Circulation of Brucellaceae,Anaplasma and Ehrlichia spp.in borderline of Iran,Azerbaijan,and Armenia

Objective:To estimate the infection of ticks to Anaplasma,Ehrlichia,Babesia,Theileria,and Brucellaceae using molecular methods in borderline of Iran,Azerbaijan,and Armenia.Methods:Totally,2022 ticks were collected from different livestock.Then,species were diagnosed under stereomicroscope according to valid morphological keys.Tick DNA was extracted followed by PCR to detect Anaplasma,Ehrlichia,Theileria,Babesia and Brucellaceae infection in ticks.Results:A total of 498 males[24.62%(95%CI 22.76%-26.57%)],741 females[36.64%(95%CI 34.54%-38.79%)],782 nymphs[38.67%(95%CI 36.55%-40.84%)]and 1 larva[0.04%(95%CI 0.00%-0.28%)]were identified.Among identified samples,we found four genera including Hyalomma,Rhipicephalus,Haemaphysalis,and Dermacentor.Molecular assay revealed that the prevalence of ticks to Anaplasma or Ehrlichia,and Brucellaceae was 22.02%(95%CI 16.01%-29.06%)and 15.03%(95%CI 9.43%-22.26%),respectively.Phylogenetic analysis showed that the identified Anaplasma sp.had the most similarity with Anaplasma centrale,Anaplasma platys,Anaplasma camelii,and Anaplasma phagocytophilum,submitted in GenBank.Furthermore,the detected Ehrlichia sp.and Brucellaceae bacterium had the most similarity with Ehrlichia ruminantium and Mycoplana peli,respectively.However,no sign of the presence of Theileria and Babesia spp.was seen in the studied samples.Conclusions:Anaplasmosis,ehrlichiosis and brucellosis should be considered as important health threats in northwestern Iran and consistent monitoring on infection of ticks and livestock should be performed regularly.

Molecular detection of Anaplasma marginale and Anaplasma ovis in sheep and goat in west highland pasture of Iran

Objective:To determine the prevalence of Anaplasma marginale(A.marginale) and Anaplasma ovis from sheep and goat in different highland pasture in west of Iran.Methods:From July 2015 to October 2015,370 blood samples of sheep and goat were collected from different regions in Hamedan province,Iran.The DNA extracted from blood and subsequently,16 S r RNA and MSP4 genes were analyzed by nested-PCR,semi nested-PCR and RFLP methods.Results:In the PCR assessment,overall 27.5%(102/370) of sheep and goat were positive for Anaplasma ovis and A.marginale infection,which was lower than reports from tropical and subtropical regions of Iran.Statistical analysis(the Chi-square test) did not show any significant relation between infection and variables such as location,tick infestation age and sex(P > 0.05).No significant correlation between the altitude and the Anaplasma species infection was found(Mann–Whitney test:P > 0.05).However,Anaplasma infection in goat significantly is more than the sheep(P = 0.008).Conclusions:The ecological changes affect the frequency and distribution of Anaplasma species.Furthermore,our results indicate that sheep as potential reservoirs of A.marginale.

Preliminary study of natural reservoirs as sentinels of <i>Anaplasma phagocytophilum</i>and <i>Ehrlichia chaffeensis</i>in Soria, northern Spain

The serum of foxes and red deer from the Province of Soria (northern Spain) was screened in indirect immunofluorescence assays to determine whether these animals could be used as sentinels of the tick-borne pathogens Anaplasma phagocytophilum and Ehrlichia chaffeensis. The results suggest that foxes and red deer would not make good sentinels in epidemiological studies on E. chaffeensis in this region, although red deer could be used as such for the study of A. phagocytophilum transmission.

Molecular Detection of <i>Anaplasma</i>and <i>Ehrlichia</i>Infection in Ticks in Borderline of Iran-Afghanistan

Anaplasmosis, a disease caused by various species of Anaplasma, poses important economic constraints to animal breeders. Ehrlichiosis is a worldwide zoonosis illness and mostly occurs in tropical and subtropical regions that are close to the vector’s distribution. Tick-borne pathogens lead to over 100,000 cases of illness in the world each year. Besides the costs of the additional veterinary care, anaplasmosis causes abortion in animals, reduction of milk production, body weight, and frequently leads to death. In this study, we investigated on infection of ticks to Anaplasma and Ehrlichia pathogens in Zabol and Zahak County in Sistan and Baluchestan Province where is bordered with Afghanistan. Totally from June 2013 to May 2014, 369 ticks were caught from goats, cows and sheep. Molecular studies on 53 of these samples which represented all specimens, showed that Ehrlichia’s DNA and Anaplasma’s DNA was found in 14 (26.4%) out of the 53 selected specimens. The results showed the infection of Rhipicephalus sanguineus and Hyalomma anatolicum with Anaplasma ovis. Also we saw infection of H. anatolicum and H. asiaticum ticks to Ehrlichia spp. This study has been intended to do a comprehensive survey of Ehrlichia and Anaplasma distribution in ticks caught from east of Iran;it was designed to investigate the presence of Anaplasma spp. and Ehrlichia spp. in Zabol and Zahak Counties, Iran. These results show that these pathogens should be controlled in such regions.

Comparative study of Anaplasma parasites in tick carrying buffaloes and cattle

A comparative study on the prevalence of Anaplasma parasite was conducted on ticks carrying buffaloes and cattle. Five hundred blood samples of both animals (250 of each) were collected during February, March and April. Thin blood smears on glass slides were made, fixed in 100% methyl alcohol and examined. Microscopic examination revealed that 205 (41%) animals had Anaplasma parasites, out of which 89, 44 and 72 animals had Anaplasma marginale, Anaplasma centrale and mixed infection respectively. Infected buffaloes and cattle were 75 and 130 respectively. The infection in female was 53 and 92 in buffaloes and cattle respectively. Twenty-two and 92 blood samples of male were found positive in buffaloes and cattle respectively. Com- parative study revealed that the cattle were 26.82% more susceptible than buffaloes. The parasite prevailing percentage in female of both animals was slightly higher than that of the male. This investigation was aimed at studying the comparative prevalence of Anaplasma parasite in tick carrying buffaloes and cattle.

黑龙江省口岸地区蜱类感染嗜吞噬细胞无形体Anaplasma phagocytophila的初步调查

为调查黑龙江省口岸蜱类携带嗜吞噬细胞无形体Anaplasma phagocytophila感染情况,运用聚合酶链式反应方法扩增嗜吞噬细胞无形体柠檬酸合成酶（gltA）基因片段,对黑龙江省各口岸地区采集的蜱标本进行检测,并对阳性结果进行序列分析.共检测蜱1 609只,29只阳性,阳性率为1.80%,结果表明,黑龙江省口岸蜱类除日本血蜱外均有自然感染嗜吞噬细胞无形体的现象.所感染的嗜吞噬细胞无形体gltA 基因与GenBank 中登录的嗜吞噬无形体gltA基因片段,相似性为82%～98%.因此,我国黑龙江省口岸地区存在蜱粒细胞无形体感染.

A survey of biochemical and acute phase proteins changes in sheep experimentally infected with Anaplasma ovis

Objective: To determine the biochemical and acute phase proteins changes in sheep experimentally infected with Anaplasma ovis(A. ovis). Methods: One Iranian sheep naturally infected with A. ovis(parasitemia 0.02%) but with no other blood parasites based on blood smear and polymerase chain reaction methods was selected as donor, and it was splenectomized to induce high level of parasitemia. Then, three weeks after splenectomy when parasitemia was 6%, donor's blood was intravenously administered to each recipient animal. Five 5-6 months old Iranian male sheep without any blood parasites were selected as recipient animals. The percent of parasites, packed cell volume, serum biochemical parameters(urea, creatinine, bilirubin, aspartate aminotransferase activity, cholesterol, total protein, albumin, globulin, Fe), acute phase proteins(haptoglobin, total iron binding capacity, fibrinogen), were evaluated in sheep before and after being experimentally infected with A. ovis(until day 38). In addition, body weights of sheep were measured on days 0, 20 and 38. Results: In recipient sheep, microscopic examination of erythrocytes revealed a significant rise of parasitemia on days 12 and 15. The lowest level of packed cell volume in sheep was seen on day 15 post infection. A significant rise existed in mean urea and bilirubin(total, direct and indirect) on days 15 and 20. The increase of indirect bilirubin level was higher than direct bilirubin. Furthermore, serum Fe significantly increased on days 20 and 23. The mean total protein concentration significantly increased on day 38. A significant increase was found in the serum globulin concentration from days 20 and 27 to 38. Maximum values of haptoglobin were observed on days 27 and 30. Moreover, aspartate aminotransferase activity(from days 20-30) and cholesterol concentration(on day 20) significantly decreased. However, no significant changes were found in other parameters. Conclusions: Experimental ovine anaplasmosis caused by A. ovis could be associated with some changes in measured parameters, which presumably could be helpful for evaluation on staging of disease.

海南中北部地区牛体表蜱种鉴定及蜱携带无形体属病原体情况调查研究

目的:研究海南牛体表蜱虫的种类及蜱携带无形体属病原体的情况。方法:本研究选取海南中北部9个县市为样本采集点,从牛体表采集蜱虫样本。通过形态学观察和分子生物学技术进行蜱种鉴定,并以16S rRNA、groEL和gltA为靶基因,利用PCR方法检测蜱虫携带无形体属病原体情况。并进一步采用系统发育分析方法,揭示海南牛体表蜱虫携带的无形体属病原体种间关系。结果:本研究共采集335只蜱虫,包括血红扇头蜱(Rhipicephalus sanguineus)44只、微小扇头蜱(Rhipicephalus microplus)291只。PCR检测结果表明,该地区蜱中存在候选博莱无形体(Candidatus Anaplasma boleense)、边缘无形体(Anaplasma marginale),阳性率分别为0.60%(2/335),0.90%(3/335)。系统发育分析结果表明,本实验获得的无形体与已知的无形体同源性较高。结论:海南中北部地区牛体表的优势蜱种为微小扇头蜱,并且蜱虫携带无形体属病原体,本研究为该地区无形体病的预防和治疗提供了重要的数据支撑。

Molecular detection of Anaplasma infections in ixodid ticks from the Qinghai-Tibet Plateau

Anaplasma species are tick-transmitted obligate intracellular bacteria that infect many wild and domestic animals and humans.The prevalence ofAnaplasma spp.in ixodid ticks of Qinghai Province is poorly understood.In this study,a total of 1104 questing adult ticks were investigated for the infection ofAnaplasma species.As a result,we demonstrated the total infection rates of 3.1,11.1,5.6,and 4.5%forA.phagocytophilum,A.bovis,A.ovis andA.capra,respectively.All of the tick samples were negative forA.marginale.The positive rates ofA.phagocytophilum,A.ovis andA.capra in different tick species were significantly different.The positive rates ofA.capra andA.bovis in the male ticks were significantly higher than that in the female ticks.Sequence analysis ofA.ovis showed 99.5-100%identity to the previous reported isolates.The sequences ofA.phagocytophilum had 100%identity to strains Ap-SHX21,JC3-3 and ZAM dog-181 from sheep,Mongolian gazelles,and dogs.Two genotypes ofA.capra were found based on 16S rRNA,citrate synthase(gltA)gene and heat shock protein(groEL)gene analysis.In conclusion,A.bovis,A.ovis,A.phagocytophilum,andA.capra were present in the ticks in Qinghai Province.Anaplasma infection is associated with tick species,gender and distribution.These data will be helpful for understanding prevalence status ofAnaplasma infections in ticks in Qinghai-Tibet Plateau.

边缘无形体MSP2的原核表达及间接ELISA抗体检测方法的建立

为建立可检测边缘无形体(Anaplasma marginale)的血清学检测方法,该研究根据GenBank收录的边缘无形体MSP2基因序列(登录号:EU526889),构建重组质粒pET28a-MSP2,原核表达获得pET28a-MSP2重组蛋白,将纯化后的蛋白作为抗原,建立间接ELISA检测方法。结果显示,该研究建立的间接ELISA方法,抗原最佳包被浓度、血清最佳稀释度和酶标二抗最佳工作浓度分别为8μg/mL、1∶400和1∶2 000,特异性、灵敏性和重复性良好。本试验成功表达并纯化了边缘无形体的MSP2蛋白,并建立了边缘无形体抗体间接ELISA方法,为边缘无形体的监测和诊断提供参考。

新疆一例牛环形泰勒虫、念珠菌和绵羊无浆体混合感染的病例初报

2021年8月17日,新疆托克逊县一头西门塔尔母牛出现高热、瘤胃胀气、肌肉震颤、呼吸急促、结膜黄染和血红蛋白尿等临床症状。为确认发病原因,采集该牛全血和体表蜱虫样品进行光学显微镜观察、血常规检测和聚合酶链式反应(PCR)检测。血涂片镜检结果显示,患牛疑似牛环形泰勒虫、念珠菌和绵羊无浆体混合感染;血常规检测显示,血红蛋白、血红细胞压积、平均血红蛋白浓度和中性粒细胞等指标下降,血小板和淋巴细胞百分比等指标上升,提示该牛机体存在贫血和炎症反应;血液样品PCR结果显示,牛环形泰勒虫、念珠菌和绵羊无浆体检测均呈阳性;蜱虫样品PCR结果显示,牛环形泰勒虫检测为阳性。综上,患牛最终被诊断为牛环形泰勒虫、念珠菌和绵羊无浆体混合感染。目前,此3种病原共同感染牛的案例极为少见。本研究为今后牛混合感染牛环形泰勒虫、念珠菌和绵羊无浆体的临床诊断提供了参考。

“Candidatus anaplasma camelii”in onehumped camels(Camelus dromedarius)in Morocco:a novel and emerging Anaplasma species?

Background:There has been a growing interest in camel anaplasmosis due to its recent emergence in this reservoir species and concerns for its zoonotic potential.The epidemiology of anaplasmosis in camels therefore remains poorly understood mostly because camels belong to marginalised poor and often transhumant populations whose interests are largely neglected.Most studies of anaplasmosis in camels have relied on microscopy and serology for diagnosis and only three studies,undertaken in Tunisia,Saudia Arabia and China,have used molecular diagnostics.The present work characterises Anaplasmataceae strains circulating in the Camelus dromedarius reservoir in Morocco using PCR.Methods:Camels(n=106)were randomly sampled from 6 regions representing different agro-ecological areas in southern Morocco.Whole blood was collected and screened using PCR methods targeting the gene groEL.Anaplasmataceae strains were characterised by sequence analysis of the gene groEL.Results:A total of 39.62%(42/106)camels screened were positive for Anaplasmataceae spp.GenBank BLAST analysis of five positive sequenced samples revealed that all strains were 100%identical to“Candidatus Anaplasma camelii”.Phylogenetic investigation and genetic characterisation of the aligned segment(650 bp)of the gene groEL confirmed high similarity with A.platys.Conclusion:This study demonstrates the circulation of a previously unidentified species of the genus Anaplasma in Morocco which is genetically close to the agent causing canine anaplasmosis but whose main reservoir is thought to be Camelus dromedarius.Trial registration number:This study is not a clinical trial and therefore a trial registration number does not apply.

山羊源牛无浆体对Balb/c小鼠感染性研究

牛无浆体(Anaplasma bovis)隶属无浆体属(Anaplasma),可感染动物单核细胞和组织巨噬细胞,对动物生产性能影响较大。用单核细胞分离方法得到山羊源牛无浆体,并将其接种至人工饲养的Balb/c小鼠,接种时分别设置高、中、低剂量组、阴性对照和空白对照。结果发现,高剂量组和中剂量组接种后3、6、9 d的血涂片检查和PCR扩增均可查到牛无浆体,9 d后转阴;低剂量组Balb/c小鼠不同时间点检查时牛无浆体均呈阴性。表明从山羊单核细胞直接分离牛无浆体可以感染Balb/c小鼠,接种9 d内可持续感染,为牛无浆体的动物感染模型的建立奠定了基础。

无浆体检测技术研究现状

无浆体是一种重要的蜱媒病原体,随着国内外学者对其关注度的提高,其对动物和人类健康的危害性也逐渐凸显,高效、准确的诊断技术显得尤其重要。实际工作中,多种分子生物学及血清学方法被用于无浆体的检测,检出率和准确性显著提高。论文综述了近5年无浆体的检测技术,对无浆体的感染诊断提供参考。

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.

四川省炉霍县牦牛源蜱传斑点热群立克次体和无形体分子检测及遗传进化分析

【目的】了解四川省炉霍县牦牛体表寄生蜱的种类及其斑点热群立克次体(Spotted fever group Rickettsia,SFGR)和无形体的感染情况。【方法】在炉霍县6个乡采集牦牛体表的蜱,经形态学初步鉴定后提取蜱基因组DNA,采用PCR技术分别扩增蜱ITS⁃2、SFGR OmpB和无形体16S rRNA基因部分片段,对阳性产物进行测序、比对及构建进化树,从而确定蜱的种类及其SFGR和无形体的感染情况。【结果】在820份蜱样本中,只包含青海血蜱(Haemaphysalis qinghaiensisus,29/820)和微小扇头蜱(Rhipicephalus micro⁃plus,791/820)。总共有408份蜱样本被检出SFGR,只检出Candidatus Rickettsia longicornii 1种SFGR,总感染率为49.8%(408/820)。本次调查的6个乡均检出SFGR,不同乡之间SFGR感染率的差异极显著(χ^(2)=111.524,P=0.000<0.01)。青海血蜱和微小扇头蜱的SFGR感染率分别为44.8%(13/29)和49.9%(395/791),差异不显著(χ^(2)=0.292,P=0.589>0.5)。有5个乡检出无形体,共检出Anaplasma bovis、A.marginale、A.platys和A.phagocytophilum 4种无形体,总感染率为30.9%(253/820),不同乡之间蜱样本无形体感染率差异极显著(χ^(2)=139.825,P=0.000<0.01)。青海血蜱和微小扇头蜱的无形体感染率分别为6.9%(2/29)和31.7%(251/791),差异极显著(χ^(2)=8.087,P=0.004<0.01)。SFGR与无形体混合感染率为18.8%(154/820)。【结论】炉霍县存在青海血蜱和微小扇头蜱,还携带SFGR和多种无形体,应做好当地蜱传斑点热群立克次体和无形体的防治工作。

海南4个地区犬体表蜱种鉴定及其无形体携带情况

为研究海南省犬体表蜱虫的种类及其携带无形体的情况,对4个地区犬蜱进行形态学、分子生物学鉴定及进化分析,且基于16S rRNA和gltA基因,通过PCR检测犬蜱携带无形体的情况。形态学及分子生物学鉴定结果表明,所有蜱类为血红扇头蜱(Rhipicephalus sanguineus),基于蜱虫线粒体16S rDNA基因的系统发育树表明,研究中所获得的HN-01和HN-02基因序列处于不同的遗传进化分支,HN-01与已知的来自美国的血红扇头蜱(MH018842)处于同一分支,HN-02与来自泰国(KC170744)的血红扇头蜱处于同一分支,与我国甘肃省、北京市所获得的血红扇头蜱(JF979377、KC203362)序列亲缘关系较远。基于扁平无形体(Anaplasma platys)gltA基因的PCR结果表明,犬蜱携带有扁平无形体,其阳性率为1.1%(6/546)。基于16S rRNA基因的PCR检测结果表明,研究中获得的犬体表蜱未携带嗜吞噬细胞无形体(Anaplasma pagocyto-philum)和牛无形体(Anaplasma bovis)。

2021年蚌埠市4种鼠传病原体检测分析

目的掌握蚌埠市鼠传病原体的分布情况,为鼠传疾病的防控工作提供科学依据。方法在蚌埠市禹会区、蚌山区、龙子湖区和淮上区野外捕鼠,采用实时荧光定量PCR法对4种鼠传病原体,包括新型布尼亚病毒、汉坦病毒、致病性钩端螺旋体和嗜吞噬细胞无形体,进行核酸检测。结果共检测200只鼠,1份样本汉坦病毒核酸检测阳性,阳性率为0.50%;18份样本致病性钩端螺旋体核酸检测阳性,阳性率为9.00%;未检出新型布尼亚病毒和嗜吞噬细胞无形体。结论2021年蚌埠市鼠类致病性钩端螺旋体感染水平较高,应做好防控工作。

中国七省(区)牛蜱的种类鉴定及其携带无形体的分子检测

为了确定牛体表寄生硬蜱的种类及其携带无形体的状况,从河南、湖南、海南、四川、贵州、广东和广西采集牛体表寄生硬蜱572只,通过形态学和分子生物学方法对硬蜱进行种类鉴定;同时用PCR方法检测硬蜱体中无形体感染情况。经形态学和分子生物学鉴定,结果 572只牛硬蜱均为微小扇头蜱,且存在A、B、C等3个分支。基于无形体的16S rRNA基因序列分析的结果表明,微小扇头蜱携带无形体,其阳性率为38.8%(222/572),其中边缘无形体、山羊无形体、扁平无形体和嗜吞噬细胞无形体分别为23.6%(135/572)、7.3%(15/572)、5.3%(42/572)、2.6%(30/572),仅在湖南的牛蜱中发现存在无形体混合感染情况。微小扇头蜱C分支的发现尚属首次,且以边缘无形体感染率最高。

蜱传人兽共患无形体和立克次体的分子生物学鉴定

目的调查承德市周边农村蜱传立克次氏体目细菌的流行现状,分析承德地区人兽共患无形体和立克次体的种类及其遗传特征,为蜱传立克次氏体病的防控提供理论依据。方法本研究从承德市三沟镇和六沟镇采集蜱并提取总DNA,使用巢氏PCR技术扩增16S rRNA基因进行立克次氏体目细菌的鉴定。扩增无形体(Anaplasma)的热休克蛋白(groEL)基因和立克次体(Rickettsia)的外膜蛋白A(ompA)基因,测序后对序列进行同源性与系统发生分析,对蜱传人兽共患无形体和立克次体病原体进行鉴定。结果在三沟镇和六沟镇共采集到336只蜱,经鉴定均为长角血蜱。蜱感染无形体的阳性率为25.3%(85/336),立克次体的阳性率为17.3%(58/336),蜱感染无形体的阳性率显著高于立克次体(P=0.01)。无形体包括山羊无形体(Anaplasma capra)和羊无形体(Anaplasma ovis),阳性率分别为8.3%(28/336)和17.0%(57/336);立克次中西伯利亚立克次体(Rickettsia sibirica)的阳性率为3.6%(12/58),饶氏立克次体(Rickettsia raoultii)的阳性率为6.3%(21/58),Candidatus Rickettsia jingxinensis的阳性率7.4%(25/58)。结论承德市长角血蜱携带有2种人兽共患无形体与3种立克次氏体,表明承德周边农村地区流行立克次氏体目细菌具有较高的遗传多样性,需加强承德地区蜱与蜱传病原体的监测与防控。