吉林口岸地区主要蜱类及蜱媒病原检测分析

本研究对吉林省口岸地区主要蜱类进行监测,并采用分子生物学方法对新发蜱媒病进行检测.结果共采集蜱类1620只,分属1科3属4种,其中,集安口岸为日本血蜱Haemaphysalis japonica和嗜群血蜱Haemaphysalis concinna 2种,以嗜群血蜱H.concinna为主;延边口岸为森林革蜱Dermacentor silvarum、日本血蜱H.japonica和嗜群血蜱H.concinna 3种,以嗜群血蜱H.concinna为主;长白口岸为森林革蜱D.silvarum 和全沟硬蜱Ixodes persulcatus 2种,以全沟硬蜱I.persulcatus为主.蜱携带病原体检测结果显示,集安口岸嗜群血蜱幼蜱中检测到伽氏疏螺旋体B.garinii和Q热贝氏斯柯氏体C.burnetii 2种病原体,最小感染率(MIR)为0.25%;若蜱和成蜱中检测到伽氏疏螺旋体B.garinii、艾氏疏螺旋体B.afzelii和Q热贝氏斯柯氏体C.burnetii 3种病原体,阳性感染率分别为2.08%、0.35%、0.69%.延边口岸嗜群血蜱中检测到伽氏疏螺旋体B.garinii、艾氏疏螺旋体B.afzelii和Q热贝氏斯柯氏体C.burnetii 3种病原体,阳性感染率分别为4.88%、1.63%和1.86%;日本血蜱中,检测到伽氏疏螺旋体B.garinii、Q热贝氏斯柯氏体C.burnetii 和北亚斑点热群立克次体R.sibirica 3种病原体,阳性感染率均为1.85%.长白口岸森林革蜱检测到伽氏疏螺旋体B.garinii、人单核细胞埃立克体E.chaffensis、Q热贝氏斯柯氏体C.burnetii、北亚斑点热群立克次体R.sibirica、黑龙江立克次体R.heilongjiangensis 5种病原体,阳性感染率分别为2.56%、5.13%、2.56%、20.51%、7.69%,其中从1只森林革蜱中同时检测出北亚斑点热群立克次体R.sibirica和伽氏疏螺旋体B.garinii的复合感染;在全沟硬蜱中,检测到伽氏疏螺旋体B.garinii、艾氏疏螺旋体B.afzelii、人单核细胞埃立克体E.chaffensis、嗜吞噬无形体A.phagocytophilum、微小巴贝西原虫B.microti、双芽巴贝西原虫B.divergens、斑点热群立克次体R.sibirica和黑龙江立克次体R.heilongjiangensis 8种病原体,阳性感染率依次为6.60%、2.90%、2.37%、3.69%、5.28%、1.85%、6.60%、1.58%.由此提示吉林口岸地区蜱类自然感染蜱媒病原存在的证据.     

全沟硬蜱、森林革蜱及嗜群血蜱经卵传播新塔拉塞维奇立克次体的检测研究

本研究旨在明确新塔拉塞维奇立克次体是否可以在全沟硬蜱、森林革蜱及嗜群血蜱3种媒介蜱中经卵传播。以在东北林区采集的牛寄生饱血雌蜱为样本,在实验室条件下人工培育,以获得3种蜱的不同时期的形态,并使用PCR方法检测各虫态中新塔拉塞维奇立克次体的感染情况,从而研究新塔拉塞维奇立克次体在蜱生活周期中的传递规律。共采集牛寄生饱血雌蜱91只,其中全沟硬蜱14只,森林革蜱55只,嗜群血蜱24只。新塔拉塞维奇立克次体感染率为85.71%,5.45%,12.50%。卵的最低感染率分别为全沟硬蜱(2%)、森林革蜱(1.3%)、嗜群血蜱(1.1%),幼蜱的最低感染率分别为全沟硬蜱(5%)、森林革蜱(1.7%)、嗜群血蜱(2.2%)。且3种蜱雌蜱、卵、幼虫均检测到的新塔拉塞维奇立克次体,序列比对一致。感染的幼蜱均可通过叮咬使小鼠感染,三种蜱都能经卵传播新塔拉塞维奇立克次体。全沟硬蜱感染率高可能是东北林区新塔拉塞维奇立克次体的主要媒介蜱。     

不同蜱种E3泛素连接酶多态性分析

E3泛素连接酶通过蛋白修饰等泛素化过程在病原体与媒介、宿主互作中发挥着关键作用,而蜱类体内E3泛素连接酶及其功能还未明确,本研究利用高通量测序手段获得了森林革蜱、嗜群血蜱E3泛素连接酶CDS,并通过基因组生物信息学检索获得了长角血蜱、全沟硬蜱和亚洲璃眼蜱E3泛素连接酶CDS.结果 发现,森林革蜱和全沟硬蜱的E3泛素连接酶同属于SH3RF3型;而长角血蜱、嗜群血蜱E3泛素连接酶属于SH3RF2型,亚洲璃眼蜱的E3泛素连接酶介于两者之间可能为中间类型.结果 表明,不同蜱类的E3泛素连接酶随系统发育呈现出一定的演化趋势和多态性特征,E3泛素连接酶的多样性及表达情况可能是影响蜱类媒介效能的关键因素之一.     

中华硬蜱TROSPA的发现与鉴定

笔者从我国莱姆病媒介蜱种中华硬蜱中鉴定出莱姆病螺旋体外膜蛋白A受体TROSPA。中华硬蜱幼蜱体内的两个cDNA克隆I．sin-1（258bp）和I．sin-2（462bp）经序列比对和分析属于TROSPA．其中,I．sin-2的氨基酸序列同肩板硬蜱I.scapularis、篦子硬蜱I.ricinus和全沟硬蜱I.persulcatus的相似度分别为88．2％、88．5％和88．8％,而I.sin-1则分别存在46％、46．1％和45．6％的相似度。中华硬蜱TROSPA的发现为解释了中华硬蜱传播莱姆病的机制以及研究中华硬蜱与病原体的相互关系奠定了基础。     

黑龙江省部分林区全沟硬蜱复合感染重要蜱媒病原的调查研究

为了研究我国重要蜱媒病的复合感染情况,本文选择黑龙江林区蜱媒疾病高发区域,以莱姆病、森林脑炎、人巴贝西原虫病、埃立克体等蜱媒病原为目标,应用聚合酶链式反应对该地区采集的全沟硬蜱可能感染的蜱媒病原情况进行检测,以探讨这些蜱媒疾病在媒介全沟硬蜱体内的复合感染状况。结果表明,该地区的全沟硬蜱感染有莱姆病、森林脑炎、人巴贝西原虫病、埃立克体、斑点热等5种疾病的病原体,感染阳性率在1.05%~10.5%之间;在这些感染个体中,将近40%的个体属于复合感染,复合感染的类型有双重感染和三重感染。其中,莱姆病螺旋体和其他病原体复合感染的比例很高;没有发现埃立克体和人巴贝西原虫的复合感染现象。我国蜱媒病原的复合感染情况,理应引起预防、临床和公共卫生相关人员的充分关注。     

莱姆病人工宿主动物模型的建立

为了建立莱姆病人工宿主模型进行实验传播研究,用从我国全沟硬蜱分离的莱姆病螺旋体Borrelia garinii CHNM4人工感染4种实验动物.结果表明,以0.1×106条/mL的剂量皮下注射3日龄昆明小鼠是比较理想的方案.由此建立的动物模型既能用作全沟硬蜱的血源动物,又能在15天内保持所感染螺旋体对全沟硬蜱的感染力,可作为人工宿主动物模型.     

吉林省延边地区蜱类分布及其携带发热伴血小板减少综合征病毒调查

本文旨在探讨吉林省延边地区自然环境和动物体表蜱类分布及其消长规律,掌握其携带发热伴血小板减少综合征布尼亚病毒(Severe fever with thrombocytopenia syndrome virus,SFTSV)状况及传播病毒能力。2016年4~9月份按月采集延边州所辖8个县(市)自然环境中生长的蜱虫和放牧动物体表蜱虫进行形态学分类,对其进行构成分析。对其中部分蜱虫分组进行SFTSV核酸检测。SFTSV核酸检测方法采用了Real time RT-PCR和RT-PCR相结合的方法。结果共采集蜱虫3 446只,其中森林革蜱763只,嗜群血蜱222只,日本血蜱639只,长角血蜱515只,全沟硬蜱1 014只,其他血蜱293只;全沟硬蜱(29.43%)和森林革蜱(22.14%)为本地优势种。长角血蜱在图们市(70.88%)和珲春市(40.59%)分布较多。对部分蜱虫(1605只)SFTSV Real time RT-PCR检测结果总最低感染率为1.81%,分种最低感染率分别为嗜群血蜱8.65%、日本血蜱4.53%、长角血蜱1.59%。序列分析结果表明,本文检测到的SFTSV病毒与我国其他省份从患者身上分离到的大部分SFTSV有高度一致性(99%)且与2012年从浙江患者血清中分离到的SFTSV、2013年韩国国家疾病预防控制中心从人身上采集的长角血蜱中分离到的SFTSV处于同一分枝,把该病毒命名为YBHC-TICK1-2016/CHINA。     

陕西省蜱莱姆病螺旋体分子流行病学研究

采用巢式PCR和RFLP方法分析研究陕西地区不同蜱种感染莱姆病螺旋体情况及基因型.结果共检测蜱样1 116只,检测出阳性138只,阳性率为12.35%.嗜群血蜱B.b.s.l.阳性率为30.06%（107/356）,其中84只为B. garinii基因型,23只为B. afzelii基因型 达吉克斯坦革蜱B.b阳性率为3.88%（29/747）,24只为B. garinii基因型,5只为B. afzelii基因型 日本血蜱B.b.s.l.阳性率为15.38%（2/13）,均为B. garinii基因型.3个地区蜱感染莱姆病螺旋体的阳性率有显著性差异（P〈0.01,χ2=36.16）,其中以富县林区阳性率（17.44%）为最高.研究证实陕西地区嗜群血蜱、达吉克斯坦革蜱和日本血蜱存在莱姆病螺旋体的感染,以伯氏疏螺旋体B.garinii基因型为主.     

我国北方蜱中人粒细胞埃立克体16S rRNA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 %     

福建省蜱类(蜱螨亚纲:蜱目)名录

本文对福建省历年采集的蜱类标本进行分类鉴定,并对文献中提及但未见标本的种类依据其分布特点进行评论.结果发现,福建省蜱类记录共2科7属30种.其中,蓖子硬蜱Ixodes ricinus应更正为中华硬蜱I.sinensis或基刺硬蜱I.spinicoxalis,金泽革蜱Dermacentor auratus应更正为台湾革蜱D.taiwanensis或丽板革蜱D.bellulus,长须血蜱Haemophysalis aponommoides、越南血蜱Hae.vietnamensis应更正为北岗血蜱Hae.kitaokai,具角血蜱Hae.cornigera应更正为台湾血蜱Hae.taiwana,二棘血蜱Hae.bispinosa应更正为待定血蜱Haemaphysalis sp..     

Intrinsic competence of three ixodid ticks (Acari) as vectors of the Lyme disease spirochete

We compared the intrinsic vector competence of Ixodes dammini Spielman et al., Dermacentor variabilis (Say), and Amblyomma americanum (L.) for the Lyme disease spirochete (Borrelia burgdorferi Johnson et al.) on Prudence Island, Rhode Island, a Lyme disease-endemic site where all three ticks occur together. Natural and experimental spirochete infection rates were determined in those ticks and their degree of contact with white-footed mice (Peromyscus leucopus), the principal reservoir host, was compared. Host-seeking nymphal and adult I. dammini and A. americanum, collected by flagging, were nearly equally abundant, as were adult D. variabilis, but only I. dammini were infected with B. burgdorferi. Larvae and nymphs of both I. dammini and D. variabilis were found infesting mice, but A. americanum never were found on this host. Furthermore, although larvae of all three tick species became infected by ingesting spirochetes while feeding on experimentally infected mice, only I. dammini remained infected following the transstadial molt. These findings suggest that of these three tick species, only I. dammini is competent as a vector of the Lyme disease spirochete.     

Accelerated infectivity of tick-transmitted Lyme disease spirochetes to vector ticks.

We determined whether the span of infectivity of Lyme disease spirochetes (Borrelia burgdorferi) to vector ticks varies with the mode of infection in laboratory mice. Noninfected larval deer ticks were permitted to feed on two strains of spirochete-infected mice that had been naturally (via tick bite) and parenterally (via needle injection) infected with B. burgdorferi 2, 4, or 8 weeks earlier, and engorged ticks were dissected and examined for spirochetes by direct immunofluorescence microscopy. After initial infection, spirochetal infectivity to ticks was less efficient in needle-infected mice than in mice infected via tick bites. Tick-transmitted spirochetes develop more rapidly from the skin of infected mice and do not induce a strong antispirochete antibody response during the early stage of infection.     

Contrasts in tick innate immune responses to Borrelia burgdorferi challenge: immunotolerance in Ixodes scapularis versus immunocompetence in Dermacentor variabilis (Acari: Ixodidae)

The blacklegged tick, Ixodes scapularis Say, transmits the Lyme disease spirochete Borrelia burgdorferi, whereas the American dog tick, Dermacentor variabilis (Say), is unable to transmit the bacterium. We compared the innate immune response of these ticks against spirochetes directly inoculated into the hemocoel cavity of ticks. In I. scapularis, some Borrelia were found associated with hemocytes, while numerous other spiral-shaped, intact bacteria remained free in the hemolymph. In contrast, in D. variabilis only remnants of the bacteria were evident in the hemolymph, indicating lysis; intact spirochetes were rare. Spirochetes were observed bound to or within the organs of both tick species, although many more spirochetes were found associated with the I. scapularis organs. The few spirochetes observed with the D. variabilis organs appeared to be dead because D. variabilis tissues rarely contained culturable bacteria, unlike I. scapularis tissues. When spirochetes were incubated with I. scapularis hemolymph plasma in vitro, bacterial survival and motility were not reduced. In contrast, incubation of spirochetes with D. variabilis hemolymph plasma resulted in > 50% of the spirochetes becoming nonmotile by 45 min. The differences in the responses of the two different tick species indicate that I. scapularis is immunotolerant when challenged with B. burgdorferi and dependent on a slow phagocytic response to clear Borrelia from the hemolymph. In contrast, D. variabilis is highly immunocompetent (i.e., innate immunity), using plasma borreliacidal factors and a rapid increase in phagocytic cells to clear the infection and limit tissue invasion.     

安徽黄山地区中华硬蜱及其宿主感染莱姆病Borrelia afzelii的检测

利用PCR和病原体分离技术对安徽黄山地区中华硬蜱及其宿主感染莱姆病螺旋体的状况进行了检测,结果从40只黄山林区的中华硬蜱中获得了一株莱姆病螺旋体分离株,寄主动物及其他中华硬蜱均未获得阳性分离结果.以16s rRNA为靶标进行PCR检测,6.78%雌性和5%雄性中华硬蜱发现有阳性感染,2只草兔(92只)和1只麂子(16只)也发现有阳性感染;若蜱和其余宿主动物未发现阳性感染.阳性分离株和扩增获得的168 rRNA序列结果一致,同GenBank中的B.afzelii的系统发育关系最近,提示该分离株螺旋体属于B.afzelii.该结果与前期的实验传播结果提示中华硬蜱是我国南方莱姆病的传播媒介.     

Ixodes scapularis ticks (Acari: Ixodidae) from Louisiana are competent to transmit Borrelia burgdorferi, the agent of Lyme borreliosis

The principal vector of Borrelia burgdorferi, the Lyme borreliosis spirochete, in the Northeast and Midwestern regions of the United States is the blacklegged tick Ixodes scapularis. Because of a favorable environment, I. scapularis is also plentiful in the South; however, a correlation with Lyme borreliosis cases does not exist in this region of the United States. Concern existed that something intrinsic to ticks found in Louisiana could mitigate their ability to transmit B. burgdorferi. Therefore, we set out to assess the ability of I. scapularis ticks from Louisiana to become infected with and transmit B. burgdorferi using mice as hosts. In the laboratory, mating adult female ticks collected in southeastern Louisiana were fed on the ears of rabbits. After oviposition and egg hatching, the resulting larvae were fed on mice that had been needle-inoculated with two different strains of B. burgdorferi sensu stricto, B31 and JD1. Larvae were found to be positive for spirochetes. Additional fed larvae were allowed to molt into the nymphal stage. Flat nymphs remained infected with B. burgdorferi. Infected nymphs were allowed to feed on na?ve mice, all of which became infected as shown by culture of ear biopsy specimens. Na?ve larvae were then fed on these same mice to assess transmissibility. The resulting engorged larvae harbored spirochetes. We have demonstrated that the I. scapularis ticks found in Louisiana are fully competent to carry and transmit B. burgdorferi infection.     

Effect of ambient temperature on competence of deer ticks as hosts for Lyme disease spirochetes.

We determined whether the temperature of extrinsic incubation affects the competence of vector ticks as hosts for Lyme disease spirochetes (Borrelia burgdorferi). Larval Ixodes dammini ticks that had engorged on spirochete-infected C3H mice were incubated continuously at various temperatures, and the gut contents of the resulting nymphs were examined for spirochetes by direct immunofluorescence microscopy. Spirochetes were present in virtually all nymphs kept at 27 degrees C or less for 6 months, in only 10% of those kept at 33 degrees C, and in none kept at 37 degrees C. Spirochetes became undetectable within 8 weeks when nymphs were warmed from 27 to 33 degrees C beginning at the time of molting. Nymphs became virtually noninfective for mice after incubation at temperatures higher than 27 degrees C for 2 weeks or longer. We conclude that ambient temperatures in excess of 27 degrees C are not permissive for transmission of the agent of Lyme disease.     

Differential spirochetal infectivities to vector ticks of mice chronically infected by the agent of Lyme disease.

We determined whether the infectivity of the Lyme disease spirochete (Borrelia burgdorferi) to vector ticks varies with the duration of infection in laboratory mice. Thus, noninfected nymphal deer ticks were permitted to feed on two strains of early (2 months after infection) and late (8 months after infection) spirochete-infected mice. The attached ticks were removed from their hosts at specified time intervals and were thereafter examined for spirochetes by direct immunofluorescence microscopy. Spirochetes can be acquired by nymphal ticks as fast as 8 h after attachment. More than 80% of the attached ticks acquired spirochetal infection within 48 h after feeding on early spirochete-infected mice. In contrast, spirochetal infectivity to ticks was less than 50% after feeding on late spirochete-infected mice. The overall infectivity of spirochete-infected mice to ticks correlated with the duration of tick attachment. In addition, there was no adverse effect on the spirochetal infectivity to ticks by high levels of host antibody against spirochetes, and no obvious differences in infectivity to ticks was observed by the site of tick feeding. We conclude that the span of spirochetal infectivity to ticks varies with the duration of infection in mice and suggest that spirochetes may persist and may be evenly distributed in the skin of infected hosts, regardless of prominent host immunity.     

首次从金华地区蜱中检测到莱姆病螺旋体

为了解金华的蜱类及蜱类宿主动物中自然感染莱姆病螺旋体的状况,用PCR法检测鼠体寄生蜱类和宿主动物脏器中莱姆病DNA片段。共采集鼠体寄生蜱41只,从寄生于1只黑线姬鼠和2只小林姬鼠(2只)的3只中华硬蜱雌性若蜱中检测到阳性莱姆病感染。后以引物VS461扩增,此3只中华硬蜱均为阳性。后经克隆测序为B.afzelli(VS461同源株)。初步认为金华地区存在莱姆病病原,尚需进一步调查和病原分离加以证实。     

我国一些常见蜱种莱姆病螺旋体的分离与鉴定

本文对我国不同地区 8种蜱感染莱姆病螺旋体的情况进行了分离培养 ,从内蒙获得 2个分离株 ,对分离株的 16srDNA基因序列进行分析比较 ,发现新分离的两株莱姆病螺旋体同我国报道的CHY13p株具有高度同源性 ,CHY13p与CHNM4有 2个碱基差异 ,CHY13与CHNM5只有 1个碱基差异 ,而CHNM4和CHNM5两株螺旋体有 2个碱基差异。可初步认定新分离莱姆病螺旋体系Borreliagarinii。