Detection of Leishmania (Leishmania) infantum RNA in fleas and ticks collected from naturally infected dogs

The occurrence of the insect vector (sand flies) with low rates of Leishmania infection, as well as autochthonous transmission in the absence of the natural vector in dogs, have been reported. These unexpected data suggest a hypothesis of other arthropods as a possible way of Leishmania transmission. The prevalence of Leishmania (Leishmania) infantum in fleas and ticks collected from dogs with canine visceral leishmaniasis (CVL), as well as parasite viability, were evaluated herein. The presence of L. (L.) infantum was assayed by PCR and ELISA in ectoparasites and biological samples from 73 dogs living in a Brazilian endemic area. As the occurrence of Leishmania DNA in ticks and fleas is expected given their blood-feeding habits, we next investigated whether parasites can remain viable inside ticks. PCR and ELISA confirmed that 83% of the dogs had CVL. Fleas and ticks (nymphs, male and female adults) were collected in 55% and 63% of the 73 dogs, respectively. Out of the 60 dogs with CVL, 80% harbored ectoparasites infected with L. (L.) infantum. The infection rates of the ectoparasites were 23% and 50% for fleas and ticks, respectively. The RNA analysis of the extract from ticks left in laboratory conditions during 7 to 10 days after removal from CVL dogs showed that parasites were alive. In addition, live parasites were also detected inside adult ticks recently molted in laboratory conditions. These findings indicate a higher infection rate of L. (L.) infantum in ticks and fleas, but they do not conclusively demonstrate whether these ticks can act as vectors of CVL, despite the fact that their rates were higher than those previously described in Lutzomyia longipalpis. The presence of viable L. (L.) infantum in ticks suggests the possible importance of dog ectoparasites in CVL dissemination.

Comparison of two methods for collecting free-living ticks in the Amazonian forest

In the present study, we compared 2 methods for collecting ixodid ticks on the verges of animal trails in a primary Amazon forest area in northern Brazil. (i) Dragging: This method was based on passing a 1-m(2) white flannel over the vegetation and checking the flannel for the presence of caught ticks every 5-10 m. (ii) Visual search: This method consisted of looking for guesting ticks on the tips of leaves of the vegetation bordering animal trails in the forest. A total of 103 adult ticks belonging to 4 Amblyomma species were collected by the visual search method on 5 collecting dates, while only 44 adult ticks belonging to 3 Amblyomma species were collected by dragging on 5 other collecting dates. These values were statistically different (Mann-Whitney Test, P = 0.0472). On the other hand, dragging was more efficient for subadult ticks, since no larva or nymph was collected by visual search, whereas 18 nymphs and 7 larvae were collected by dragging. The visual search method proved to be suitable for collecting adult ticks in the Amazon forest: however, field studies should include a second method, such as dragging in order to maximize the collection of subadult ticks. Indeed, these 2 methods can be performed by a single investigator at the same time, while he/she walks on an animal trail in the forest. (C) 2010 Elsevier GmbH. All rights reserved.

Diagnoses of fipronil resistance in Brazilian cattle ticks (Rhipicephalus (Boophilus) microplus) using in vitro larval bioassays

Fipronil is a phenylpyrazolic insecticide that is widely used in agriculture and has been recently used to control the cattle tick, Rhipicephalus (Boophilus) microplus. Because of the serious problems associated with resistance to the available acaricides, this product has been used as an important alternative to control acaricide-resistant ticks. The objective of this work was to analyse the fipronil sensitivity of ticks that were collected from farms with a history of fipronil use by larval bioassays. A total of 11 Brazilian tick populations were studied: one population from Rio Grande do Sul, one population from Mato Grosso do Sul and nine populations from Sao Paulo. To validate the assays, susceptible reference strains, POA (Porto Alegre, Brazil) and Mozo (Dilave, Uruguay), and ticks from six different farms that never used fipronil were tested. The resistance of various tick populations to technical grade fipronil (95.3%) was primarily evaluated using the larvae immersion test (LIT) and the larval packet test (LPT), when a sufficient number of larvae was collected. Using the LIT, the resistance ratios (RR(50)) of the tick populations from Rio Grande do Sul and Mato Grosso do Sul were 14.9 and 2.6, respectively, and the populations derived from Sao Paulo had RR(50)s ranging from 2.5 to 6.9. Four populations were evaluated with the LPT, and two populations displayed lower RR(50), while other populations displayed higher RR(50) than those determined by the LIT. This article reports the first cases of fipronil resistance in Brazil and highlights the LIT as a more sensitive technique for the evaluation of fipronil resistance in R. (B) microplus ticks. We suggest the use of the LIT as an evaluation tool for monitoring fipronil resistance in the control programmes of R. (B) microplus. (C) 2010 Elsevier B.V. All rights reserved.

Relation of genetic phylogeny and geographical distance of tick-borne encephalitis virus in central Europe

Tick-borne encephalitis virus (TBEV) is the most important arboviral agent causing disease of the central nervous system in central Europe. In this study, 61 TBEV E gene sequences derived from 48 isolates from the Czech Republic, and four isolates and nine TBEV strains detected in ticks from Germany, covering more than half a century from 1954 to 2009, were sequenced and subjected to phylogenetic and Bayesian phylodynamic analysis to determine the phylogeography of TBEV in central Europe. The general Eurasian continental east-to-west pattern of the spread of TBEV was confirmed at the regional level but is interlaced with spreading that arises because of local geography and anthropogenic influence. This spread is reflected by the disease pattern in the Czech Republic that has been observed since 1991. The overall evolutionary rate was estimated to be approximately 8x10(-4) substitutions per nucleotide per year. The analysis of the TBEV E genes of 11 strains isolated at one natural focus in Zd`ar Kaplice proved for the first time that TBEV is indeed subject to local evolution.

Characterization of proteinases from the midgut of Rhipicephalus (Boophilus) microplus involved in the generation of antimicrobial peptides

Background: Hemoglobin is a rich source of biologically active peptides, some of which are potent antimicrobials (hemocidins). A few hemocidins have been purified from the midgut contents of ticks. Nonetheless, how antimicrobials are generated in the tick midgut and their role in immunity is still poorly understood. Here we report, for the first time, the contribution of two midgut proteinases to the generation of hemocidins. Results: An aspartic proteinase, designated BmAP, was isolated from the midgut of Rhipicephalus (Boophilus) microplus using three chromatographic steps. Reverse transcription-quantitative polymerase chain reaction revealed that BmAP is restricted to the midgut. The other enzyme is a previously characterized midgut cathepsin L-like cysteine proteinase designated BmCL1. Substrate specificities of native BmAP and recombinant BmCL1 were mapped using a synthetic combinatorial peptide library and bovine hemoglobin. BmCL1 preferred substrates containing non-polar residues at P2 subsite and polar residues at P1, whereas BmAP hydrolysed substrates containing non-polar amino acids at P1 and P1`. Conclusions: BmAP and BmCL1 generate hemocidins from hemoglobin alpha and beta chains in vitro. We postulate that hemocidins may be important for the control of tick pathogens and midgut flora.

TICK INNATE IMMUNITY

Ticks are blood feeding parasites transmitting a wide variety of pathogens to their vertebrate hosts. The vector competence of ticks is tightly linked with their immune system. Despite its importance, our knowledge of tick innate immunity is still inadequate and the limited number of sufficiently characterized immune molecules and cellular reactions are dispersed across numerous tick species. The phagocytosis of microbes by tick hemocytes seems to be coupled with a primitive complement-like system, which possibly involves self/nonself recognition by fibrinogen-related lectins and the action of thioester-containing proteins. Ticks do not seem to possess a pro-phenoloxidase system leading to melanization and also coagulation of tick hemolymph has not been experimentally proven. They are capable of defending themselves against microbial infection with a variety of antimicrobial peptides comprising lysozymes, defensins and molecules not found in other invertebrates. Virtually nothing is known about the signaling cascades involved in the regulation of tick antimicrobial immune responses. Midgut immunity is apparently the decisive factor of tick vector competence. The gut content is a hostile environment for ingested microbes, which is mainly due to the antimicrobial activity of hemoglobin fragments generated by the digestion of the host blood as well as other antimicrobial peptides. Reactive oxygen species possibly also play an important role in the tick-pathogen interaction. The recent release of the Ixodes scapularis genome and the feasibility of RNA interference in ticks promise imminent and substantial progress in tick innate immunity research.

Exogenous insulin stimulates glycogen accumulation in Rhipicephalus (Boophilus) microplus embryo cell line BME26 via PI3K/AKT pathway

Ticks are obligatory blood-feeding arthropods and important vectors of both human and animal disease agents. Besides its metabolic role, insulin signaling pathway (ISP) is widely described as crucial for vertebrate and invertebrate embryogenesis, development and cell survival. In such cascade, Phosphatidylinositol 3-OH Kinase (PI3K) is hierarchically located upstream Protein Kinase B (PKB). To study the insulin-triggered pathway and its possible roles during embryogenesis we used a culture of embryonic Rhipicephalus microplus cells (BME26). Exogenous insulin elevated cell glycogen content in the absence of fetal calf serum (FCS) when compared to cells without treatment. Moreover, in the presence of PI3K inhibitors (Wortmannin or LY294002) these effects were blocked. We observed an increase in the relative expression level of PI3K`s regulatory subunit (p85), as determined by qRT-PCR. In the presence of PI3K inhibitors these effects on transcription were also reversed. Additionally, treatment with Wortmannin increased the expression level of the insulin-regulated downstream target glycogen synthase kinase 3 beta (GSK3 beta). The p85 subunit showed elevated transcription levels in ovaries from fully engorged females, but was differentially expressed during tick embryogenesis. These results strongly suggest the presence of an insulin responsive machinery in BME26 cells, and its correlation with carbohydrate/glycogen metabolism also during embryogenesis. (C) 2009 Published by Elsevier Inc.

Propagation of a Brazilian isolate of Anaplasma marginale with appendage in a tick cell line (BME26) derived from Rhipicephalus (Boophilus) microplus

Anaplasma marginale is a tick-borne pathogen of cattle responsible for the disease anaplasmosis. Data suggest that Rhipicephalus (Boophilus) microplus and R. annulatus may be the major tick vectors of A. marginale in tropical and subtropical regions of the world. In this work we demonstrated the first infection and propagation of a Brazilian isolate of A. marginale (UFMG1) in the BME26 cell line derived originally from embryos of R. (Boophilus) microplus. The establishment of A. marginale infection in a cell line derived from R. (Boophilus) microplus is relevant for studying the A. marginale/tick interface. (C) 2008 Elsevier B.V. All rights reserved.

Selection of an ivermectin-resistant strain of Rhipicephalus microplus (Acari: Ixodidae) in Brazil

Resistance to ivermectin (IVM) in field Populations of Rhipicephalus microplus of Brazil has been observed since 2001 In this work, four selection methods (infestations with: (I) IVM-treated larvae, (2) larvae from IVM-treated adult female ticks, (3) larvae from IVM-treated adult female ticks on an IVM-treated host, and (4) larvae obtained from W-treated females that produced eggs with a high eclosion rate) were used oil a field population with an initial ivermectin (IVM) resistance ratio at LC50 (RR50) of 1 37 with the objective to obtain experimentally a highly-resistant strain After ten generations, using these methods combined, the final RR50 was 8 06 This work shows for the first time that it was possible to increase IVM resistance in R. microplus in laboratory conditions. The establishment of a drug resistant R microplus strain is a fundamental first step for further research into the mechanisms of ivermectin-resistance in R. microplus and potentially methods to control this resistance (C) 2009 Elsevier B V All rights reserved

Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) resistance to fipronil in Uruguay evaluated by in vitro bioassays

Rhipicephalus (Boophilus) microplus obtained from four local populations in Uruguay (2007-2008) were subjected to various bioassay techniques to determine the presence of fipronil resistance within the country. Resistance ratios (RRs) obtained by larval immersion test varied between 3.3 and 3635 for tick populations subjected to treatment with fipronil for the last 3-7 years. The highest RR was observed in the population which received fewer treatments. Using discriminating concentration (8 ppm) for larval immersion test, all field strains were correctly diagnosed as fipronil-resistant. This study presents the first diagnoses of cattle tick resistance to fipronil in Uruguay`s field populations. It also highlights the importance of the possible conflict between programs to control agricultural pests and cattle ticks. The findings provide valuable information for selection and adoption of new control alternatives to manage drug resistance exhibited by cattle ticks. (C) 2009 Elsevier BM. All rights reserved.