Clinical features of chronic kidney disease in dogs with the serological presence of Leptospira spp.,Ehrlichia canis,and Anaplasma phagocytophilum

Chronic kidney disease is commonly diagnosed in dogs,and clinical signs may be aggravated when infected agents are involved.In this case report,33 dogs with chronic kidney disease were clinically evaluated and serologically tested for Leptospira spp.,Ehrlichia canis,and Anaplasma phagocytophilum.The seroprevalence for Leptospira spp.was 39.4%.The most frequent serovars found were Pyrogenes,Canicola,Bratislava and Australis,with serological titers between 1:100 to 1:800.Clinical signs included fever,depression,decreased body condition,vomiting and hema‑turia.Signifcant laboratory fndings were anemia,leukocytosis,thrombocytopenia,increased liver enzymes,urea and creatinine,hyperbilirubinemia and hyperphosphatemia.All leptospira seronegative dogs were positive for one or both monitored homoparasites(i.e.,E.canis and A.phagocytophilum);only three leptospira seropositive dogs were positive for one or both hemoparasites.Findings also suggest that endemic hemoparasites of dogs should be moni‑tored in dogs with a kidney condition for a better clinical picture of the patients and therapeutic approach.

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.

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.

Molecular Detection and Identification of Candidatus Ehrlichia Hainanensis,A Novel Ehrlichia Species in Rodents from Hainan Province,China

Ehrlichia(Anaplasmataceae family)are obligatory intracellular bacteria that infect humans and animals.They are hosted by mammals such as canines,bovines and wild rodents,and are vectored by ticks.In this study,we collected 121 rodent samples comprising 67 Niviventer fulvescens,27 Rattus tanezumi,24 Chiromyscus sp.,2 Rattus nitidus and 1 Leopoldamys edwardsi from Hainan province,which includes the second largest island in China.The presence and genetic diversity of Ehrlichia species was evaluated and characterized by amplification and sequencing of 16S rRNA,groEL and gltA genes.An Ehrlichia species was detected in 5 of the 67 Niviventer fulvescens samples(7.46%).The 16S rRNA,groEL and gltA genes showed the highest identity to known Ehrlichia sequences(99.20%,89.87%and 83.86%,respectively).In the phylogenetic trees they formed a cluster distinct from all other species.We propose that this species is a putative novel Ehrlichia species,which we suggest be named Candidatus Ehrlichia hainanensis.Its pathogenicity to humans remains to be further researched,and molecular surveillance in local populations is needed.

Molecular detection of <i>Ehrlichia canis</i>in ticks population collected on dogs in Meshkin-Shahr, Ardebil Province, Iran

Canine monocytic ehrlichiosis is a disease which can cause death in dogs and cats by the Ehrlichia canis and transmitted by hard ticks. The main vector is Rhipicephalus sanguineus which is a common tick in Iran and common ectoparasite on dogs. Few researches has been done to show Ehrlichiosis situation in dog’s ticks. Animal husbandry in Meshkin-Shahr County from Ardebil province (North-West Iran) is the main job of people. Specimens were collected on dogs’ ears, neck, shoulder and toes and they were transferred to the Entomology Laboratory, School of Public Health, Tehran University of Medical Sciences. After DNA extraction, Ehrlichia agent was detected by nested-PCR, 16 s rRNA amplification. Determination of sequence homologies have been done in Gen-Bank. 146 ticks were identified which included 29.44% female and 47.94% male. Rhipicephalus sanguineus were the most prevalent ticks. Ehrlichia spp and Anaplasma spp were found in 43.84% of all the specimens containing Anaplasma ovis, and Ehrlichia sp. and Herlichia canis. Rhipicephalus sanguineus is widely spread and the most common ticks on dogs. As far as we know, this is the first report of E. canis in vector from Iran. Nested PCR showed that hard ticks can contain Anaplasma ovis, Ehrlichia spp and E. canis. These results warrant studying on vector competence of ticks for the Ehrlichiosis agents.

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.

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.

Diagnostic Evaluation of Ehrlichia canis Human Infections

Ehrlichiosis is an infection of the group of Vector Borne Diseases (VBD), caused by different echrlichia species (spp). Ehrlichiae are primarily recognized as causers of animal diseases, later as human diseases—Humane Monocytic (HME) and Humane Granulocytic (HGE) ehrlichiosis. The dogs, cats and rodents have natural reservoirs of ehrlichial agents. Ehcrlichiae are spread to human’s trough, the bites of infected ticks, sometimes resulting from contact with animals, possible to the transfusion. In period from 2008 to 2013, we analyzed 250 patients suspect of VBD. On examination, there were included clinical, laboratory and epidemiological methods. The confirmation of Ehrlichia canis was in serum of 64 patients, thanks to Indirect Immune fluorescent Assay (IIF). The other laboratory and serological methods (Elisa, IIF, and Western blood) are utilized for differential diagnosis, and detection of co-infective forms of diseases. Ehrlichia canis infection has been reported in humans as a cause of serious illnesses characterized by non specific symptoms: fever, headache, musculoskeletal pain, enlarged liver, neurological, hematological dysfunction. Co-infections with E. canis due to causers of different VBD have been documented in humans. In 53 cases, we detected co-infective participation of E. canis with Rickettsia conorii in 24 cases, Coxiella burneti in 6, and Borrelia burgdorferi in 23 cases. The preferred drug for both human ehrlichiosis is doxycycline, except children to 8 years, pregnant females. Ehrlichiosis is a difficult infectious disease to diagnose and prognose because of dominant non specific symptoms and co-infections.

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.

反刍动物埃立克体微滴数字PCR方法的建立及初步应用

为建立反刍动物埃立克体(E.ruminantium)准确定量的微滴数字PCR方法(ddPCR),本研究以E.rum-inantium pCS20基因为靶基因,选取保守区域设计引物和探针,建立了E.ruminantium ddPCR方法。利用本研究建立的ddPCR方法检测E.ruminantium、牛巴贝斯虫、牛双芽巴贝斯虫、环形泰勒虫、无形体、犬埃立克体、牛埃立克体、马埃立克体和立氏埃立克体等,结果显示仅E.ruminantium出现特异性扩增,而与其他寄生虫均无交叉反应,特异性强;将pUC57-pCS20重组质粒标准品10倍倍比稀释(1×10^(-1)拷贝/μL~1×10^(5)拷贝/μL)后,利用该ddPCR方法检测,结果显示,该方法对重组质粒标准品的检测限为1.8拷贝/μL,比相应荧光定量PCR方法的敏感性高10倍,本实验建立的ddPCR方法敏感性高;批内和批间重复性试验结果变异系数(CV)均小于2%,重复性好。利用建立的ddPCR方法对50只钝眼蜱样品检测,结果显示,阳性样品反刍动物埃立克体核酸的最低拷贝数为26拷贝/μL,ddPCR和荧光定量PCR检测试剂盒的检出率均为16.0%(8/50)。本研究首次建立的ddPCR方法敏感性高、特异性强、重复性好,可作为E.ruminantium准确定量检测的有效手段。

犬埃立克体病原特点及其致病性研究进展

犬埃立克体(Ehrlichia canis,E.canis)引起的传染性疾病对犬或人类生命健康具有严重威胁,其可通过皮肤表面的伤口或节肢动物的叮咬侵入机体,进一步引起机体一系列反应,导致犬埃立克体相关疾病。此外,由犬埃立克体引起的疾病的临床症状与较多疾病的临床症状相似,在快速诊断上带来了一定程度的困难。犬埃立克体为严格的胞内寄生病原体,革兰氏染色为阴性,吉姆萨染色为紫色。目前已能成功分离该病原,并实现体外传代培养。犬埃立克体可诱导宿主细胞自噬,夺取宿主细胞的营养物质利于自身存活和增殖,并可诱导机体发生免疫反应,产生大量致炎因子,引起机体发热和对组织器官造成不同程度的损伤。犬埃立克体存在一种特殊的免疫逃避机制,能避免被宿主溶酶体或其他免疫因子清除,并在巨噬细胞内生长繁殖,破坏宿主细胞内的抗炎因子,进而破坏宿主的免疫功能。然而,犬埃立克体夺取或破坏宿主细胞营养成分或存活的具体机制尚不清楚,且如何逃避宿主免疫系统“追踪”有待研究。治疗方面,犬埃立克体仅对多西环素(doxycycline)等部分抗生素敏感,预防该病的最佳方法仍是定期清除体外寄生虫。笔者对犬埃立克体的病原特性、流行病学、致病机制等主要方面及部分病原与宿主细胞间作用的研究现状进行了综述。

新疆部分地区鼠类无形体和埃立克体的调查及16srRNA序列分析

目的调查新疆部分地区鼠类携带无形体和埃立克体的状况。方法对新疆博乐、石河子、乌鲁木齐3个地区捕获的鼠类(沙鼠、褐家鼠、田鼠)401份,采集肺脏、脾脏提取总DNA,通过巢式PCR扩增无形体和埃立克体16SrRNA片段并与GenBank中相应基因序列进行比对分析。结果在沙鼠样本中扩增到无形体和埃立克体16SrRNA片段,而在其它两种鼠类未扩增到目的片段,扩增片段经测序、比对后确定为Anaplasma phagocytophilum和Erhlichia chaffeenisis。在312份沙鼠检测样本中,检出无形体17份,占5.45%;埃立克体48份,占15.38%;无形体和埃立克体均检出12份,占3.84%。结论新疆地区存在无形体和埃立克体病原,其主要存在与荒漠和半荒漠地带生存的沙鼠中,新疆存在这两种病原的自然疫源地,而且两种病原可以共同存在于同一宿主动物。

野外采集标本中人埃立克体16S rRNA基因扩增方法及应用

本文应用特异引物 ,通过多对半巢式PCR从采自内蒙古大兴安岭的蜱标本中扩增出人粒细胞埃立克体和查菲埃立克体的 16SrRNA全基因 ,一方面提供了这 2种埃立克体存在于中国的证据 ,另一方面为今后进行埃立克体病的病原、媒介和宿主的调查研究提供了一个可行的方法。并从一只大林姬鼠的脏器中检测到EC

PCR检测蜱中查菲埃立克体DNA及其序列分析

应用从查菲埃立克体１６ＳｒＲＮＡ基因序列高变区构建的特异引物，进行ＰＣＲ，检测蜱标本中病原体ＤＮＡ。结果从云南采集的龟形花蜱、从福建采集的越原血蜱和卵形硬蜱扩增出３８９ｂｐ的特异ＤＮＡ片段。收集龟形花蜱和越原血蜱的特异ＰＣＲ产物，通过与Ｔ载体连接，进行克隆和序列测定。其ＤＮＡ序列与美国查菲埃立克体分离株对应位置相差一个核苷酸，与其它种埃立克体的同源性为８０．７％～９６．１％。这是首次在我国发现埃立克体存在的病原线索。

应用半巢式PCR检测我国北方一些蜱种中的查菲埃立克体

目的 :了解我国北方一些蜱种中是否携带查菲埃立克体 (Ehrlichiachaffeensis,简写为EC)。方法 :应用半巢式PCR检测蜱的带菌率 ,利用 16SrRNA基因测序方法鉴定所测序列。结果 :从内蒙古莫尔道嘎林业局采集的全沟硬蜱和森林革蜱中扩增出查菲埃立克体的 16SrRNA基因 ,阳性率分别是 39.0 6 %和 10 .0 0 % ;并从新疆精河采集的全沟硬蜱和草原革蜱中也测到相同的序列 ,最小阳性率分别为 5.79%和 1.6 7%。对莫尔道嘎采集的蜱标本进行 12 2 0bp的EC16SrRNA基因分析 ,结果与美国 1株查菲埃立克体 (GenBankU2 350 3)的相应序列只差 1个碱基。结论 :所使用的两套半巢式PCR扩增引物 。

我国犬埃立克体病病原分离与鉴定 Ⅱ.我国病原株与其他埃立克体间的遗传距离分析

应用clustalw 计算机分析软件,对我国南方某养犬基地的埃立克体病原株(Gzh981 和Gzh982)与基因库中其他相关埃立克体16SrRNA 基因进行了匹配分析。结果表明,我国的2 种分离株分别与E. platys和 E. canis 的遗传距离最小(0.001)。在系统发育中, Gzh981 和 E. platys、HGE、E. equi、E.phagocytophila 同属一群;Gzh982 和E. canis、E. ew ingii、E. chaffeensis、E. m uris同属一群。

我国北方蜱中人粒细胞埃立克体16SrRNA基因的检测

本文应用人粒细胞埃立克体病 ( HGE)的病原体的 1 6S r RNA基因序列的特异引物对采自我国北方地区的一些蜱标本进行扩增 ,首次从内蒙古大兴安岭采集的全沟硬蜱、森林革蜱和嗜群血蜱 ,以及从新疆精河采集的全沟硬蜱和草原革蜱中扩增出该病原体的 1 6S r RNA基因片段。所测出的 967bp序列与美国 HGE株 ( Gen Bank U0 2 52 1 )的同源性为 1 0 0 %

人粒细胞无形体病的诊治进展

人粒细胞无形体病是由人嗜粒细胞无形体引起的一种经蜱传播的新发的自然疫源性疾病。该病主要临床表现为发热、消化道及血液系统症状等,少数严重患者可因多脏器功能衰竭而死亡。近年来,在美国、欧洲、非洲等地均有相关病例报道,我国也有病例发现。该病日益受到人们的关注和重视,本文就人粒细胞无形体病的诊断与治疗研究的进展作一综述。

江西省赣州市蜱携带立克次体和埃立克体的调研

目的 了解江西省赣州市蜱的种类及其携带的病原体,为蜱传疾病的防控提供科学依据。方法 在野外用布旗法和在动物体表采集蜱,进行种类鉴定。用PCR法扩增立克次体(Rickettsia sp.)gltA基因和埃立克体(Ehrlichia sp.)16S rRNA基因片段并测序,进行系统进化分析。结果 江西省赣州市4县采集的395只蜱的种类包括2属4种,分别为血蜱属的长角血蜱(Haemaphysalislongicornis)、嗜群血蜱(H.concinna)、具角血蜱(H.cornigera)和扇头蜱属的微小扇头蜱(Rhipicephalus microplus)。395只蜱分为43批检测立克次体和埃立克体,共有18批阳性。系统进化分析显示,龙南县22批蜱中检测到16批阳性,来源于具角血蜱的LN1615株与扇头蜱立克次体(R.rhipicephali)处于同一分支,来源于微小扇头蜱的LN1620株与马赛立克次体(R.massiliae)处于同一分支,其余的12株来源于具角血蜱和2株来源于微小扇头蜱,与日本立克次体(R.japonica)处于同一分支。崇义县CY1602株来源于嗜群血蜱与R.raoultii处于同一分支。于都县YD1606株为来源于长角血蜱的埃立克体,与2010年Yonaguni206(HQ697589)、Yonaguni138(HQ697588)和2009年HLAE178(GU075695)处于同一分支。安远县微小扇头蜱检测均为阴性。结论 本文对江西省赣州市采集的蜱进行了鉴定,首次在江西省赣州市的蜱中检测到日本立克次体、扇头蜱立克次体、马赛立克次体、R.raoultii和埃立克体,为江西省蜱种类的调查及其传播疾病的预防控制提供根据。

黑龙江全沟硬蜱中检测出类似人粒细胞埃利希体的病原体DNA

目的 寻找我国蜱中人粒细胞埃利希体感染存在的病原学证据。方法 应用从人粒细胞埃利希体 16SrRNA基因构建的特异引物进行半套式PCR ,检测蜱标本中埃利希体DNA。然后对PCR扩增产物进行克隆和序列测定 ,与已知序列进行同源性比较。结果 从黑龙江采集的全沟硬蜱 (Ixodespersulcatus)中扩增出特异DNA片段 ,计算最小阳性率为 0 8%。对 919bp的扩增产物进行序列分析 ,证实为埃利希体DNA ,与美国人粒细胞埃利希体分离株对应序列比较 ,相差 4个核苷酸。结论 这是首次证明我国有类似人粒细胞埃利希体的病原体存在。