Immunosuppressive effects of sialostatin L1 and L2 isolated from the taiga tick Ixodes persulcatus Schulze

Tick saliva contains immunosuppressants which are important to obtain a blood meal and enhance the infectivity of tick-borne pathogens. In Japan, Ixodes persulcatus is a major vector for Lyme borreliosis pathogens, such as Borrelia garinii, as well as for those causing relapsing fever, such as B. miyamotoi. To date, little information is available on bioactive salivary molecules, produced by this tick. Thus, in this study, we identified two proteins, I. persulcatus derived sialostatin L1 (Ip-sL1) and sL2 (Ip-sL2), as orthologs of I. scapularis derived sL1 and sL2. cDNA clones of Ip-sL1 and Ip-sL2 shared a high identity with sequences of sL1 and sL2 isolated from the salivary glands of I. scapularis. Semi-quantitative PCR revealed that Ip-sL1 and Ip-sL2 were expressed in the salivary glands throughout the life of the tick. In addition, Ip-sL1 and Ip-sL2 were expressed even before the ticks started feeding, and their expression continued during blood feeding. Recombinant Ip-sL1 and Ip-sL2 were developed to characterize the proteins via biological and immunological analyses. These analyses revealed that both Ip-sL1 and Ip-sL2 had inhibitory effects on cathepsins L and S. Ip-sL1 and Ip-sL2 inhibited the production of IP-10, TNFα, and IL-6 by LPS-stimulated bone-marrow-derived dendritic cells (BMDCs). Additionally, Ip-sL1 significantly impaired BMDC maturation. Taken together, these results suggest that Ip-sL1 and Ip-sL2 confer immunosuppressive functions and appear to be involved in the transmission of pathogens by suppressing host immune responses, such as cytokine production and dendritic cell maturation. Therefore, further studies are warranted to investigate the immunosuppressive functions of Ip-sL1 and Ip-sL2 in detail to clarify their involvement in pathogen transmission via I. persulcatus.     

伯氏疏螺旋体在全沟硬蜱体内的分布与经卵传递

本实验首次证实全沟硬蜱可经卵传递伯氏疏螺旋体,2只卵巢感染螺旋体的雌蜱的子代感染率分别为59%和29%。伯氏疏螺旋体主要分布于全沟硬蜱的中肠内,饥饿和饱血蜱的中肠感染率相近,分别为30.9%和29.0%:而两者的全身感染与中肠感染之比则分别为2.9%和15.0%。     

Detection and genetic characterization of a putative novel Borrelia genospecies in Ixodes apronophorus / Ixodes persulcatus / Ixodes trianguliceps sympatric areas in Western Siberia

Four genospecies from the Borrelia burgdorferi sensu lato complex were detected in Ixodes persulcatus and Ixodes pavlovskyi ticks from Siberia and genetically characterized. The presence of Borrelia spp. in Ixodes apronophorus and Ixodes trianguliceps ticks found in Asia has never been studied. In this study, genetic diversity of B. burgdorferi s.l. was investigated in three I. persulcatus / I. trianguliceps / I. apronophorus sympatric habitats in Western Siberia. Three groups of samples were examined: (i) ticks that were taken from rodents and molted in a laboratory; (ii) non-molted ticks collected from rodents; (iii) specimens from small mammals. Expectedly, Borrelia afzelii and Borrelia bavariensis were detected in I. persulcatus and in small mammals from the studied locations. Borrelia bavariensis was first found in molted I. apronophorus and I. trianguliceps. Identical genovariants of B. bavariensis were found in I. apronophorus, I. trianguliceps, and I. persulcatus. In addition, a new Borrelia genovariant was discovered in non-molted and molted I. apronophorus and non-molted I. persulcatus and I. trianguliceps, as well as in small mammals. This new genovariant was genetically characterized using MLST and single locus sequence analysis, which indicated that the new Borrelia genovariant significantly differs from all known Borrelia species. We propose the name "Candidatus Borrelia sibirica" for this putative new species.     

全沟硬蜱经期传播莱姆病螺旋体的研究

目的：研究全沟硬蜱经期传播莱姆病螺旋体的能力。方法：以莱姆病螺旋体感染的KM鼠作为宿主动物，利用培养和PCR方法检测全沟硬蜱对吸血感染的莱姆病螺旋体的保持能力以及蜕化后若蜱对敏感动物的感染能力。结果：全沟硬蜱幼蜱攻击感染KM鼠后获得97.5%的阳性感染，并能保持所感染的莱姆病螺旋体至若蜱。幼蜱蜕化后,45.0%的若蜱仍携带姆病螺旋体并可通过叮咬感染敏感KM鼠。结论：全沟硬蜱具备经期传播莱姆病螺旋体的能力。     

全沟硬蜱在黑龙江小兴安岭莱姆病螺旋体传播中的作用

目的：探讨全沟硬蜱在莱姆病螺旋体传播中的作用。方法：用布旗法在林区草地采蜱，并分类鉴定；用直接荧光抗体法检查蜱类中肠带菌情况，并分离菌株。结果：全沟硬蜱为当地优势蜱种，人群发病与该蜱叮咬率密切相关；该蜱中肠带菌率为４０％，并从１４组（７０只）蜱中分离出２株莱姆病螺旋体；新分离株的单克隆抗体反应与国内分离株Ｍ７相同，而与美国菌株Ｂ３１有区别。结论：当地全沟硬蜱是莱姆病螺旋体的主要生物媒介。     

Identification and functional analysis of ferritin 2 from the Taiga tick Ixodes persulcatus Schulze

Ferritin 2 (FER2) is an iron storage protein, which has been shown to be critical for iron homeostasis during blood feeding and reproduction in ticks and is therefore suitable as a component for anti-tick vaccines. In this study, we identified the FER2 of Ixodes persulcatus, a major vector for zoonotic diseases such as Lyme borreliosis and tick-borne relapsing fever in Japan, and investigated its functions. Ixodes persulcatus-derived ferritin 2 (Ip-FER2) showed concentration-dependent iron-binding ability and high amino acid conservation, consistent with FER2s of other tick species. Vaccines containing the recombinant Ip-FER2 elicited a significant reduction of the engorgement weight of adult I. persulcatus. Interestingly, the reduction of engorgement weight was also observed in Ixodes ovatus, a sympatric species of I. persulcatus. In silico analyses of FER2 sequences of I. persulcatus and other ticks showed a greater similarity with I. scapularis and I. ricinus and lesser similarity with Hyalomma anatolicum, Haemaphysalis longicornis, Rhipicephalus microplus, and R. appendiculatus. Moreover, it was observed that the tick FER2 sequences possess conserved regions within the primary structures, and in silico epitope mapping analysis revealed that antigenic regions were also conserved, particularly among Ixodes spp ticks. In conclusion, the data support further protective tick vaccination applications using the Ip-FER2 antigens identified herein.     

Protective immunity against Ixodes ricinus induced by a salivary serpin

Iris is a specific elastase inhibitor expressed in the salivary glands of the hard tick Ixodes ricinus. It belongs to the superfamily of serpins and interferes with both haemostasis and the immune response of the host. In this study, we first show that Iris is expressed in nymphs but not in the female midgut nor in males. We also show that Iris is present in the saliva. To examine its potency as anti-tick vaccine candidate, we set up three models of I. ricinus infestation on immunized animals: nymphs on mice, and adults and nymphs on rabbits. We report the rise of neutralizing antibodies following immunization of rabbits and mice. This comes with a significant protective immunity against ticks in rabbits only, resulting in a 30% mortality rate and a diminution of weight gain in both nymphs and adults and a prolongation of blood feeding time in adults. This is the first report on an anti-tick vaccine trial on I. ricinus using a protein able to interact with both host immunity and haemostasis, as a vaccinating antigen.     

Ixodes persulcatus,a major vector of Alphaproteobacteria in Russia

Ixodes persulcatus Schulze, 1930 (I. persulcatus) is distributed from the western to the eastern borders of Russia where it is found in the taiga and in mixed forests of the European type. This tick is a known vector of viral (tick-borne encephalitis virus), spirochetal (Borrelia spp.), and protozoan (Babesia spp.) agents. In this work, we analyzed the phylogenetic position of I. persulcatus within the Prostriata based on its 18S rRNA gene. Furthermore, we review the relationship of this tick with 9 Alphaproteobacteria from the genera Rickettsia, Ehrlichia, and Anaplasma which are found in I. persulcatus in the territory of Russia. These observations suggest that I. persulcatus may be one of the more important vectors and reservoirs of Alphaproteobacteria and other microorganisms in Russia.     

Phylogeographical structure of the tick Ixodes persulcatus: A novel view

The tick Ixodes persulcatus Schulze, 1930, has a wide distribution from the Baltic to the Far East and is a vector of a number of human pathogens. Thus, the study of the genetic structure and evolution of this species is of great epidemiological importance. rRNA genes were used as genetic markers to identify the phylogeographical structure of the ticks. The sequences of gene fragments of 28S (expansion segment D3) and mitochondrial 12S rRNA for 25 and 76 ticks, respectively, that had been collected in various regions of Russia in 2007–2011, were obtained. The sequences of the 28S rRNA D3 segment were identical for all ticks within the studied area. Analysis of the sequences of the mitochondrial 12S rRNA fragment revealed 4 haplotypes with one occurring at a frequency of 0.96. It is shown that the ‘deep’ population structure of I. persulcatus (McLain et al., 2001) was erroneous because of the inclusion of contaminating fungi sequences of 28S rRNA in the phylogenetic analysis. This was, possibly, due to the use of universal PCR primers that amplify the DNA of a wide range of eukaryotes, particularly of fungi which are common in samples of ticks. The influence of PCR conditions on the preferential amplification of the DNA of different organisms is also demonstrated.     

Natural hybridization of the ticks Ixodes persulcatus and Ixodes pavlovskyi in their sympatric populations in Western Siberia

Hybridization of ticks of the genus Ixodes has been described for several species under laboratory conditions although no molecular genetics evidence confirming interspecific hybridization in nature is available. We have designed a real time PCR targeted on nuclear (ITS2) and mitochondrial (cox1) markers to accurately identify tick species and to detect interspecific hybrids of Ixodes persulcatus and Ixodes pavlovskyi in their sympatric populations in Western Siberia. A survey of 783 individual ticks from a suburb of Tomsk showed that 44.2% of ticks belong to I. pavlovskyi species and 55.8% to I. persulcatus, based on the mtDNA data. Results obtained with the nuclear marker were not consistent, indicating that approximately 10% of the ticks were hybrids and about 5% revealed mtDNA introgression. Both hybridization and introgression have been shown to occur bidirectionally but more efficiently in the mating pair female I. pavlovskyi × male I. persulcatus than vice versa. The existence of the first generation hybrids and backcrosses challenges the existing view about effective reproductive barriers between I. pavlovskyi and I. persulcatus. While using only mitochondrial markers can lead to errors in determining tick species, we propose to use nuclear or both markers instead. The results obtained in the present paper and published earlier suggest that hybridization between closely related tick species in their sympatric zones is common rather than exceptional. The role of hybrid populations of vectors in the evolution of transmitted pathogens is also discussed.     

Suppressive effect of dietary raffinose on T-helper 2 cell-mediated immunity

The effects of the dietary oligosaccharide raffinose on immune responses, with special reference to its anti-allergic functions, were examined in vivo. First, feeding a diet supplemented with 50 g raffinose/kg to BALB/c mice significantly (P<0.05) increased interleukin (IL) 12 secretion from isolated Peyer's patch (PP) cells in vitro compared with feeding control diet. When isolated PP cells were used as antigen-presenting cells (APC) for CD4+ T-splenocytes isolated from ovalbumin (OVA)-specific T-cell receptor transgenic (Tg) mice in the presence of OVA as antigen, significantly (P<0.05) higher levels of interferon-gamma were observed in the cultures using APC from raffinose-fed mice than those cultures using APC from control mice. Second, the diet containing 50 g raffinose/kg or control diet was fed to OVA Tg mice, and subsequently, OVA was added to each diet to prime T cells in vivo. CD4+ T-cells from the mesenteric lymph nodes of the raffinose-fed mice secreted significantly (P<0.05) higher levels of IL-2 and significantly (P<0.05) lower levels of IL-4 following in vitro antigenic stimulation compared with those of the control mice. These present results suggest that feeding raffinose may suppress differentiation of na?ve T-helper (Th) cells into Th2 cells in the mesenteric lymphoid nodes. Last, feeding raffinose suppressed rises of serum immunoglobulin E levels in the Tg mice treated with long-term ingestion of OVA. In conclusion, it is suggested that dietary raffinose suppresses serum immunoglobulin E response through suppression of Th2-type immune response against oral antigen in the lymphoid organs located in or near the intestine.     

Occurrence of Borrelia burgdorferi in Ixodes ricinus in The Netherlands

In a 1989 surveillance to study the infection rate of Ixodes ricinus ticks with Borrelia burgdorferi, a total of 1838 nymphal and adult Ixodes ricinus ticks were collected from 20 locations in The Netherlands. By means of a culture method using modified Barbour-Kelly-Stoenner medium nymphs were examined in pools of 4 and adults were examined individually. With 63 out of 75 isolates propagation and serological identification were possible. All of these 63 isolates were identified as Borrelia burgdorferi by means of an indirect immunofluorescence assay. In nymphs the minimal infection rate was 2.4%. The rate of infection for adults was shown to be 14.3%. These results are in agreement with the percentages found by other investigators in West-Europe. In all locations examined Ixodes ricinus were found to be infected with Borrelia burgdorferi, although the infection rate varied. The results indicate that the infection is widespread among ticks in The Netherlands and the potential risk of acquiring Lyme borreliosis after a tick bite is serious.     

Occurrence of Borrelia burgdorferi sensu stricto, Borrelia garinii and Borrelia afzelii in the Ixodes ricinus ticks from Eastern Slovakia

A total of 2816 unfed adults nymphs of Ixodes ricinus ticks were collected from vegetation in Koice (Eastern Slovakia) from 1994 to 1997. Prevalence of Borrelia burgdorferi s. 1. in I. ricinus ticks, detected by dark field microscopy, varied and depended upon the year and the habitat of the collected ticks. The lowest prevalence was observed in 1994 (4.8%). During 1995 it increased to 17.2% and during the next two years decreased to 15.5% and 14.2%. The rate of infection varied from 2.1 to 23.3% within 10 examined habitats of the Koice area. A different value of relative density of ticks was observed in various habitats. It ranged from 9–212 ticks per collecting hour within one flagged area (600m²) which is 1.5–35.5 ticks per 100m². Eight isolates were obtained from the infected ticks. Electrophoresis and immunoblotting with 6 monoclonal antibodies were used for the identification of Borrelia strains. Three tick isolates were identified as B. burgdorferi s. s. and the other three isolates were found to be B. garinii. One strain reacted as a mixed culture of B. burgdorferi s. s., and B. garinii. The strain originated from the Vihorlat Mountains habitat and was detected by PCR-SSCP as B. burgdorferi s. s. with a small amount of B. afzelii. The obtained results emphasize the epidemiological importance not only of B. garinii and B. afzelii but also of B. burgdorferi s. s. in Central Europe.     

美发摩丝对小鼠免疫功能的抑制

目的：研究美发摩丝对小鼠免疫功能的影响。方法：选择体质量18－22g的雌性昆明种小鼠10只／组,以美发摩丝12．5mg/kg,250mg/kg,1000mg/.kg灌胃染毒3周后进行体液免疫,细胞免疫,非特异性免疫及免疫脏器质量测试,结果：250mg/kg,1000mg/kg剂量组的胸腺和脾脏质量明显低于对照组（P＜0．01）,PFC对数值,血清溶血素抗体积数,足跖肿胀度及碳廓清指数均低于对照组（P＜0．05,P＜0．01）。结论：美发摩丝对小鼠的免疫功能有抑制作用。     

Borrelia species in Ixodes affinis and Ixodes scapularis ticks collected from the coastal plain of North Carolina

Ixodes affinis and I. scapularis are tick species that are widely distributed in the coastal plain region of North Carolina. Both tick species are considered enzootic vectors for spirochetal bacteria of the genus Borrelia and specifically for B. burgdorferi s.s., the pathogen most often attributed as the cause of Lyme disease in the USA. Laboratory testing of individual I. affinis and I. scapularis ticks for the presence of Borrelia DNA was accomplished by PCR, targeting 2 regions of the 16S-23S intergenic spacer. In I. affinis, Borrelia DNA was detected in 63.2% of 155 individual ticks. B. burgdorferi s.s. and B. bissettii were identified by DNA sequencing in 33.5% and 27.9% I. affinis, respectively. Statistical differences were found for sex distribution of Borrelia DNA between I. affinis females (76.8%) and I. affinis males (55.6%) where B. burgdorferi s.s. was more prevalent in females (44.6%) than in males (27.3%). In I. scapularis, 298 individually tested ticks yielded no Borrelia PCR-positive results. This study found a higher incidence of Borrelia spp. in I. affinis collected in coastal North Carolina as compared to previous reports for this tick species in other Southern states, highlighting the potential importance of I. affinis in the maintenance of the enzootic transmission cycle of B. burgdorferi s.l. in North Carolina. The lack of Borrelia DNA in I. scapularis highlights the need for additional studies to better define the transmission cycle for B. burgdorferi s.s. in the southeastern USA and specifically in the state of North Carolina.     

High prevalence of Borrelia lusitaniae in Ixodes ricinus ticks in Tunisia

To investigate whether ticks of the genus Ixodes are infected by Borrelia burgdorferi complex, 490 unfed Ixodes ricinus ticks were collected by flagging in three different areas of Tunisia in 1998. DNAs extracted from 81 adults, 60 nymphs and 38 larvae were analysed after genic amplification of the noncoding spacer between the two copies of the rrl–rrf genes of B. burgdorferi sl. The prevalence of B. burgdorferi sl. in adults, nymphs and larvae was found to be 34, 33.3 and 2.6%, respectively. All DNAs (n = 61) but one were identified as belonging to different genotypes of B. lusitaniae by analysis of the restriction fragment length polymorphism of amplification products. In addition, 290 adults, 14 nymphs and 7 larvae were used to inoculate BSK-H medium to isolate spirochetes. Fifteen strains were isolated from adult ticks in the humid areas of Tunisia, whereas only one was obtained from larvae. Isolates were identified as B. lusitaniae (15/16) and B. garinii (1/16). These results provide new evidence for the existence of Lyme borreliosis in North Africa.     

The Ixodes ricinus tick as a vector of Borrelia burgdorferi in Slovenia

In many parts of Slovenia, the vegetation, climate and animals form an ideal natural environment for the persistence of different tick species. In this study, Ixodes ricinus ticks, the prevalent tick species in our country, are identified as the vectors of Borrelia burgdorferi in Slovenia. Ticks were collected by flagging between May and October 1990. All the ticks collected from different parts of Slovenia were I. ricinus and each one was examined individually. The ticks were tested to determine the presence of B. burgdorferi by indirect immunofluorescent assay. Among the 496 ticks examined for borreliae there were 85 adults and 411 nymphs. Twenty (23.5%) adults and 18 (4.4%) nymphs were found positive for Borreliae. This study discusses the importance of I. ricinus ticks in the role of Lyme borreliosis in Slovenia.     

Prevalence of Borrelia miyamotoi in Ixodes Ticks in Europe and the United States

Borrelia miyamotoi, a relapsing fever-related spirochete transmitted by Ixodes ticks, has been recently shown to be a human pathogen. To characterize the prevalence of this organism in questing Ixodes ticks, we tested 2,754 ticks for a variety of tickborne pathogens by PCR and electrospray-ionization mass spectrometry. Ticks were collected from California, New York, Connecticut, Pennsylvania, and Indiana in the United States and from Germany and the Czech Republic in Europe from 2008 through 2012. In addition, an isolate from Japan was characterized. We found 3 distinct genotypes, 1 for North America, 1 for Europe, and 1 for Japan. We found B. miyamotoi infection in ticks in 16 of the 26 sites surveyed, with infection prevalence as high as 15.4%. These results show the widespread distribution of the pathogen, indicating an exposure risk to humans in areas where Ixodes ticks reside.     

Genetic variability of Rickettsia spp. in Ixodes persulcatus/Ixodes trianguliceps sympatric areas from Western Siberia,Russia: Identification of a new Candidatus Rickettsia species

Rickettsia spp. are the causative agents of a number of diseases in humans. These bacteria are transmitted by arthropods, including ixodid ticks. DNA of several Rickettsia spp. was identified in Ixodes persulcatus ticks, however, the association of Ixodes trianguliceps ticks with Rickettsia spp. is unknown. In our study, blood samples of small mammals (n = 108), unfed adult I. persulcatus ticks (n = 136), and I. persulcatus (n = 12) and I. trianguliceps (n = 34) ticks feeding on voles were collected in two I. persulcatus/I. trianguliceps sympatric areas in Western Siberia. Using nested PCR, ticks and blood samples were studied for the presence of Rickettsia spp. Three distinct Rickettsia species were found in ticks, but no Rickettsia species were found in the blood of examined voles. Candidatus Rickettsia tarasevichiae DNA was detected in 89.7% of unfed I. persulcatus, 91.7% of engorged I. persulcatus and 14.7% of I. trianguliceps ticks. Rickettsia helvetica DNA was detected in 5.9% of I. trianguliceps ticks. In addition, a new Rickettsia genetic variant was found in 32.4% of I. trianguliceps ticks. Sequence analysis of the 16S rRNA, gltA, ompA, оmpB and sca4 genes was performed and, in accordance with genetic criteria, a new Rickettsia genetic variant was classified as a new Candidatus Rickettsia species. We propose to name this species Candidatus Rickettsia uralica, according to the territory where this species was initially identified. Candidatus Rickettsia uralica was found to belong to the spotted fever group. The data obtained in this study leads us to propose that Candidatus Rickettsia uralica is associated with I. trianguliceps ticks.     

全沟硬蜱为内蒙大兴安岭莱姆病螺旋体的主要生物媒介

我们于1990年5～8月在内蒙大兴安岭,对莱姆病螺旋体的生物媒介和人群感染进行了调查,全沟硬蜱(Ixodes persulcatus)是当地蜱类的优势种,用直接荧光抗体法检查全沟硬蜱的中肠涂片,发现其带菌率为40%;从46组(380只)全沟硬蜱中分离出19株莱姆病螺旋体。分离出的菌株在单克隆反应上与美国菌株不同。用间接荧光抗体法检测308名居民血清,阳性率为13.3%。以上研究证明,大兴安岭是莱姆病疫区,全沟硬蜱是将莱姆病螺旋体传播到人和动物的主要生物媒介。