Characterisation of divergent flavivirus NS3 and NS5 protein sequences detected in Rhipicephalus microplus ticks from Brazil

Transcripts similar to those that encode the nonstructural (NS) proteins NS3 and NS5 from flaviviruses were found in a salivary gland (SG) complementary DNA (cDNA) library from the cattle tick Rhipicephalus microplus.Tick extracts were cultured with cells to enable the isolation of viruses capable of replicating in cultured invertebrate and vertebrate cells. Deep sequencing of the viral RNA isolated from culture supernatants provided the complete coding sequences for the NS3 and NS5 proteins and their molecular characterisation confirmed similarity with the NS3 and NS5 sequences from other flaviviruses. Despite this similarity, phylogenetic analyses revealed that this potentially novel virus may be a highly divergent member of the genus Flavivirus. Interestingly, we detected the divergent NS3 and NS5 sequences in ticks collected from several dairy farms widely distributed throughout three regions of Brazil. This is the first report of flavivirus-like transcripts inR. microplus ticks. This novel virus is a potential arbovirus because it replicated in arthropod and mammalian cells; furthermore, it was detected in a cDNA library from tick SGs and therefore may be present in tick saliva. It is important to determine whether and by what means this potential virus is transmissible and to monitor the virus as a potential emerging tick-borne zoonotic pathogen.

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

In vitro acaricidal activity of neem (Azadirachta indica) seed extracts with known azadirachtin concentrations against Rhipicephalus microplus

The effect of four extracts from neem seeds (Azadirachta indica) containing 2000, 5000, 9000 and 10,000 ppm of azadirachtin A (AZA), quantified by high-performance liquid chromatography (HPLC) and diluted to 1.25%; 2.5%; 5.0%; 10.0% and 12.8% was verified by in vitro tests with engorged females and larvae of the cattle tick Rhipicephalus micro plus. The results from the bioassays with the engorged females showed that the main toxic effect of the extracts was reduction of the reproductive parameters, with a sharp drop in the number of eggs laid and the hatching rate, mainly when the extracts were diluted to 10.0% and 12.8%. The product effectiveness (PE) calculations for all the solutions tested showed that the AZA solution at 10,000 ppm (N10) was the most effective. However, statistical analysis of the PE data obtained for the proportional AZA concentrations in the different diluted extracts showed significance (P<0.05) of the effects included in the model (extract dilution, principle effect (classificatory) of the assay (extract) and the interaction between the two), indicating significant variations due to the dilution, the test and the interaction between the two factors in the tests with engorged females. For solutions N2, N5, and N9, it was not possible to estimate LC(90) values in the dilution range tested. The lowest LC(50) was observed for extract N5, and although extract N10 was the only extract for which the LC(90) could be estimated within the range tested, the LC(50) was higher than for N5 and N9. These results suggest that substances other than AZA present in the extracts influenced the efficacy, especially up to a certain LC range. In the tests with larvae, no mortality was observed, indicating zero effectiveness of all the extracts tested. The results of the tests with engorged females showed that the neem extracts had acaricide activity, inhibiting egg laying and the larval hatching rate. Complementary studies are necessary to develop new methods to isolate and/or identify other substances besides AZA contained in this plant, to enable using products made from it as acaricides. (C) 2011 Elsevier B.V. All rights reserved.

Resistance of cattle of various genetic groups to the tick Rhipicephalus microplus and the relationship with coat traits

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Influence of the physiological state on infestation by Rhipicephalus microplus in dairy cows

The purpose of this study was to identify the influence of physiological state, season, breed, number of lactations, dairy productivity, and stocking rate on infestation by Rhipicephalus microplus in dairy cows. Two hundred cows were selected through proportional representative sampling at the Seropédica Experimental Station (Pesagro-Rio), Brazil. Fully or partially engorged R. microplus females measuring between 4.5 and 8.0. mm were counted on the right side of each animal. The tick infestation prevalence data were analyzed in relation to the risk factors, using multiple logistic regression. Associations between prevalence and its possible influencing determinants were measured using odds ratios. The average tick count of cows during the peripartum and lactation periods was significantly higher (p<0.05; OR=4.82) than the count in dry cows. Taurine animals showed significantly higher infestation (p<0.05; OR=3.28) than pure zebuine animals. Among cross-bred animals, the infestation was higher (p<0.05) in F1 animals (1/2 taurine × 1/2 zebuine) than in Girolando animals (5/8 zebuine × 3/8 taurine). Primiparous cows (p<0.05) had significantly heavier infestations than multiparous cows. Dairy production showed a positive correlation with tick infestation (p<0.03; OR=2.94), such that the most productive animals were the most parasitized ones. Animals kept at high densities were significantly more heavily infested (p<0.05, OR=6.32) than animals kept at low density. First-lactation and high-productivity taurine animals were more vulnerable to R. microplus, thus comprising a high-risk group in dairy herds. © 2012 Elsevier GmbH.

Analysis of the risk factors relating to cows' resistance to Rhipicephalus microplus ticks during the peripartum

This study aimed to evaluate season, breed, number of lactations and milk production as risk factors relating to Rhipicephalus microplus infestation in dairy cows during the peripartum. Eighty-four animals were randomly selected through proportional stratified sampling. All engorged and partially engorged female R. microplus specimens measuring 4.5-8.0 mm were counted during the 5 weeks before calving, calving week and 5 weeks after calving. The peripartum had a significant effect [calving (p < 0.05; prevalence ratio (PR)= 3.12) and post calving (p < 0.05; PR = 2.02)] on R. microplus infestation. Although the average tick count was higher during the rainy season than during the dry season, there was no significant difference (p = 0.055; PR = 0.63). The average R. microplus count was significantly higher (p < 0.05; PR = 2.10) in Bos taurus animals, followed by F1 (p < 0.05; PR = 1.64) and Girolando (p < 0.05; PR = 1.39). The average R. microplus count was significantly higher (p < 0.05; PR = 0.97) in first-lactation animals, followed by those at the second, third and, fourth or subsequent lactation. Milk production showed a negative correlation with R. microplus count, such that high-production animals were significantly (p = 0.003; PR = 2.04) more vulnerable to infestation than were low-production animals. First-lactation and high-production B. taurus animals had greatest vulnerability to R. microplus infestation over the peripartum period, and constituted the at-risk group in the dairy herd.u

Applicability of in vitro bioassays for the diagnosis of ivermectin resistance in Rhipicephalus microplus (Acari: Ixodidae)

The applicability of laboratory bioassays to diagnose ivermectin (IVM) resistance in Rhipicephalus microplus was evaluated. Adult immersion tests (AITs), larval immersion tests (LITs) and larval packet tests (LPTs) were performed to characterise the effects of ivermectin toxicity on adults and larvae of a susceptible reference strain. The AIT was determined to be a reasonable assay but requires a large number of individuals to attain interpretable results. The LIT and LPT were validated with an IVM resistant strain, revealing resistance ratios (RRs) of 6.73 and 1.49, respectively. In a field survey, nine different populations of cattle tick from the states of Sao Paulo and Mato Grosso do Sul, Brazil, were analysed with the LIT. Populations without previous exposure to ivermectin exhibited RRs between 0.87 and 1.01. Populations previously exposed to IVM showed RRs between 1.83 and 4.62. The LIT was more effective at discriminating between resistant and susceptible populations than the LPT. The use of the LIT is recommended for the diagnosis of ivermectin resistance in R microplus. (C) 2011 Elsevier B.V. All rights reserved.

Resistência aos pesticidas piretróides em populações de Rhipicephalus microplus e aos piretróides e organofosforados em Haematobia irritans colhidas em rebanhos de corte no Estado de São Paulo.

A crescente resistência aos pesticidas em populações do carrapato Rhipicephalus microplus e da mosca-dos-chifres Haematobia irritans tem provocado prejuízos aos pecuaristas do Brasil. Testes fenotípicos, que detectam a suscetibilidade de diferentes populações desses parasitas a vários pesticidas estão disponíveis e deveriam ser usados com maior frequência, como critério na seleção de bases com melhor eficácia. Testes que detectam mutações específicas devido ao uso de pesticidas piretroides e organofosforados foram recentemente desenvolvidos e servem para monitorar a ocorrência de altos níveis de resistência, tendo em vista que a resistência ocorre somente em parasitas submetidos a tratamentos constantes com o mesmo princípio químico. Assim neste experimento foi investigado o uso dos pesticidas nas fazendas de gado de corte no estado de São Paulo e a resistência fenotípica e genotípica dos carrapatos e mosca-dos-chifres colhidos nesses rebanhos. Além disso, os testes genotípicos foram validados para as populações de parasitas e padronizados para serem usados na rotina do Laboratório de Sanidade Animal da Embrapa Pecuária Sudeste. Foram testadas dez populações de moscas e dez de carrapatos, Resistência aos pesticidas piretróides em populações de Rhipicephalus microplus e aos piretróides e organofosforados em Haematobia irritans colhidas em rebanhos de corte no Estado de São Paulo colhidas em propriedades rurais no estado de São Paulo. As moscas foram submetidas aos testes com papéis de filtro impregnados com cipermetrina e diazinon, e os carrapatos, ao Teste do Pacote de Larvas (LPT) impregnado com cipermetrina. Os resultados de mortalidade dos parasitas para cada diluição do inseticida foram anotados em planilhas e analisadas pelo procedimento Probit do programa Statistical Analisys System (SAS), para a obtenção das concentrações letais para 50% (CL50) e para 90% (CL90) e o cálculo do Fator de Resistência (FR) das populações de parasitas. As moscas que sobreviveram às maiores concentrações dos pesticidas foram submetidas à extração do DNA para a pesquisa de mutação específica para resistência a cipermetrina (KDR e sKDR) e diazinon (G262A). As larvas de carrapatos sobreviventes foram submetidas à extração de DNA individualmente e testadas para a mutação do tipo KDR. Das 17 propriedades amostradas para moscas e carrapatos, 11 tinham rebanhos compostos por animais Bos taurus indicus (10 propriedades de Nelore e uma de Guzerá), três de Bos t. taurus (uma de Caracu e duas de Angus) e três de bovinos cruzados. Os responsáveis pelos rebanhos taurinos afirmaram que o principal problema era o carrapato, no caso da raça Angus, e a mosca-dos-chifres, no caso da Caracu. Para os rebanhos de animais cruzados, os dois ectoparasitas eram igualmente difíceis de combater e, para os zebuínos, a mosca-dos-chifres era o principal problema. Os inseticidas do grupo dos piretroides foram os mais usados nos rebanhos estudados (11/17), seguidos da combinação piretroide + fosforado (4/17) e abamectina (2/17). As populações de H. irritans mostraram FR entre 0,75 e 6.434,26 para a cipermetrina e, apesar disso, apenas 3,79% das moscas genotipadas (n=686) apresentavam o alelo mutante para KDR e nenhuma para sKDR. Para o diazinon, os FR variaram entre 1,00 e 103,00 e das 587 moscas testadas, nenhuma apresentou o alelo mutante para resistência. Os testes com os carrapatos mostraram FR entre 1,00 e 718,52. A mutação KDR só foi detectada em 3,6% (com o genótipo heterozigoto SR) e 0,48% (com o genótipo homozigoto RR) das 631 larvas testadas. Podemos concluir que, no estado de São Paulo, existem populações de H. irritans e R. microplus resistentes aos grupos pesticidas piretroides e organofosforados, sendo predominante nas populações avaliadas a resistência aos piretroides. Os pecuaristas deverão ser instruídos no sentido de resguardar ao máximo esses princípios químicos, uma vez que poderão se tornar ineficientes em um curto período de tempo no controle da maioria das populações de ectoparasitas presentes nos rebanhos bovinos paulistas.

Avaliação da ocorrência da bactéria simbionte Wolbachia spp. em populações do carrapato Rhipicephalus microplus e em populações de moscas com a finalidade de desenvolver metodologia para controle biológico desses parasitas.

2016

Diagnóstico molecular de tristeza parasitária bovina em rhipicephalus microplus da região tropical do méxico / molecular diagnosis of cattle tick fever agents in rhipicephalus microplus from tropical region of mexico

2016

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.

BYC, an atypical aspartic endopeptidase from Rhipicephalus (Boophilus) microplus eggs

An aspartic endopeptidase was purified in our laboratory from Rhipicephalus (Boophilus) microplus eggs [Logullo, C., Vaz, I.S., Sorgine, M.H., Paiva-Silva, G.O., Faria, F.S., Zingali, R.B., De Lima, M.F., Abreu, L., Oliveira, E.F., Alves, E.W, Masuda, H., Gonzales, J.C., Masuda, A., and Oliveira, P.L., 1998. Isolation of an aspartic proteinase precursor from the egg of a hard tick, Rhipicephalus (Boophilus) microplus. Parasitology 116, 525-532]. Boophilus yolk cathepsin (BYC) was tested as component of a protective vaccine against the tick, inducing a significant immune response in cattle [da Silva, VI., Jr., Logullo, C., Sorgine, M., Velloso, F.F., Rosa de Lima, M.F., Gonzales, J.C., Masuda, H., Oliveira, P.L., and Masuda, A., 1998. Immunization of bovines with an aspartic proteinase precursor isolated from Rhipicephalus (Boophilus) microplus eggs. Vet. Immunol. Immunopathol. 66,331-341]. In this work, BYC was cloned and its primary sequence showed high similarity with other aspartic endopeptidases. In spite of this similarity, BYC sequence shows many important differences in relation to other aspartic peptidases, the most important being the lack of the second catalytic Asp residue, considered to be essential for the catalysis of this class of endopeptidases. When we determined BYC cleavage specificity by LC-MS, we found out that it presents a preference for hydrophobic residues in P1 and P1` in accordance to most aspartic endopeptidases. Also, when analyzed by circular dicroism, BYC presented high beta sheet content, also a characteristic of aspartic endopeptidases. On the other hand, although both native and recombinant BYC are catalytically active, they present a very low specific activity, what seems to indicate that this peptidase will digest its natural substrate, vitellin, very slowly. We speculate that such a slow Vn degradative process might constitute an important strategy to preserve egg protein content to the hatching larvae. (c) 2007 Elsevier Inc. All rights reserved.

Spotted fever group Rickettsia infecting ticks (Acari: Ixodidae) in the state of Santa Catarina, Brazil

During 2006-2008, a total of 260 adult ticks were collected from domestic and wild animals in different regions of the state of Santa Catarina (SC), Brazil, including areas where human cases of Brazilian spotted fever have been reported. Collected ticks belonging to nine species (Amblyomma aureolatum, Amblyomma cajennense, Amblyomma dubitatum, Amblyomma longirostre, Amblyomma ovale, Amblyomma tigrinum, Dermacentor nitens, Rhipicephalus microplus and Rhipicephalus sanguineus) were tested by polymerase chain reaction (PCR) for rickettsial infection. Overall, eight (3.1%) ticks were found to be infected with Rickettsia species. After sequencing the PCR products, we determined that the sequences generated from three A. aureolatum, one A. ovale and one R. sanguineus from the municipality of Blumenau, one A. ovale from the municipality of Águas Mornas and one A. ovale from the municipality of Urussanga were identical to the corresponding partial rickettsial ompA gene sequence of Rickettsia parkeri strain Atlantic rainforest. The sequence generated from one A. longirostre from Blumenau was 100% identical to the corresponding partial rickettsial ompA gene sequence of Rickettsia amblyommii strain AL. Because R. parkeri strain Atlantic rainforest was recently shown to have caused two cases of human spotted fever in other states of Brazil, the role of this rickettsial agent as a possible etiological agent of spotted fever in SC is discussed.

Esterase profile in the postembryonic development of Rhipicephalusmicroplus

The objective of this work was to analyze the pattern of esterase activity in the development stages of Rhipicephalus microplus by nondenaturing polyacrylamide gel electrophoresis using specific staining for esterase. The electrophoretical results revealed the presence of nine regions displaying esterase activity, stained with both alpha-naphthyl acetate and beta-naphthyl acetate, and classified as alpha-beta-esterase. Stage-specific esterases were found, with the first nymphal and larval stages showing the greatest esterase activity throughout the development. An esterase called EST-4 was detected only in males and was considered sex-specific. There are differences in the esterase profile among the different postembryonic development stages of R. microplus.