An insight into the sialotranscriptome of the brown dog tick, Rhipicephalus sanguineus

Background: Rhipicephalus sanguineus, known as the brown dog tick, is a common ectoparasite of domestic dogs and can be found worldwide. R. sanguineus is recognized as the primary vector of the etiological agent of canine monocytic ehrlichiosis and canine babesiosis. Here we present the first description of a R. sanguineus salivary gland transcriptome by the production and analysis of 2,034 expressed sequence tags (EST) from two cDNA libraries, one consctructed using mRNA from dissected salivary glands from female ticks fed for 3-5 days (early to mid library, RsSGL1) and the another from ticks fed for 5 days (mid library, RsSGL2), identifying 1,024 clusters of related sequences. Results: Based on sequence similarities to nine different databases, we identified transcripts of genes that were further categorized according to function. The category of putative housekeeping genes contained similar to 56% of the sequences and had on average 2.49 ESTs per cluster, the secreted protein category contained 26.6% of the ESTs and had 2.47 EST's/clusters, while 15.3% of the ESTs, mostly singletons, were not classifiable, and were annotated as ""unknown function"". The secreted category included genes that coded for lipocalins, proteases inhibitors, disintegrins, metalloproteases, immunomodulatory and antiinflammatory proteins, as Evasins and Da-p36, as well as basic-tail and 18.3 kDa proteins, cement proteins, mucins, defensins and antimicrobial peptides. Comparison of the abundance of ESTs from similar contigs of the two salivary gland cDNA libraries allowed the identification of differentially expressed genes, such as genes coding for Evasins and a thrombin inhibitor, which were over expressed in the RsSGL1 (early to mid library) versus RsSGL2 (mid library), indicating their role in inhibition of inflammation at the tick feeding site from the very beginning of the blood meal. Conversely, sequences related to cement (64P), which function has been correlated with tick attachment, was largely expressed in the mid library. Conclusions: Our survey provided an insight into the R. sanguineus sialotranscriptome, which can assist the discovery of new targets for anti-tick vaccines, as well as help to identify pharmacologically active proteins.

Experimental Infection of Amblyomma aureolatum Ticks with Rickettsia rickettsii

We experimentally infected Amblyomma aureolatum ticks with the bacterium Rickettsia rickettsii, the etiologic agent of Rocky Mountain spotted fever (RMSF). These ticks are a vector for RMSF in Brazil. R. rickettsii was efficiently conserved by both transstadial maintenance and vertical (transovarial) transmission to 100% of the ticks through 4 laboratory generations. However, lower reproductive performance and survival of infected females was attributed to R. rickettsii infection. Therefore, because of the high susceptibility of A. aureola turn ticks to R. rickettsii infection, the deleterious effect that the bacterium causes in these ticks may contribute to the low infection rates (< 1%) usually reported among field populations of A. aureolatum ticks in RMSF-endemic areas of Brazil. Because the number of infected ticks would gradually decrease after each generation, it seems unlikely that A. aureolatum ticks could sustain R. rickettsii infection over multiple successive generations solely by vertical transmission.

Wide Dispersal and Possible Multiple Origins of Low-Copy-Number Plasmids in Rickettsia Species Associated with Blood-Feeding Arthropods

Plasmids are mobile genetic elements of bacteria that can impart important adaptive traits, such as increased virulence or antibiotic resistance. We report the existence of plasmids in Rickettsia (Rickettsiales; Rickettsiaceae) species, including Rickettsia akari, ""Candidatus Rickettsia amblyommii,"" R. bellii, R. rhipicephali, and REIS, the rickettsial endosymbiont of Ixodes scapularis. All of the rickettsiae were isolated from humans or North and South American ticks. R. parkeri isolates from both continents did not possess plasmids. We have now demonstrated plasmids in nearly all Rickettsia species that we have surveyed from three continents, which represent three of the four major proposed phylogenetic groups associated with blood-feeding arthropods. Gel-based evidence consistent with the existence of multiple plasmids in some species was confirmed by cloning plasmids with very different sequences from each of two ""Ca. Rickettsia amblyommii"" isolates. Phylogenetic analysis of rickettsial ParA plasmid partitioning proteins indicated multiple parA gene origins and plasmid incompatibility groups, consistent with possible multiple plasmid origins. Phylogenetic analysis of potentially host-adaptive rickettsial small heat shock proteins showed that hsp2 genes were plasmid specific and that hsp1 genes, found only on plasmids of ""Ca. Rickettsia amblyommii,"" R. felis, R. monacensis, and R. peacockii, were probably acquired independently of the hsp2 genes. Plasmid copy numbers in seven Rickettsia species ranged from 2.4 to 9.2 per chromosomal equivalent, as determined by real-time quantitative PCR. Plasmids may be of significance in rickettsial evolution and epidemiology by conferring genetic plasticity and host-adaptive traits via horizontal gene transfer that counteracts the reductive genome evolution typical of obligate intracellular bacteria.

The Immunomodulatory Action of Sialostatin L on Dendritic Cells Reveals Its Potential to Interfere with Autoimmunity

Sialostatin L (SialoL) is a secreted cysteine protease inhibitor identified in the salivary glands of the Lyme disease vector Ixodes scapularis. In this study, we reveal the mechanisms of SialoL immunomodulatory actions on the vertebrate host. LPS-induced maturation of dendritic cells from C57BL/6 mice was significantly reduced in the presence of SialoL. Although OVA degradation was not affected by the presence of SialoL in dendritic cell cultures, cathepsin S activity was partially inhibited, leading to an accumulation of a 10-kDa invariant chain intermediate in these cells. As a consequence, in vitro Ag-specific CD4(+) T cell proliferation was inhibited in a time-dependent manner by SialoL, and further studies engaging cathepsin S(-/-) or cathepsin L(-/-) dendritic cells confirmed that the immunomodulatory actions of SialoL are mediated by inhibition of cathepsin S. Moreover, mice treated with SialoL displayed decreased early T cell expansion and recall response upon antigenic stimulation. Finally, SialoL administration during the immunization phase of experimental autoimmune encephalomyelitis in mice significantly prevented disease symptoms, which was associated with impaired IFN-gamma and IL-17 production and specific T cell proliferation. These results illuminate the dual mechanism by which a human disease vector protein modulates vertebrate host immunity and reveals its potential in prevention of an autoimmune disease. The Journal of Immunology, 2009, 182: 7422-7429.

Cellular and molecular characterization of an embryonic cell line (BME26) from the tick Rhipicephalus (Boophilus) microplus

The cellular and molecular characteristics of a cell line (BME26) derived from embryos of the cattle tick Rhipicephalus (Boophilus) microplus were studied. The cells contained glycogen inclusions, numerous mitochondria, and vesicles with heterogeneous electron densities dispersed throughout the cytoplasm. Vesicles contained lipids and sequestered palladium meso-porphyrin (Pd-mP) and rhodamine-hemoglobin, suggesting their involvement in the autophagic and endocytic pathways. The cells phagocytosed yeast and expressed genes encoding the antimicrobial peptides (microplusin and defensin). A cDNA library was made and 898 unique mRNA sequences were obtained. Among them, 556 sequences were not significantly similar to any sequence found in public databases. Annotation using Gene Ontology revealed transcripts related to several different functional classes. We identified transcripts involved in immune response such as ferritin, serine proteases, protease inhibitors,. antimicrobial peptides, heat shock protein, glutathione S-transferase, peroxidase, and NADPH oxidase. BME26 cells transfected with a plasmid carrying a red fluorescent protein reporter gene (DsRed2) transiently expressed DsRed2 for up to 5 weeks. We conclude that BME26 can be used to experimentally analyze diverse biological processes that occur in R. (B.) microplus such as the innate immune response to tick-borne pathogens. (C) 2008 Elsevier Ltd. All rights reserved.

Proteins in the saliva of the Ixodida (ticks): Pharmacological features and biological significance

The saliva of ticks (Suborder Ixodida) is critical to their survival as parasites. A tick bite should result in strong responses from the host defence systems (haemostatic, immune and inflammatory) but tick saliva appears to have evolved to counter these responses. We review current knowledge of tick saliva components, with emphasis on those molecules confirmed to be present in the secreted saliva but including some that have only been confirmed to be present in salivary glands. About 50 tick saliva proteins that are well described in the literature are discussed. These saliva components include enzymes, enzyme inhibitors, amine-binding proteins and cytokine homologues that act as anti-haemostatic, anti-inflammatory or immuno-modulatory agents. Sequence comparisons are illustrated. The importance of tick saliva and the significance of the findings to date are also discussed. (C) 2006 Elsevier Ltd. All rights reserved.

Phlébotomes de Bolivie: III. Description de Lutzomyia andersoni n. sp. (Diptera, Psychodidae)

Les auteurs décrivent le mâle et la femelle de Lutzomyia andersoni n.sp., nouvelle espèce de phlébotome, non anthropophile, du groupe walkeri martins, Williams et Falcão, 1987 très apparentée à L. sericea Floch et Abonnenc, 1944.

Use of plastic tips in artificial feeding of Dermacentor (Anocentor) nitens females Neumann, 1897 (Acari: Ixodidae)

The establishment of laboratory colonies of ticks is often hampered by their lack of adaptation to alternative hosts. The aim of this study was to artificially feed partially engorged Dermacentor (Anocentor) nitens females through plastic tips, and to identify what are the optimal conditions of application of this technique to get as much as possible close to the natural conditions. The technique of artificial feeding through plastic tips allowed the engorgement of D. nitens ticks to a final weight within the normal range for the species. (C) 2014 Published by Elsevier GmbH.

Questing Dermacentor reticulatus harbouring Babesia canis DNA associated with outbreaks of canine babesiosis in the Swiss Midlands

In 2011 and 2012, outbreaks of clinical canine babesiosis were observed in 2 areas of the Swiss Midlands that had no history of this disease so far. In one area, cases of canine babesiosis occurred over 2 consecutive tick seasons. The outbreaks involved 29 dogs, 4 of which died. All dogs were infected with large Babesia sp. as diagnosed in Giemsa-stained blood smears and/or PCR. These were identified as B. canis (formerly known as B. canis canis) by subsequent partial sequencing of the 18S rRNA gene of Babesia sp. Interestingly, the sequence indicated either a genotype with heterogeneity in the ssrRNA gene copies or double infection with different B. canis isolates. None of the dogs had a recent travel history, but one had frequently travelled to Hungary and had suffered twice from clinical babesiosis 18 and 24 months prior to the outbreak in autumn 2011. Retrospective sequencing of a stored blood DNA sample of this dog revealed B. canis, with an identical sequence to the Babesia involved in the outbreaks. For the first time in Switzerland, the partial 18S rRNA gene of B. canis could be amplified from DNA isolated from 19 out of 23 adult Dermacentor reticulatus ticks flagged in the same area. The sequence was identical to that found in the dogs. Furthermore, one affected dog carried a female D. reticulatus tick harbouring B. canis DNA. Our findings illustrate that, under favourable biogeographic and climatic conditions, the life-cycle of B. canis can relatively rapidly establish itself in previously non-endemic areas. Canine babesiosis should therefore always be a differential diagnosis when dogs with typical clinical signs are presented, regardless of known endemic areas.

Occurrence and molecular characterization of Cryptosporidium spp. isolated from domestic animals in a rural area surrounding Atlantic dry forest fragments in Teodoro Sampaio municipality, State of São Paulo, Brazil

The aim of this study was to assess the occurrence of Cryptosporidium in domestic animals in rural properties surrounding rain forest fragments within the municipality of Teodoro Sampaio, southeastern Brazil. Conventional sucrose flotation method followed by molecular characterization of the parasites by sequencing PCR products amplified from SSU rRNA gene were used. Stool samples were collected from domestic animals raised as pets and livestock in all rural properties surrounding three forest fragments. Samples from cattle (197), equine (63), pigs (25), sheep (11), and dogs (28) were collected from 98 rural properties. The frequency of occurrence of Cryptosporidium within each animal species was 3.0% (6/197) among cattle and 10.7% (3/28) among dogs. Cryptosporidium was not detected in stool samples from equine, sheep, and pigs. All sequences obtained from the six samples of calves showed molecular identity with Cryptosporidium andersoni while all sequences from dog samples were similar to C. canis. The frequency of occurrence of Cryptosporidium in these domestic animal species was low. The absence of C. parvum in the present study suggests that the zoonotic cycle of cryptosporidiosis may not be relevant in the region studied. The presence of Cryptosporidium species seldom described in humans may be, otherwise, important for the wild fauna as these animals are a source of infection and dissemination of this protozoan to other animal species. The impact and magnitude of infection by C. andersoni in wild ruminants and C. canis in wild canids have to be assessed in future studies to better understand the actual importance of these species in this region.

Differential expression of genes in salivary glands of male Rhipicephalus (Boophilus)microplus in response to infection with Anaplasma marginale

Background: Bovine anaplasmosis, caused by the rickettsial tick-borne pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae), is vectored by Rhipicephalus (Boophilus) microplus in many tropical and subtropical regions of the world. A. marginale undergoes a complex developmental cycle in ticks which results in infection of salivary glands from where the pathogen is transmitted to cattle. In previous studies, we reported modification of gene expression in Dermacentor variabilis and cultured Ixodes scapularis tick cells in response to infection with A. marginale. In these studies, we extended these findings by use of a functional genomics approach to identify genes differentially expressed in R. microplus male salivary glands in response to A. marginale infection. Additionally, a R. microplus-derived cell line, BME26, was used for the first time to also study tick cell gene expression in response to A. marginale infection. Results: Suppression subtractive hybridization libraries were constructed from infected and uninfected ticks and used to identify genes differentially expressed in male R. microplus salivary glands infected with A. marginale. A total of 279 ESTs were identified as candidate differentially expressed genes. Of these, five genes encoding for putative histamine-binding protein (22Hbp), von Willebrand factor (94Will), flagelliform silk protein (100Silk), Kunitz-like protease inhibitor precursor (108Kunz) and proline-rich protein BstNI subfamily 3 precursor (7BstNI3) were confirmed by real-time RT-PCR to be down-regulated in tick salivary glands infected with A. marginale. The impact of selected tick genes on A. marginale infections in tick salivary glands and BME26 cells was characterized by RNA interference. Silencing of the gene encoding for putative flagelliform silk protein (100Silk) resulted in reduced A. marginale infection in both tick salivary glands and cultured BME26 cells, while silencing of the gene encoding for subolesin (4D8) significantly reduced infection only in cultured BME26 cells. The knockdown of the gene encoding for putative metallothionein (93 Meth), significantly up-regulated in infected cultured BME26 cells, resulted in higher A. marginale infection levels in tick cells. Conclusions: Characterization of differential gene expression in salivary glands of R. microplus in response to A. marginale infection expands our understanding of the molecular mechanisms at the tick-pathogen interface. Functional studies suggested that differentially expressed genes encoding for subolesin, putative von Willebrand factor and flagelliform silk protein could play a role in A. marginale infection and multiplication in ticks. These tick genes found to be functionally relevant for tick-pathogen interactions will likely be candidates for development of vaccines designed for control of both ticks and tick-borne pathogens.

Francisella-like endosymbionts of ticks

Ticks affect human and animal health both directly by their blood feeding and indirectly by transmission of many disease-causing bacteria, such as Rickettsia, Ehrlichia, Borrelia, Coxiella, Cowdria, Anaplasma, Aegyptionella, and Tularemia, as well as many viruses (Piesman and Gage, 1996). In addition to these infectious agents, ticks harbor bacterial endosymbionts, such as Wolbachia persica, which was first isolated from the soft tick now classified as Argus arboreus (Suitor and Weiss, 1961).

Occurrence of ticks (Acari: Ixodidae) in the municipality of Goiatins, Tocantins

Due to a suspected human case of Brazilian Lyme-like disease in the city of Goiatins, Tocantins State, an epidemiological survey was carried out in eight counties in this region during September 2007 and February 2008, where 1,890 ticks were collected from domestic animals and from the environment. A total of eight tick species were identified: Rhipicephalus sanguineus, Rhipicephalus (Boophilus) microplus, Dermacentor nitens, Amblyomma cajennense, Amblyomma oblongoguttatum, Amblyomma ovale, Amblyomma parvum and Amblyomma tigrinum. The last four species were described for the first time in this region. Although human parasitism by ticks is frequently described in Goiatins, no ticks collected from humans were analyzed. The Study of ixodids in this region contributes with the survey of Brazilian ticks, as well as the elucidation of the possible transmission of the agent that caused the Brazilian Lyme-like disease case in Goiatins.

Rickettsial infection in domestic mammals and their ectoparasites in El Valle de Anton, Cocle, Panama

The present research evaluated the presence of Rickettsia spp. on ectoparasites of horses and dogs (using PCR techniques), and their sera (using immunofluorescence assay) in El Valle de Anton town in Panama. A total of 20 horses and 20 dogs were sampled, finding four species of ectoparasites on dogs (the ticks Rhipicephalus sanguineus, Amblyomma ovale, Amblyomma oblongoguttatum, and the flea Ctenocephalides felis), and two tick species on horses (Amblyomma cajennense and Dermacentor nitens). DNA of Rickettsia amblyommii was found in pools of A. cajennense, D. nitens, and R. sanguineus, while Rickettsia fells was detected in C. felis pools. Overall, 70% (14/20) and 65% (13/20) of the horses and dogs, respectively, were seroreactive (titer >= 64) to spotted fever group rickettsiae. Sera from six dogs and five horses reacted to R. amblyommii antigens with titers at least four-fold higher than those for the other antigens tested (Rickettsia bellii, Rickettsia parked, Rickettsia rhipicephali, R. felis, and R. rickettsii). These serological results, coupled with our molecular findings, suggest that these dogs and horses were infected by Rickettsia amblyommii. More studies need to be realized afford to identify the Rickettsia species responsible for other serological and molecular positive results, and their ecological importance. (C) 2010 Elsevier B.V. All rights reserved.

Infection by Spotted Fever Rickettsiae in People, Dogs, Horses and Ticks in Londrina, Parana State, Brazil

Spotted fever is a disease caused by bacteria from the genus Rickettsia of the spotted fever group (SFG). Rickettsia rickettsii is likely the main agent of Brazilian spotted fever (BSF). With the objective of gathering information on the circulation of SFG rickettsiae in Londrina, Parana state, ticks from dogs and horses and also blood from dogs, horses and humans were collected in a neighbourhood of the city which presented potential for circulation of rickettsiae between hosts and vectors. Amblyomma cajennense, Dermacentor nitens, and Rhipicephalus sanguineus ticks were subjected to Polymerase Chain Reaction targeting a fragment of the Rickettsia gltA gene. This specific gene encodes the enzyme citrate synthase of Rickettsia spp., and results on all ticks were negative. Human and animal sera were tested by Indirect Immunofluorescence Assay in which R. rickettsii and R. parkeri were used as antigens. Sera from 4.7% human, 2.7% canine and 38.5% equine were positive for R. rickettsii. For R. parkeri, 0.9% human, 2.7% canine and 11.5% equine samples were positive. All samples reactive to R. parkeri also reacted to R. rickettsii. An epidemiological questionnaire was applied, but there were no statistically significant results. Comparison of our serological results with previous studies in Brazil, among BSF endemic and non-endemic areas, indicates that there is no established rickettsial infection in the study area, a statement corroborated with our molecular analysis. Nonetheless, as humans of the present study are highly exposed to tick infestations, health education within the population is needed to obtain efficient tick control. Zoonoses and Public Health 416 (C) 2011 Blackwell Verlag GmbH . Zoonoses Public Health. 58 (2011) 416-423