Fiorotti Jéssica, Gôlo Patrícia S, Marciano Allan Felipe, Camargo Mariana G, Angelo Isabele C, Bittencourt Vânia R E P
Post-Graduate Program in Veterinary Sciences, Federal Rural University of Rio de Janeiro.
Post-Graduate Program in Veterinary Sciences, Federal Rural University of Rio de Janeiro; Department of Animal Parasitology, Federal Rural University of Rio de Janeiro.
J Vis Exp. 2019 Jun 1(148). doi: 10.3791/59899.
Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight loss, depreciation of the animals' leather and also can act as a vector of several pathogens. Due to the exorbitant costs to control these parasites, damage to the environment caused by the inappropriate use of chemical acaricides, and the increased resistance against traditional parasiticides, alternative control of ticks, by the use of entomopathogenic fungi, for example, has been considered an interesting approach. Nevertheless, few studies have demonstrated how the tick's immune system acts to fight these entomopathogens. Therefore, this protocol demonstrates two methods used for entomopathogen inoculation into engorged females and two techniques used for hemolymph collection and hemocytes harvesting. Inoculation of pathogens at the leg insertion in the tick female's body allows evaluation of females biologic parameters unlike the inoculation between the scutum and capitulum, which frequently damages Gené's organ. Dorsal hemolymph collection yielded a higher volume recovery than collection through the legs. Some limitations of tick hemolymph collection and processing include i) high rates of hemocytes' disruption, ii) hemolymph contamination with disrupted midgut, and iii) low hemolymph volume recovery. When hemolymph is collected through the leg cutting, the hemolymph takes time to accumulate at the leg opening, favoring the clotting process. In addition, fewer hemocytes are obtained in the collection through the leg compared to the dorsal collection, even though the first method is considered easier to be performed. Understanding the immune response in ticks mediated by entomopathogenic agents helps to unveil their pathogenesis and develop new targets for tick control. The inoculation processes described here require very low technological resources and can be used not only to expose ticks to pathogenic microorganisms. Similarly, the collection of tick hemolymph may represent the first step for many physiological studies.
蜱是专性吸血的体外寄生虫，微小扇头蜱在兽医学中具有重要意义，因为它会导致贫血、体重减轻、动物皮革质量下降，还可作为多种病原体的传播媒介。由于控制这些寄生虫的成本过高、化学杀螨剂使用不当对环境造成破坏以及对传统杀寄生虫剂的抗性增加，因此，例如使用昆虫病原真菌来替代控制蜱被认为是一种有趣的方法。然而，很少有研究表明蜱的免疫系统如何对抗这些昆虫病原体。因此，本方案展示了两种将昆虫病原体接种到饱血雌蜱体内的方法以及两种用于采集血淋巴和收获血细胞的技术。在蜱雌蜱身体的腿部插入处接种病原体，与在盾板和假头之间接种不同，后者经常会损害基因器官，这样可以评估雌蜱的生物学参数。通过背部采集血淋巴的回收率高于通过腿部采集。蜱血淋巴采集和处理存在一些局限性，包括：i）血细胞破坏率高；ii）血淋巴被破损的中肠污染；iii）血淋巴回收率低。当通过切断腿部采集血淋巴时，血淋巴需要时间在腿部开口处积聚，这有利于凝血过程。此外，与背部采集相比，通过腿部采集获得的血细胞较少，尽管第一种方法被认为更容易操作。了解昆虫病原体介导的蜱免疫反应有助于揭示其发病机制并开发蜱控制的新靶点。这里描述的接种过程所需的技术资源非常少，不仅可用于使蜱暴露于致病微生物。同样，蜱血淋巴的采集可能是许多生理学研究的第一步。
