3种次级内共生菌的感染率及共同感染率较低。研究结果可为桑粉虱内共生菌的鉴定和功能研究等提供参考。

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。

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.

Investigation of anaplasmosis in Yiyuan County,Shandong Province,China

Objective:To investigate the situation of anaplasmosis in Yiyuan county.Shandong Province. Methods:A total of 26 blood samples from febrile patients suspected of anaplasmosis,48 blood samples from healthy farmers,8 from dogs,and 10 from goats and 170 ticks were collected in the same area during 2005-2007,and detected by serological and molecular methods.Results: Eight confirmed cases and 6 probable cases were determined using serologic and molecular methods.The seroprevalence of Anaplasma phagocytophilum(A.phagocytophilum) was 26.7% in healthy cases.Nine out of 10 sheep samples and 7 out of 8 dog samples reacted positively to the A.phagocytophilum antigen.PCR amplification and sequencing of the 16SrRNA of,4. phagocytophilum gene showed that some samples from patients,goats and ticks were 100% identical.The seroprevalence of Rickettsia typhi was 22.9%,Orientia tsutsugamushi 6.3%, Rickettsia sibirica 27.1%,Coxiella burnetii 18.8%,Bartonella henselae 31.3%,and Borrelia burgdorferi 41.6%.Conclusions:It is important to make differential diagnosis of febrile patients and to apply treatment with specific antibiotics.It is needed to enforce essential prevention and control measures including tick control and to improve sanitation conditions.

Severe scrub typhus infection: Clinical features, diagnostic challenges and management

Scrub typhus infection is an important cause of acute undifferentiated fever in South East Asia. The clinical picture is characterized by sudden onset fever with chills and non-specific symptoms that include headache, myalgia, sweating and vomiting. The presence of an eschar, in about half the patients with proven scrub typhus infection and usually seen in the axilla, groin or inguinal region, is characteristic of scrub typhus. Common laboratory findings are elevated liver transaminases, thrombocytopenia and leukocytosis. About a third of patients admitted to hospital with scrub typhus infection have evidence of organ dysfunction that may include respiratory failure, circulatory shock, mild renal or hepatic dysfunction, central nervous system involvement or hematological abnormalities. Since the symptoms and signs are non-specific and resemble other tropical infections like malaria, enteric fever, dengue or leptospirosis, appropriate laboratory tests are necessary to confirm diagnosis. Serological assays are the mainstay of diagnosis as they are easy to perform; the reference test is the indirect immunofluorescence assay(IFA) for the detection of Ig M antibodies. However in clinical practice, the enzyme-linked immuno-sorbent assay is done due to the ease of performing this test and a good sensitivity and sensitivity when compared with the IFA. Paired samples, obtained at least two weeks apart, demonstrating a ≥ 4 fold rise in titre, is necessary for confirmation of serologic diagnosis. The mainstay of treatment is the tetracycline group of antibiotics or chloramphenicol although macrolides are used alternatively. In mild cases, recovery is complete. In severe cases with multi-organ failure, mortality may be as high as 24%.

Metabolic integration during the evolutionary origin of mitochondria

Although mitochondria provide eukaryotic cells with certain metabolic advantages, in other ways they may be disadvantageous. For example, mitochondria produce reactive oxygen species that damage both nucleocytoplasm and mitochondria, resulting in mutations, diseases, and aging. The relationship of mitochondria to the cytoplasm is best understood in the context of evolutionary history. Although it is clear that mitochondria evolved from symbiotic bacteria, the exact nature of the initial symbiosis is a matter of continuing debate. The exchange of nutrients between host and symbiont may have differed from that between the cytoplasm and mitochondria in modern cells. Speculations about the initial relationships include the following. (1) The pre-mitochondrion may have been an invasive, parasitic bacterium. The host did not benefit. (2) The relationship was a nutritional syntrophy based upon transfer of organic acids from host to symbiont. (3) The relationship was a syntrophy based upon H2 transfer from symbiont to host, where the host was a methanogen. (4) There was a syntrophy based upon reciprocal exchange of sulfur compounds.The last conjecture receives support from our detection in eukaryotic cells of substantial H2S-oxidizing activity in mitochondria, and sulfur-reducing activity in the cytoplasm.

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.

An unusual cause of optic neuritis:rickettsiosis disease

Optic neuritis(ON) may be associated to a range of autoimmune or infectious diseases.We report herein a case of ON induced by Rickettsia conorii.A 53-year-old woman presented with a recent decrease in visual acuity and headache.ON was diagnosed on the basis of ophthalmologic examination and flash visual evoked potentials.Etiological investigation made in our department eliminated first autoimmune disorders(vasculitis and connective tissue diseases).Rickettsial optic neuritis was confirmed by detection of specific antibodies in serum and the negativity of other serologic tests.An association between corticosteroids and cyclines was prescribed with improvement of visual acuity.

First molecular identification of multiple tick‑borne pathogens in livestock within Kassena‑Nankana,Ghana

The risk of pathogen transmission continues to increase significantly in the presence of tick vectors due to the trade of livestock across countries.In Ghana,there is a lack of data on the incidence of tick-borne pathogens that are of zoonotic and veterinary importance.This study,therefore,aimed to determine the prevalence of such pathogens in livestock using molecular approaches.A total of 276 dry blood spots were collected from cattle(100),sheep(95)and goats(81)in the Kassena-Nankana Districts.The samples were analyzed using Polymerase Chain Reaction(qPCR)and conventional assays and Sanger sequencing that targeted pathogens including Rickettsia,Coxiella,Babesia,Theileria,Ehrlichia and Anaplasma.An overall prevalence of 36.96%was recorded from the livestock DBS,with mixed infections seen in 7.97%samples.Furthermore,the prevalence of infections in livestock was recorded to be 19.21%in sheep,14.13%in cattle,and 3.62%in goats.The pathogens identified were Rickettsia spp.(3.26%),Babesia sp.Lintan(8.70%),Theileria orientalis(2.17%),Theileria parva(0.36%),Anaplasma capra(18.48%),Anaplasma phagocytophilum(1.81%),Anaplasma marginale(3.26%)and Anaplasma ovis(7.25%).This study reports the first molecular identification of the above-mentioned pathogens in livestock in Ghana and highlights the use of dry blood spots in resource-limited settings.In addition,this research provides an update on tick-borne pathogens in Ghana,suggesting risks to livestock production and human health.Further studies will be essential to establish the distribution and epidemiology of these pathogens in Ghana.

Mediterranean Spotted Fever Complicated with Thrombosis of the Left Central Retinal Vein: A Case Report and Literature Review

We describe a case of Mediterranean spotted fever complicated with thrombosis of the left central retinal vein. A 41-year-old woman patient living in the city Scodra was referred to our hospital in October 2017 for high fever 40°C, muscular and articular pains, severe headache, maculopapular rash, chills, photophobia and visual loss in his left eye. Ocular examination showed vision acuity: OD 8/10, OS 2/10. Funduscopic examination complemented with fluorescein angiography showed: optic nerve papilla with clear contours, diffuse hemorrhages in the contest off retinal vein thrombosis, white retinal lesions, vascular sheathing and macular cystoids edema. R. conorii antibodies were identified by ELISA anti Rickettsia conorii IgM, IgG and Weil-Felix Ox-19 test. Investigation of other infective agents and the immunological panel were negative. After treatment with doxycycline 200 mg/day for 10 days vision acuity was OD: 8/10 OS: 6/10;FO: There are less hemorrhages in fluorescein angiograph and OCT showed a reduced macular cystoids edema. Mediterranean spotted fever should be considered in the differential diagnosis of a patient who presents with an acute febrile disease accompanied by maculopapular rash especially in the seasons of spring, summer or autumn.

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.

Serological Evidence of Human Coinfection by Brazilian Spotted Fever and Bartonellosis

Brazilian spotted fever and bartonellosis are zoonotic, emerging and under diagnosed diseases. Pets may be co-infected by multiple pathogens and become transmissions sources to humans. The study reports the first case of active co-infection by Brazilian spotted fever and bartonellosis based on serological evidence. The authors aim to demonstrate the importance of performing systematic syndromic investigations on nonspecific febrile syndromes, guided by the epidemiological history and considering the possibility of co-infection by zoonosis sharing the same ecological niche.

Zoonotic Pathogens Detected in Ticks in Kenyan Game Reserves

Little is known on tick-borne pathogens and their role in disease in game reserves in Kenya. Ticks were collected by sterile forceps from restrained cattle hide and placed into labeled falcon tubes. Ticks were screened for pathogens by High Resolution Melting (HRM) analysis and sequencing of specific RT-PCR products of Anaplasma, Ehrlichia, and Rickettsia species. A total of 317 ticks (281 adult ticks and 36 nymphs) comprising seven species were collected around the Tsavo National Reserve (TNR) in Taita Taveta County with Amblyomma gemma being the most commonly collected species (n = 135, 42.6%). From near Shimba Hill game reserve (SHNR), a total of 240 adult’s ticks were sampled, representing eight species, with again Amblyomma gemma being the most sampled species (n = 156, 65%). From Tsavo, a total of three pools of Rhipicephalus appendiculatus were positive for Theileria parva, two pools of Rhipicephaline evertsi for Anaplasma platys and one pool of Amblyomma variegatum nymphs for Rickettsia africae. Rickettsia africae, which causes African tick-bite fever, was detected in two pools of Am. variegatum and one pool of Amblyomma gemma collected near Shimba Hill game reserve. Rickettsia sp. and Anaplasma sp. were detected in Am. gemma and Rh. evertsi respectively. Rickettsia aeschlimannii was detected in a pool of Am. gemma. These findings highlight the risk of transmission of zoonotic pathogens to humans in regions with high human-wildlife interfaces. Of specific importance, we provide evidence of R. aeschlimannii in A. gemma for the first time, representing a potential new R. aeschlimannii vectors.

Molecular detection and phylogenetic characterization of Rickettsia in ticks collected from leopard tortoise(Geochelone pardalis)in rural Zambia

In sub-Saharan Africa,limited studies have investigated zoonotic pathogens that may be harboured by ticks infesting reptiles such as tortoises.Here,we report the presence of pathogenic Rickettsia in ticks(Amblyomma marmoreum)collected from the leopard tortoise(Geochelone pardalis)in rural Zambia.Using polymerase chain reaction,56%(49/87)of ticks were positive for the Rickettsia outer membrane protein(ompB)gene.Multi-locus sequence and phylogenetic analysis based on the ompB,ompA,and citrate synthase(gltA)genes showed that the ticks carried R.africae,and other Rickettsia spp.closely related to R.raoultii,R.massiliae,R.tamurae and R.monacensis.Given the proximity between humans,livestock,and wildlife in these habitats,there exists a considerable risk of transmission of zoonotic Rickettsia to human populations in this rural setting.These results call for heightened awareness and further research into the dynamics of tick-borne diseases in regions where humans and animals coexist,particularly in the context of tortoise-associated ticks as vectors.Understanding and addressing these potential disease vectors is crucial for effective public health measures and the prevention of Rickettsia zoonoses.