Development of Paratransgenic Ticks for Disease Control

描述：(申请人提供)：壁虱传播的疾病越来越多 在人类和动物身上被诊断出。其中一些是由于以前的 已知的疾病，如落基山斑疹热(Walker 1995)。但其他人 是由于新出现的病原体造成的。仅在斑点热组(SFG)中， 在过去的15年里，已经描述了8种新的人类病原体(Statos等人)。 1997年；Nilsson等人。1999年；Fournier等人。2000)，但它们也包括 病毒、嗜热菌和巴贝斯虫(Dawson et al.1991年；Thomford et al.1994年； Bakken等人。1994年；Telford等人1991年)。新颖、高效、具体、 通过以下方式干扰疾病传播的环境可接受的方法 扁虱是迫切需要的。使用携带共生体的副转基因扁虱 原核生物表达抗微生物物质的原核生物，如已经通过 恰加斯病病媒Rhodnius prolixus的共生体(Durvesula等人 1997)，可以提供一种安全有效的方式来减少疾病传播 滴答滴答。实现这一目标的一个主要障碍是缺乏文化。 扁虱共生体的系统。我们实验室收集的扁虱数量最多。 细胞系。我们已经成功地利用这些技术分离出与扁虱相关的 立克次体(Munderloh等人1998年；Weller等人。1998年；Palmer等人。1999年； Simser等人。2001a，b)来自落基山脉的落基山树林(MOAA) 扁虱(R.parockli DAE100R)和蓖麻扁虱(Rmoreli T2)。我们有 用光学和电子显微镜表征这些微生物，通过使用 并对165株特异性抗体进行了聚合酶链式反应和核苷酸序列分析 RDNA和其他关键基因。我们现在正在定义文化，以 促进共生体的基因操作。Ourlong-Termaim是 天蚕素A对立克次体的稳定转化昆虫的Ponn基因 (Hultmark等人)1983年)。用转化的立克次体感染硬蜱，以及 干扰病原体传播。我们计划将目标对准非功能性 孔雀花根结线虫的Rompa基因作为同源转化位点，避免了 与重要基因的破坏有关的有害影响，例如rpoB 基恩(Troyer等人)1999年)。我们将把最近的进展成功地 以昆虫传播的立克次体转化为指南(rachek等人。1998年； Troyer等人。1999年)。并应用转座体技术(Eicentre)。 具体来说，我们将1.优化产生菌的培养条件。 孔雀在硬蜱细胞培养中的行为，在硬蜱和哺乳动物细胞中的行为 光镜和电子显微镜培养。2.对培养的R. 硬蜱中的孔雀II型组织嗜性和经腹/腹侧向 通过，并对天蚕素A敏感。最后，我们将努力实现3.稳定 抗菌肽A转化孔雀绿球藻的研究然后我们将测试 体外和扁虱体内抗菌活性的转染体，以及 通过序列分析对它们进行表征。

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.