Estudo de carrapatos associados com aves no entorno do tinguá, Nova Iguaçu, Rio de Janeiro, Brasil; detecção de Rickettsia spp., e o desenvolvimento de métodos moleculares para análise de interações carrapato-microrganismo

Abstract

Ticks parasites of wild birds may serve as vectors of pathogens of major importance for wildlife and public health. Thus, the present study examined the prevalence and intensity of infestation of ticks captured from wild birds in three regions of the Atlantic Forest, with different levels of human impact, located in Nova Iguaçu, State of Rio de Janeiro, Brazil, with the objective of highlighting the possible relationship between prevalence of infestation and preservation of forest areas in the region. Collections were conducted from May 2011 to April 2012, resulting in the capture of 625 birds represented by 98 species divided into 26 families and seven orders, which equated to 14.2% of the richness of bird species in the state. A total of 17.44% (n= 109, 109/625) of the captured birds were parasitized by 458 ticks, of which 88.2% (n = 404, 404/458) were in the larval stage (LL) and 11.8% (n= 54, 54/458) in the nymphal stage. Among the ticks collected, 99% (n = 453) were represented by the genus Amblyomma: Amblyomma longirostre (LL = 345; NN = 34), Amblyomma nodosum (NN = 13), Amblyomma calcaratum (NN = 3), Amblyomma varium (NN = 2); Amblyomma coelebs (NN = 1), Amblyomma naponense (LL = 1), Amblyomma ovale (LL = 1) and Amblyomma sp. (LL = 53). The genus Haemaphysalis was represented by four larvae and a single nymph, corresponding to 1% of the ticks collected. In addition, new species of host birds were reported in Brazil, along with an unprecedented case of super infestation with larvae of A. longirostre on Xiphorhynchus guttatus. Molecular analysis (sequencing of mitochondrial 16S rDNA), provided validation of morphological identification of larvae of A. longirostre at the species level without to need to apply methods of clarification commonly used. The detection and identification of pathogens and their tick hosts are increasingly dependent upon molecular biological methods, which have as their first step the extraction of DNA from the tick and the microorganisms contained therein. In an attempt to standardize the extraction of DNA from ticks and their asscoaited microbiota, the present study compared six methods of DNA extraction (Boiling in sterile water, InstaGene®, QIAamp® DNA Mini Kit, Phenol:chloroform, Diatomaceous earth and modified Hotshot). To date, samples subjected to extraction with the kit and Phenol:chloroform have demonstarted superior results (in terms of quantity and quantity of extracted DNA), when compared to the other methods. In silico analysis of sequences encoding the gltA, htrA and ompB genes of tick associated rickettsial species provided the basis for a novel PCR-RFLP method which allowed differential identification of the Rickettsia species reported in Brazil. The method was evaluated using larvae and nymphs of Amblyomma longirostre, Amblyomma ovale and Amblyomma varium collected from birds captured in this study. The bacterium "Candidatus Rickettsia amblyommii" was identified in 100% (17/17) of A. longirostre larvae examined, while the other two species of ticks were universally PCR negative. The basic method employs the restriction endonucleases MspI and RsaI, can be performed during a single working day. It offers a convenient and cost effective means to perform large scale analysis of tick populations, and should be of benefit to researchers who lack the financial or technical resources necessary for nucleotide sequence based identification. In conclusion, this study expands the understanding of the ecological relationships between ticks and birds in a major biome of the Neotropics, with new perspectives in the context of molecular techniques applied to investigations of parasitism involving tick parasites of wild birds