Development of Paratransgenic Ticks for Disease Control

描述：（由申请人提供）：蜱传疾病越来越多 在人类和动物中诊断。有些是由于以前的复苏 已知的疾病，如落基山斑疹热（步行者1995）。但其他人 是由于新出现的病原体。在斑点热组（SFG）中， 在过去的15年中，已经描述了8种新的人类病原体（Stenos et al. 1997; Nilsson et al. 1999; Fournier et al. 2000），但它们也包括 病毒、艾希立克次体和巴贝氏体（Dawson等1991; Wenford等1994; Bakken等人，1994; Telford等人，1991）。新颖、高效、特异， 环境可接受的方法，通过以下方式干扰疾病传播： 迫切需要蜱虫。利用带有共生体的副转基因蜱 表达抗微生物物质的原核生物，如已经用 南美锥虫病载体Rhodnius prolixus（Durvasula等人 1997年），可以提供一种安全有效的方法来减少疾病的传播， 滴答声。实现这一目标的一个主要障碍是缺乏文化 蜱虫共生体的系统我们的实验室收集了世界上最多的 细胞系我们已经成功地用这些来分离蜱相关的 立克次体（Munderloh等，1998; Weller等，1998; Palmer等，1999; Simser等，2001 a，B）来自落基山森林中的孤星星蜱（MOAa） tick（R.孔雀属DAE 100 R）和蓖麻蜱（Rmoreli T2）。我们有 通过光学和电子显微镜表征这些微生物， 特异性抗体，以及通过PCR和核苷酸序列分析165 rDNA和其他关键基因。我们现在正在定义文化， 促进共生体的遗传操纵。我们的长期目标是 用抗菌肽A稳定转化立克次体昆虫ponn基因 （Hultmark等人，1983年）。用转化的立克次体感染蜱，以及 干扰病原体传播。我们计划把目标锁定在 rompA基因。孔雀作为同源转化的场所，避免 与生命基因（如rpoB）破坏有关的有害作用 基因（troyer et al.1999）。我们将利用最近的进展， 以虫媒立克次体的转化为指导（Rachek等，1998; Troyer等人，1999年）。并应用转座体技术（Epicentre）。 具体来说，我们将1。优化了产酶条件。 孔雀在蜱类细胞培养中的行为，Exanine及其在蜱类和哺乳动物细胞中的行为 通过光学和电子显微镜进行培养。2.我们将分析培养的R。 蜱中孔雀的组织嗜性和跨节/跨卵 传代和对天蚕素A的敏感性。最后，我们将努力实现3。稳定 转化R.孔雀与天蚕素A.然后我们将测试 用于体外和蜱中的抗微生物活性的transfonglycine，和 通过序列分析来表征它们。

DESCRIPTION: (provided by the applicant): Tick-borne diseases are increasingly diagnosed in humans and animals. Some are due to the resurgence of previously known illnesses, like Rocky Mountain spotted fever (Walker 1995). but others are due to new, emerging pathogens. Among the spotted fever group (SFG) alone, 8 new human pathogens have been described in the last 15 years (Stenos et al. 1997; Nilsson et al. 1999; Fournier et al. 2000), but they also include viruses, ehilichias and Babesia (Dawson et al. 1991; Thomford et al. 1994; Bakken et al. 1994; Telford et al 1991). Novel, efficient, specific and environmentally acceptable methods that interfere with disease transmission by ticks are urgently needed. Using paratransgenic ticks that carry symbiotic prokaryotes expressing an antimicrobial substance, as has been achieved with the symbiote of the Chagas disease vector, Rhodnius prolixus (Durvasula et aL 1997), could offer a safe and effective way to reduce disease transmission by ticks. A major obstacle to accomplishing this goal has been the lack of culture systems for tick symbiotes. Our laboratory has the largest collection of tick cell lines. We have successfully used these to isolate tick-associated rickettsiae (Munderloh et al. 1998; Weller et al. 1998; Palmer et al. 1999; Simser et al. 2001a,b) from the Lone Star tick (MOAa), the Rocky Mountain wood tick (R. peacockli DAE100R), and the Castor Bean tick (Rmoreli T2). We have characterized these microbes by light and electron microscopy, by using specific antibodies, as well as by PCR and nucleotide sequence analysis of 165 rDNA and other key genes. We are now in the process of defining the cultures to facilitate genetic manipulation of the symbiotes. Ourlong-termaim is the stabletransformation of Rickettsiapeacockii with cecropinA. an insect ponn gene (Hultmark et al. 1983). Infection of ticks with the transformed rickettsia, and interference with pathogen transmission. We plan to target the non-functional rompA gene of R. peacockli as a site for homologous transformation, avoiding deleterious effects associated with disruption of a vital gene, e.g. the rpoB gene (troyer et al. 1999). We will take the recent advances in successful transformation of insect-borne rickettsiae as a guide (Rachek et al. 1998; Troyer et al. 1999). and also apply transposome technology (Epicentre). Specifically, we will 1. optimize culture conditions for production of R. peacockii in tick cell culture, exanine its behavior in tick and mammalian cell culture by light and electron microscopy. 2. We will analyze cultured R. peacockii in ticks in terms of tissue tropism and transstadial/transovanal passage, and sensitivity to Cecropin A. Finally, we will work towards 3. stable transformation of R. peacockii with cecropinA. We will then test the transfonnants for antimicrobial activity in vitro and in ticks, and characterize them by sequence analysis.