Pyronaridine combined with diminazene aceturate inhibits Babesia in vitro and in vivo

Objective:To evaluate the combination therapy of pyronaridine tetraphosphate and diminazene aceturate against Babesia in vitro and in vivo.Methods:Bioinformatic analysis was performed using atom pair fingerprints.An in vitro combination test was performed against Babesia bovis and Theileria equi.Moreover,the in vivo chemotherapeutic efficacy of pyronaridine tetraphosphate in combination with diminazene aceturate was investigated against the growth of Babesia microti in mice using a fluorescence inhibitory assay.Results:Pyronaridine tetraphosphate and diminazene aceturate exhibited nearly similar molecular weights.The in vitro combination of pyronaridine tetraphosphate and diminazene aceturate was synergistic on Babesia bovis and additive on Theileria equi.In addition,5 mg/kg pyronaridine tetraphosphate combined with 10 mg/kg diminazene aceturate inhibited Babesia microti growth significantly compared with those observed after treatment with 25 mg/kg diminazene aceturate alone from day 6 post treatment to day 12 post treatment.The combination therapy also normalized the hematological parameters of infected mice.Conclusions:An oral dose of pyronaridine tetraphosphate combined with a subcutaneous dose of diminazene aceturate inhibits Babesia in vitro and in mice,suggesting it might be a new paradigm for the treatment of babesiosis.

在中国新发现的一种羊的巴贝斯虫(Babesia)未定种

在中国甘肃某地自然感染发病绵羊的血液涂片中，观察到一种大型的巴贝斯虫（Ｂａｂｅｓｉａ）。虫体形态具多形性，有双梨籽形、单梨籽形、圆环形、棒状、不规则形、逗点形和三叶形。典型双梨籽形虫体的大小为１．８～２．５μｍ×０．９～１．８μｍ，平均（２．２１±０．１２）μｍ×（１．１７±０．１８）μｍ。个别红细胞中寄生有４个或８个虫体。该虫种特征性的形态是三叶形。它是相连的双梨籽形虫体两尖端的核质延伸（出芽），并生出原生质，发育成一个完整的梨籽形虫体，与母体共同排列形成三叶形的。该虫种可引起绵羊严重发病和死亡。本研究对自然感染发病羊的临床症状和死亡羊的病理变化进行了观察。该虫种的形态大小和致病力，与莫氏巴贝斯虫（Ｂ．ｍｏｔａｓｉ）和粗糙巴贝斯虫（Ｂ．ｃｒａｓｓａ）明显不同。

Detection of Babesia bovis in blood samples and its effect on the hematological and serum biochemical profile in large ruminants from Southern Punjab

Objective:To determine the presence of Babesia bovis(B.bovis) in large ruminants in southern Punjab and its effect on hematological and serum biochemical profile of host animals.Methods:Blood samples were collected from 144 large ruminants,including 105 cattle and 39 buffaloes,from six districts in southern Punjab including Multan,Layyah,Muzaffar Garh,Bhakar,Bahawalnagar and Vehari.Data on the characteristics of animals and herds were collected through questionnaires.Different blood(hemoglobin,glucose) and serum(ALT,AST,LDH,cholesterol)parameters of calves and cattle were measured and compared between parasite positive and negative samples to demonstrate the effect of B.bovis on the blood and serological profile of infected animals.Results:27 out of 144 animals.from 5 out of 6 sampling districts,produced the541-bp fragment specific for B.bovis.Age of animals(P=0.02).presence of ticks on animals(P=0.04)and presence of ticks on dogs associated with herds(P=0.5) were among the major risk factors involved in the spread of bovine babesiosis in the study area.ALT concentrations were the only serum biochemical values that significantly varied between parasite positive and negative cattle.Conclusions:This study has reported for the first time the presence of B.bovis in large ruminant and the results can lead to the prevention of babesiosis in the region to increase the livestock output.

Inhibition of <i>Ehrlichia canis</i>and <i>Babesia canis</i>transmission among ticks fed together on dogs vaccinated with Bm86 antigen

GAVAC (Heber Biotec S.A, Havana, Cuba) is a commercially available vaccine developed with the Rhipicephalus (Boophilus) microplus Bm86 recombinant antigen. Bm86 is a “concealed” antigen that is present in the plasmatic membrane of tick gut epithelial cells with unknown function so far. It is well known that after vaccination in the last fifteen years in Cuba, there was a significant decrease of babesiosis (Babesia bovis and Babesia bigemina) and anaplasmosis (Anaplasma marginale) in cattle. A reduced transmission capacity of ticks fed on tick-immune animals and humans has been reported for several tick-borne pathogens. Recent experiments have demonstrated that an anti-tick vaccine may contribute to the control of tick-borne pathogens not only by decreasing the exposure of susceptible hosts to ticks, but also by reducing the vector capacity of ticks. In this study, the potential of Bm86 vaccination to interfere with pathogen transmission among ticks was evaluated by using as experimental model the brown dog tick Rhipicephalus sanguineus and the tickborne Babesia canis and Ehrlichia canis pathogens. Dogs, vaccinated and not vaccinated, were infested with pathogen-infected ticks and noninfected nymphs of R. sanguineus. After feeding, the pathogen transmission to newly molted adults from co-feeding uninfected nymphs was studied by conventional PCR and qPCR. Results suggest that the anti-Bm86 antibodies could be able to block the transmission of B. canis and/or E. canis from infected to non-infected ticks.

田鼠巴贝虫(Babesia mocroti)的超微结构观察

目的观察田鼠巴贝虫的超微结构，了解田鼠巴贝虫在宿主红细胞中发育的形态变化。方法运用扫描电镜观察田鼠巴贝虫侵入宿主红细胞的过程，运用透射电镜观察田鼠巴贝虫发育的形态变化过程及宿主红细胞形态变化情况。结果扫描电镜下观察到田鼠巴贝虫裂殖子大小在406~981nm之间，虫体发育过程分为裂殖子、分裂中的裂殖子、滋养体3个阶段，裂殖子是通过红细胞膜上微孔进入宿主红细胞，分裂中的裂殖子可使红细胞变形，滋养体是通过溶解红细胞膜游离红细胞。透射电镜观察到田鼠巴贝虫裂殖子的核膜为双层膜结构，虫体中有核糖体、微管、内质网、线粒体和溶酶体等完整的细胞器，分裂中的裂殖子在侵入后期可见食物空泡，滋养体的外膜和核不规则，胞浆中含有颗粒和空泡，宿主红细胞的电子密度随着虫体的发育而逐渐变稀疏。结论田鼠巴贝虫是含有完整细胞器的单细胞有机体，具有一般细胞所有的基本结构，它能完成多细胞动物所具有的生命机能，并以宿主红细胞中的血红蛋白作为生存氧料。

Inhibitory effects of methanolic Olea europaea and acetonic Acacia laeta on growth of Babesia and Theileria

Objective:To evaluate the antipiroplasmic activities of methanolic extract of Olea europaea(MOE)and acetonic extract of Acacia laeta(AAL)against Babesia and Theileria parasites in vitro and evaluate the chemotherapeutic effects of these extracts against Babesia(B.)microti in vivo.Methods:Fluorescence assay using SYBR Green 1 nucleic acid stain was used to detect inhibitory effects of the two extracts as well as the combination effects of the two extracts with diminazene aceturate and atovaquone on four Babesia species and Theileria equi in vitro while for in vivo experiments,8-weekold female BALB/c mice were injected intraperitoneally with 1× 107 B.microti-iRBCs and treated orally at a dose of 150 mg/kg of both extracts.Results:The half maximal inhibitory concentration(IC50)values of AAL against B.bovis,B.bigemina,B.divergens,B.caballi,and Theileria equi were lower than those of MOE extracts.Toxicity assay on Madin-Darby bovine kidney,mouse embryonic fibroblast(NIH/3T3),and human foreskin fibroblast cell lines showed that MOE and AAL affected only the viability of Madin-Darby bovine kidney cell line with half maximal effective concentrations(EC50)of(794.7±41.9)and(873.9±17.5)μg/mL,respectively.The oral treatments of MOE and AAL at 150 mg/kg inhibited the growth of B.microti in mice by 80.4% and 64.4%,respectively.The MOE and diminazene aceturate combination showed a higher chemotherapeutic effect than that of monotherapy.Conclusions:MOE and AAL have the potential to be an alternative remedy for treating piroplasmosis.Furthermore,the combination therapy of MOE + DA was more potent against B.microti infection in mice than their monotherapies.

Differential IL10 mRNA Profiles Associated to <i>Babesia bovis</i>and <i>B. bigemina</i>Infection Levels in Persistently Infected Animals

This work aimed to find quantitative phenotypic traits that can be used to discriminate the levels of resistance/susceptibility to B. bovis and B. bigemina in two groups of cattle presenting the highest (H) or lowest (L) infection levels and Rhipicephalus microplus ticks count. The animals were selected from a previous study of 50 Canchim (5/8 Charolais/zebu) heifers raised in an endemic area for these parasites. These animals were evaluated regarding their TNFα, IL10, IFN-γ, IL12 and iNOS mRNA levels. No differences were found between these groups regarding TNFα, IFN-γ, IL12β or iNOS transcripts. However, the IL10 transcripts were significantly higher in the H group compared to the L group. Moreover, significant correlation coefficients were observed between B. bovis loads and both IL10 and IFN-γ transcripts, while no correlations were found for B. bigemina loads and all tested immune-related transcripts, suggesting that differential IL10 mRNA profiles were closely associated to B. bovis loads. Our results have contributed to a better understanding of the immune responses against Babesia infection, as we demonstrated that the IL10 cytokine levels might also influence or be influenced by parasitemia levels in persistently infected animals.

田鼠巴贝西虫Babesiamicroti截短型分泌抗原的重组表达及其在诊断上的应用

田鼠巴贝西虫分泌抗原l（Babesiamicrotisecretedantigen1，BmSA1）是一个重要的诊断抗原候选分子，但全长蛋白的表达量少，也不宜纯化。本研究在此基础上，通过生物信息学分析，选取N-末端信号肽后，C-末端跨膜区前，包含CPSF73-100_C功能区的228个氨基酸所编码基因片段，重组高效表达了一个可溶性截短型抗原，解决了完整蛋白重组表达纯化的困难。纯化的截短型抗原作为酶联免疫试验（ELISA）诊断抗原，可清晰地区分阴性及阳性鼠血清，并与其他病原感染无交叉反应，显示其诊断的特异性。鼠人工感染田鼠巴贝西虫系列血清检测表明，该抗原可检测早期感染（5天）和感染（55天）以后的样本，显示其对不同时期感染的宿主均具有诊断价值。结果表明，重组B．microti截短型抗原可作为一种诊断制剂检测田鼠巴贝西虫抗体。

羊巴贝斯虫病流行病学的研究进展

羊巴贝斯虫病是一种由硬蜱传播的血液原虫病,呈全球性分布,主要临床症状为发热、四肢无力、贫血、腹泻、反刍减弱、厌食、精神不振、虚弱等,严重可导致死亡。本文阐述了巴贝斯虫病病原的种类,并对感染羊的莫氏巴贝斯虫、绵羊巴贝斯虫、粗糙巴贝斯虫、泰氏巴贝斯虫和叶状巴贝斯虫的流行病学情况进行了总结,为羊巴贝斯虫病的防控提供一定的依据。

河北省牛梨形虫流行病学调查及遗传进化分析

为了解我国河北省牛梨形虫的种类、感染率、遗传进化及季节流行性情况,本研究共收集河北省7个地级市的牛血液样品564份,以提取的牛全血液基因组DNA为模板,采用套式PCR对牛梨形虫18S r RNA基因进行扩增检测,并鉴定虫种,选取10份不同地区牛犁形虫阳性样品测序,并构建其18S r RNA基因系统进化树分析其遗传进化特征。结果显示,我国河北省牛梨形虫总感染率为18.6%(105/564),其中张家口地区牛梨形虫感染率最高,为83.3%(20/24),极显著高于河北省其他地区(P<0.01),唐山、沧州、石家庄及承德的牛梨形虫感染率分别为27.4%(32/117)、22.1%(17/77)、25.0%(20/80)和19.0%(16/84),秦皇岛和衡水地区牛梨形虫感染率为0。感染梨形虫的虫种主要为双芽巴贝斯虫、牛巴贝斯虫和瑟氏泰勒虫,感染率最高的为双芽巴贝斯虫(12.2%,69/564)。18S r RNA基因的进化树分析结果显示,10份牛梨形虫18S r RNA基因均呈地区性聚集;统计学分析结果显示,8月份河北省牛梨形虫感染率最高(29.7%),显著高于其他月份(P<0.05),4月~8月间牛梨形虫感染率呈逐渐上升的趋势,8月份达到顶峰后感染率逐渐下降。本研究首次对河北省牛梨形虫进行了流行病学调查,并分析了其遗传进化特征,结果表明河北省存在牛梨形虫的感染,尤其是张家口地区,该地区应在夏季应加强对牛梨形虫的重点防控。本研究为河北省牛梨形虫的防制提供了科学依据。

乌鲁木齐市3例疑似犬巴贝斯虫病的诊断及病原体的分子鉴定

犬巴贝斯虫病是一种由犬巴贝斯虫引起的、能够造成寄主出现包括消瘦、贫血、黄疸、发热甚至死亡等一系列临床症状的血液原虫传染病。乌鲁木齐市3例疑似犬巴贝斯虫病患犬通过临床检查、血常规、血涂片镜检、血清生化检查、分子生物学检查(PCR)进行诊断,并选择3例患犬中强阳性的病例进行测序分型。结果显示,3例患犬血小板数目均不同程度地减少,其中两例患犬的红细胞数目和血红蛋白也有所减少。血涂片镜检发现,患犬红细胞内疑似存在犬巴贝斯虫虫体。3例患犬犬巴贝斯虫特异性引物核酸检测结果均呈阳性,病原体经分子生物学分析及测序鉴定为犬巴贝斯虫。

Co-infections with Babesia microti and Plasmodium parasites along the China-Myanmar border

Background:Babesiosis is an emerging health risk in several parts of the world.However,little is known about the prevalence of Babesia in malaria-endemic countries.The area along the China-Myanmar border in Yunnan is a main endemic area of malaria in P.R.China,however,human infection with Babesia microti(B.microti)is not recognized in this region,and its profile of co-infection is not yet clear.Methods:To understand its profile of co-infections with B.microti,our investigation was undertaken in the malaria-endemic area along the China-Myanmar border in Yunnan between April 2012 and June 2013.Four parasite species,including B.microti,Plasmodium falciparum(P.falciparum),P.vivax,and P.malariae,were identified among 449 suspected febrile persons detected by nested polymerase chain reaction(PCR)assay based on small subunit ribosomal ribonucleic acid(RNA)genes of B.microti and Plasmodium spp.Results:Of all the collected samples from febrile patients,mono-infection with B.microti,P.vivax,P.falciparum,and P.malariae accounted for 1.8%(8/449),9.8%(44/449),2.9%(13/449),and 0.2%(1/449),respectively.The rate of mixed infections of B.microti with P.falciparum or P.vivax are both 0.2%(1/449),and mixed infections of P.falciparum and P.vivax accounted for 1.1%(5/449).Conclusions:This report supports the hypothesis that babesiosis caused by B.microti is emerging along the China-Myanmar border in the Yunnan province,P.R.China,but it was ignored because of low parasitemia or mixed infection with Plasmodium spp.More sensitive and specific diagnosis methods are needed to find the rapid response mechanism of emergency for babesiosis and malaria co-prevalence areas.

A case of human infection with a novel Babesia species in China

Background:Babesiosis is an uncommon but emerging tick-borne disease caused by the genus Babesia.In this case study,we report a case of human infection with a novel Babesia sp.in China.Findings:The patient in question had been suffering from repetitive occurrences of mild fever of unknown origin and fatigue for 10 years.Ring forms,tetrads,and one or two dots of chromatin or trophozoite-like organisms were observed in the patient’s thin blood smears and bone marrow smears.Using a confocal laser-scanning microscope,it was observed that the patient’s serum had reactivity with the surface proteins of the B.microti strain.Electron microscopy revealed oval red blood cells with 1~2μm of knob protrusions in the cellular membrane.The results of the Babesia-specific nested PCR assay for 18S rRNA confirmed the presence of Babesia infection.The construction of a phylogenetic relationship showed clustering with B.microti and B.duncani,which was identified as a novel Babesia species and named as Babesia sp.XXB/HangZhou.Azithromycin,doxycycline,and moxifloxacin hydrochloride were shown to relieve symptoms but were not as effective after continuous usage.After atovaquone(Mepron®)administration,the patient recovered from fever and tested negative for detection of Babesia-specific genes.Conclusion:Babesia sp.XXB/HangZhou is a novel Babesia species,which causes mild babesiosis in an immunocompetent patient.

卵形巴贝斯虫与中华泰勒虫双重PCR检测方法的建立

为建立一种能快速对卵形巴贝斯虫(Babesia ovata)和中华泰勒虫(Theileria sinensis)同时进行检测的双重PCR方法。根据GenBank已报道的卵形巴贝斯虫AMA1基因和中华泰勒虫MPSP基因设计合成了2对特异性引物,通过条件优化,建立了双重PCR检测方法。结果显示:双重PCR可特异扩增出卵形巴贝斯虫和中华泰勒虫目的条带,片段大小分别为500 bp和986 bp。该方法具有较好的特异性,对卵形巴贝斯虫和中华泰勒虫的最低检出浓度为16 fg/μL。对采集的90份牛血液样本进行双重PCR检测,卵形巴贝斯虫阳性率为30%(27/90),中华泰勒虫阳性率为16.67%(15/90),混合感染率为10%(9/90)。结果表明,双重PCR方法可用于卵形巴贝斯虫和中华泰勒虫的快速诊断和流行病学调查。

云南部分地区牛羊寄生蜱媒梨形虫的流行病学研究

为了了解云南省玉溪市、昆明市、楚雄市和普洱市牛羊寄生蜱媒梨形虫的流行情况,试验从上述4个地区牛羊体表共采集455份蜱样本,基于形态学和cox1基因序列对寄生蜱进行物种鉴定,通过扩增梨形虫18S rDNA基因及序列分析对寄生蜱媒梨形虫进行鉴定,并统计不同地区、不同宿主寄生蜱媒梨形虫的阳性率,分析寄生蜱感染巴贝斯虫和泰勒虫的风险因素(所在地区、宿主种类)。结果表明:采集的蜱均为微小扇头蜱。在455份蜱样本中,共鉴定到5株巴贝斯虫,其中4株为双芽巴贝斯虫,1株未定种;共鉴定到60株泰勒虫,其中54株为东方泰勒虫,6株未定种。选取2株双芽巴贝斯虫和4株东方泰勒虫构建遗传进化树,其中双芽巴贝斯虫CX-370和CX-369与双芽巴贝斯虫参考株(GenBank登录号为KY805825.1、AY603402.1)和巴贝斯虫参考株(GenBank登录号为KP410277.1)位于的同一分支,未定种的巴贝斯虫CH-367为独立分支且与上述虫株汇聚为一支,东方泰勒虫PE-423、CJ-374、KM-106和PE-394与东方泰勒虫参考株(GenBank登录号为MH208640.1、MH208639.1、MH208636.1)位于同一分支且与未定种的泰勒虫PE-417、KM-114汇聚为一支,未定种的泰勒虫PE-399、PE-390、PE-420与环形泰勒虫参考株(GenBank登录号为OQ411264.1)位于同一分支。4个地区牛羊寄生蜱媒梨形虫总阳性率为14.29%(65/455),其中巴贝斯虫总阳性率为1.10%(5/455),泰勒虫总阳性率为13.19%(60/455);玉溪市、昆明市、楚雄市和普洱市牛羊寄生蜱媒梨形虫阳性率分别为21.43%(3/14)、18.56%(31/167)、2.08%(4/192)和32.93%(27/82),牛羊寄生蜱媒巴贝斯虫阳性率分别为7.14%(1/14)、0(0/167)、2.08%(4/192)、0(0/82),牛羊寄生蜱媒泰勒虫阳性率分别为14.29%(2/14)、18.56%(31/167)、0(0/192)、32.93%(27/82)。4个地区牛寄生蜱媒梨形虫阳性率为16.89%(63/455),而羊寄生蜱媒梨形虫阳性率为2.44%(2/82);其中牛和羊寄生蜱媒巴贝斯虫阳性率分别为1.34%(5/373)和0(0/82),泰勒虫阳性率分别为15.55%(58/373)和2.44%(2/82)。寄生蜱所在地区不是影响其感染梨形虫的重要风险因素,而寄生蜱宿主种类是影响其感染梨形虫的重要风险因素,牛寄生蜱相对于羊寄生蜱更易感染泰勒虫。说明云南省玉溪市、昆明市、楚雄市和普洱市4个地区牛羊寄生优势蜱种为微小扇头蜱,牛羊寄生蜱存在巴贝斯虫和泰勒虫感染;4个地区中,普洱市的泰勒虫阳性率最高,玉溪市的巴贝斯虫阳性率最高;牛寄生蜱媒泰勒虫阳性率高于羊寄生蜱。

犬及犬寄生蜱的巴贝虫检测和系统发育分析

目的 了解江西南昌某警犬基地犬及犬寄生蜱的巴贝虫感染情况,并分析阳性样本的巴贝虫系统发育情况。方法 2022年8—10月在南昌某警犬基地,采集犬血液标本及犬体表蜱,采用靶向巴贝虫属18S rRNA、ITS-1、COX1、HSP70基因的特异性引物进行PCR检测及序列比对分析。结果 调查宿主犬52只,采集到犬血样标本47份,寄生蜱264只,经鉴定均为血红扇头蜱,分成110个待测蜱标本。2份蜱标本检测出巴贝虫DNA,阳性率为1.82%;而犬血液标本未检出。基于巴贝虫属18S rRNA、ITS-1、COX1、HSP70基因扩增的序列比对和系统发育分析表明,本研究中鉴定出的2株巴贝虫与韦氏巴贝虫(Babesia vogeli)聚集在同一分支。结论 南昌某警犬基地犬体表蜱存在韦氏巴贝虫(B.vogeli)感染,对周边动物有潜在感染的风险,应加强预防和控制措施。

辽吉两省东部山区鼠类微小巴贝斯虫和伯氏疏螺旋体感染调查

目的调查辽吉两省东部山区鼠类微小巴贝斯虫和伯氏疏螺旋体感染情况。方法采用聚合酶链式反应(polymerase chain reaction,PCR)对从辽宁省宽甸地区和吉林省集安林区捕获的野鼠进行微小巴贝斯虫和伯氏疏螺旋体检测。结果对129份集安和宽甸两地野鼠脾DNA样本分别进行微小巴贝斯虫和伯氏疏螺旋体检测,检出阳性共15份,阳性率11.63%,微小巴贝斯虫和伯氏疏螺旋体的阳性率分别为6.20%(8/129)和5.43%(7/129),二者感染率差异无统计学意义(χ^(2)=0.000,P=1.000)。宽甸、集安两地不同鼠种野鼠检出微小巴贝斯虫和伯氏疏螺旋体的阳性率差异无统计学意义。结论我国辽吉两省东部山区野鼠存在微小巴贝斯虫和伯氏疏螺旋体感染,提示该地区存在感染风险。

犬巴贝斯虫抗体胶体金检测卡制备与评价

试验旨在快速检测犬巴贝斯虫抗体,利用胶体金免疫层析技术制备犬巴贝斯虫抗体胶体金检测卡并评价其功能性。将羊抗鼠免疫球蛋白G (IgG)和犬巴贝斯虫表面抗原Bg P47重组蛋白(Bg P47 Ag)包被于NC膜上分别做为质控线和检测线,胶体金标记的鼠IgG和鼠抗犬IgG均匀喷点在聚酯纤维膜上作为标记垫,将化学品配制的玻纤溶液均匀涂在样品垫上,并用吸水纸和PVC底板制备犬巴贝斯虫抗体胶体金检测卡,对其特异性、灵敏度、均一性、加速稳定性、实时稳定性进行评价,并与ELISA、PCR检测结果进行对比。结果显示,该检测卡不与犬莱姆抗体(lyme Ab)阳性犬血清、犬瘟热病毒抗体(CDV Ab)阳性犬血清、犬细小病毒抗体(CPV Ab)阳性犬血清、犬无形体抗体(Anaplasma Ab)阳性犬血清、犬三联疫苗阳性免疫犬血清样本发生交叉反应;可检测浓度稀释至1∶256倍的样本;有效期可达到24个月。研究表明,该检测卡可快速、准确地检测犬巴贝斯虫抗体,为动物临床诊断和治疗提供参考。

High prevalence of Babesia microti in small mammals in Beijing

Background: Babesiosis is an emerging tick-borne zoonotic infectious disease.Babesia microti is responsible for most cases of human babesiosis globally.It is important to investigate the prevalence of B.microti in the mammalian host population of a specific region in order to elucidate mechanisms of pathogen transmission and to define geographic areas where humans face the greatest risk of exposure.The aim of this study is to understand the prevalence and genotypes of B.microti in the small mammals that are found in Beijing,China.Methods:: We trapped small mammals from all of the 16 urban,suburban,and outer suburban districts of Beijing during the years 2014,2017 and 2018.Genomic DNA was extracted from the heart tissues individually and the Babesia 18S rRNA gene was detected by PCR.The genotypes of B.microti were identified based on sequence alignments and phylogenetic analysis.The morphology of the parasites was observed under light microscopy.The risk factors were analyzed statistically based on both univariate analyses and multivariate logistic regression.Results: A total of 1391 small mammals were collected.Positive infection of B.microti was detected in 12.1%(168/1391)of small mammals from 15 out of the 16 districts.Both Kobe-type and U.S.-type B.microti,accounting for 9.5%and 2.7%,respectively,were identified.Classic diverse morphologic forms of B.microti were observed.Specific types of ecological habitats including shrub areas,broad-leaved forest,and cropland were revealed to be risk factors associated with B.microti infection.Conclusions: This study demonstrated the wide prevalence of B.microti infection in eight species of small mammals in Beijing,with Kobe-type more prevalent than U.S.-type.This study provides fundamental information for the development of informed prevention and control measures by public health authorities;the data gathered indicates a need for further monitoring of both clinical diseases in individuals presenting with babesiosis-like symptoms,as well as the infection status of ticks in high risk areas.

Isolation and in vitro cultivation of Babesia venatorum(Apicomplexa:Babesiidae),a zoonotic hemoprotozoan from Ixodes persulcatus ticks in China

The emerging Babesia venatorum upsurges as a potential health threat occurring in China and other endemic countries.Few attempts to isolate and culture the Babesia species had been conducted in vitro.We collected the questing Ixodes persulcatus from identified endemic areas and allowed them to feed on the severe combined immunodeficiency(SCID)mice.The positive mice were chosen to provide positive erythrocytes with asexual B.venatorum for continuous culture in mouse or human erythrocytes in vitro,with RPMI 1640 medium and appropriate serum.With B.venatorum in the SCID mice,erythrocytes were cultured in vitro for confirmation by morphological observations with transmission electron microscopes.Sequences of B.venatorum were then identified by way of conventional PCR amplification.Parasitemia counts monitored the growth of B.venatorum on thin blood smears and real-time quantitative PCR in parallel.As expected,B.venatorum positive mice were harvested by sufficient attacks of I.persulcatus ticks.The erythrocytes of the infected mice were then inoculated and successfully cultured in donated erythrocytes from humans and mice in RPMI 1640 culture medium.Active growth of B.venatorum was well demonstrated in human erythrocytes with 3.3 times parasite-load when compared with a mouse under similar conditions.With the increased subcultures,a prolonged period of detectable parasitemia with much higher peak parasitemia and shorter time to reach peak parasitemia were observed in the following subcultures.A new strategy for isolation and in vitro culture of B.venatorum has been provided with a continuous supply of sufficient pathogens to satisfy human babesiosis's testings and clinical therapies.