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.

Antimicrobial activity in the tick Rhipicephalus (Boophilus) microplus eggs: Cellular localization and temporal expression of microplusin during oogenesis and embryogenesis

Arthropods display different mechanisms to protect themselves against infections, among which antimicrobial peptides (AMPs) play an important role, acting directly against invader pathogens. We have detected several factors with inhibitory activity against Candida albicans and Micrococcus luteus on the surface and in homogenate of eggs of the tick Rhipicephalus (Boophilus) microplus. One of the anti-M. luteus factors of the egg homogenate was isolated to homogeneity. Analysis by electrospray mass spectrometry (ESI-MS) revealed that it corresponds to microplusin, an AMP previously isolated from the cell-free hemolymph of X (B.) microplus. Reverse transcription (RT) quantitative polymerase chain reactions (qPCR) showed that the levels of microplusin mRNA gradually increase along ovary development, reaching an impressive highest value three days after the adult females have dropped from the calf and start oviposition. Interestingly, the level of microplusin mRNA is very low in recently laid eggs. An enhance of microplusin gene expression in eggs is observed only nine days after the onset of oviposition, achieving the highest level just before the larva hatching, when the level of expression decreases once again. Fluorescence microscopy analysis using an anti-microplusin serum revealed that microplusin is present among yolk granules of oocytes as well as in the connecting tube of ovaries. These results, together to our previous data. suggest that microplusin may be involved not only in protection of adult female hemocele, but also in protection of the female reproductive tract and embryos, what points this AMP as a considerable target for development of new methods to control R. (B.) microplus as well as the vector-borne pathogens. (c) 2009 Elsevier Ltd. All rights reserved.

Efficacy of the Bm86 antigen against immature instars and adults of the dog tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Hepatozoon canis infection associated with dog ticks of rural areas of Rio de Janeiro State, Brazil

Hepatozoon canis is a tick-borne protozoan that infects dogs and has been reported throughout the world. Manifestation of H. canis infection varies from being sub-clinical in apparently healthy dogs to severe illness. The main vector of the infection is the dog tick, Rhipicephalus sanguineus although other species may also transmit this agent. H. canis has been reported previously in Brazil, but mostly as an occasional finding during laboratory exams and always associated with other diseases. The prevalence of H. canis in dogs of rural areas of Brazil has been little studied. For this study, 250 dogs from seven counties of Rio de Janeiro state were examined. All the dogs were from rural areas, near forest. of the dogs examined, 26 dogs were from Seropedica, 82 from Itaguai, 41 from Paracambi, 26 from Mangaratiba, 32 from Barra do Pirai, 32 from Pirai and 11 from Miguel Pereira. Blood smears from the peripheral blood of the ear were taken and ticks found on the dogs were collected for identification in the laboratory. Using blood smear evaluation, H. canis was identified in 39.2% of the animals examined. Other hemoparasites identified were Babesia canis (5.2%) and Ehrlichia canis (4.8%). Four tick species were found parasitizing the dogs: Amblyomma cajennense (23.6%), R. sanguineus (12.4%), Amblyomma aureolatum (2.8%) and Amblyomma ovale (2.0%). There was a positive correlation between the presence of A. cajennense and H. canis infection. (C) 2001 Elsevier B.V. B.V. All rights reserved.

Azadirachta indica A. Juss (neem) induced morphological changes on oocytes of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) tick females

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evaluation of cytotoxic effects of fipronil on ovaries of semi-engorged Rhipicephalus sanguineus (Latreille, 1806) (Acari : Ixodidae) tick female

The ovary of the Rhipicephalus sanguineus dog tick female is structurally formed by oocytes in five different stages of development (from I to V) and attached to the gonad by the pedicel. The present study evaluated possible toxic effects of the acaricide fipronil (Frontline (R)) on ovaries of semi-engorged ticks. Sixty partially fed females of R. sanguineus tick were distributed into four groups of 15 specimens each: I - non-treated; II, III and IV - treated with 1, 5 and 10 ppm of fipronil, respectively. The acaricide induced structural changes in the oocytes of individuals of the different treated groups ranging from the presence of a few small vacuoles to cellular death. In conclusion, germinative cells of semi-engorged R. sanguineus tick female are affected by different concentrations of fipronil leading to the reduction of tick fertility. (c) 2008 Elsevier Ltd. All rights reserved.

Determination of LC50 of permethrin acaricide in semi-engorged females of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Western blot analysis of tick antigens from a Rhipicephalus sanguineus unfed larval extract and identification of antigenic sites in tick sections using immunohistochemistry. A comparative study between resistant and susceptible host species

Most parasite-host relationships are characterized by the development of resistance by the host, thus limiting the number of parasites. However, some cases are very unusual. In the relationship of the domestic dog with the brown dog-tick Rhipicephalus sanguineus this does not occur, whereas guinea pigs develop efficient resistance. Sera from domestic dogs, crab-eating foxes and guinea pigs collected before and after infestation with R. sanguineus ticks, and after immunization with a whole tick adult or larval homogenate, were used in Western blot analysis to compare and identify potential important antigens from a tick larval homogenate. The same sera were tested in an indirect immunohistochemistry assay in an attempt to compare relevant antigenic sites on histological tick sections. The immunoblotting displayed antigens recognized only by the guinea pigs, as well as several shared antigens between host species, depending on the kind of immunization. Immunohistochemistry revealed probable antigenic sites on the cells and tissues of ticks, which varied depending on the kind of immunization (infestation or vaccination) and the animal species involved.

Ehrlichiosis in anemic, thrombocytopenic, or tick-infested dogs from a hospital population in South Brazil

Ehrlichia canis has a worldwide distribution, but clinical manifestations may vary geographically. We selected 129 dogs to determine prevalence of ehrlichiosis in dogs with anemia, thrombocytopenia, or ticks presented to a Veterinary Teaching Hospital in South Brazil. Of the 129 dogs, 68 carried the brown dog tick (Rhipicephalus sanguineus), 61 had thrombocytopenia (platelet count <150,000/μl), and 19 had anemia (PCV < 22%). Twenty dogs fulfilled more than one inclusion criteria. Ehrlichiosis was diagnosed by positive amplification of ehrlichial DNA by PCR using primers ECC and ECB that amplify a sequence of the 16S rRNA gene. Presence of E. canis was confirmed by cleavage of the amplified DNA using endonucleases HaeIII and AvaI. Fourteen of 68 (21%) dogs with ticks had ehrlichiosis, whereas 12 of 61 (20%) dogs presented with thrombocytopenia and 4 of 19 (21%) anemic dogs had ehrlichiosis. Similar results were obtained in dogs with thrombocytopenia and anemia (one of eight positive) and in dogs with thrombocytopenia and ticks (two of seven positive). All four dogs with anemia and ticks, and the dog that fulfilled all inclusion criteria yield no amplification of ehrlichial DNA by PCR. Based on our results, one in each five dogs infested by the brown dog tick, with anemia or thrombocytopenia had ehrlichosis. Contrary to widespread believe, ehrlichiosis was not the main cause for thrombocytopenia in our region. © 2003 Elsevier B.V. All rights reserved.

Ultrastructure of the synganglion in the larvae and nymphs of tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Degenerative process and cell death in salivary glands of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) semi-engorged female exposed to the acaricide permethrin

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Eventos externos e internos da infecção de larvas e ninfas de Rhipicephalus sanguineus por Metarhizium anisopliae

Examinaram-se a adesão, a germinação, a penetração e a colonização de larvas e ninfas de Rhipicephalus sanguineus por Metarhizium anisopliae, assim como as lesões infringidas pelo fungo nas respectivas fases do ciclo de vida do ácaro. Realizaram-se infecções experimentais em 11 grupos contendo 250 larvas e 11 grupos contendo 75 ninfas de R. sanguineus, por meio de banho, durante três minutos sob agitação manual, em suspensão contendo 10(8) conídios/ml do fungo. Nos grupos-controles, o banho foi realizado usando o veículo da suspensão. Larvas e ninfas foram processadas para um estudo histopatológico e de microscopia eletrônica de varredura nos seguintes tempos após a infecção: uma e 18 horas, e um, dois, três, quatro, cinco, seis, sete, nove e 11 dias. A germinação dos conídios ocorreu em até 18 horas pós-inoculação, e o fungo penetrou nas larvas e ninfas através do tegumento, dois e três dias após a infecção, respectivamente. Após penetração, o fungo invadiu o corpo das larvas e ninfas, promovendo uma colonização difusa, sem preferência aparente por tecidos específicos. Lesões significativas não foram observadas. A morte das larvas e ninfas ocorreu no terceiro e quarto dias pós-infecção, e a esporulação do patógeno sobre o cadáver foi iniciada no sexto dia pós-infecção.

Distribution of Rickettsia rickettsii in ovary cells of Rhipicephalus sanguineus (Latreille1806) (Acari: Ixodidae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Molecular and serological detection of Ehrlichia canis and Babesia vogeli in dogs in Colombia

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Molecular and parasitological survey of Hepatozoon canis (Apicomplexa : Hepatozoidae) in dogs from rural area of São Paulo state, Brazil

Hepatozoon canis is a protozoan that infects dogs and is transmitted by the ingestion of the brown dog tick, Rhipicephalus sanguineus. Two distinct species of Hepatozoon genus can infect dogs, H. canis and H. americanum. Routine tests to detect the disease are based on direct examination of gametocytes on Giemsa-stained blood smears. The objectives of this study were the investigation of infection prevalence in rural area dogs, the comparison of diagnostics by blood smear examination and polymerase chain reaction (PCR), and the association of infection with tick infestation. Blood smears, collected by puncture of the cephalic vein and ear margin capillary bed from 150 dogs, were examined. This technique detected 17 positive animals (11.3%), with 14 (9.3%) in peripheral blood and seven (4.7%) in cephalic vein blood. PCR tests detected 80 (53.3%) positive animals. R. sanguineus and Amblyomma spp. were found in 36 of the dogs (24%), in equal proportions. The identified species for Amblyomma genus were A. cajennense and A. ovale. Data analysis showed that PCR was much more sensitive when compared to blood smear examination. Hepatozoon species was previously identified as closely related to H. canis.