Caracterização do controle de Haematobia irritans e Rhipicephalus (Boophilus) microplus no Triângulo Mineiro e Alto Paranaíba, Minas Gerais

O controle eficaz de Haematobia irritans (mosca-dos-chifres) e Rhipicephalus (Boophilus) microplus (carrapato-do-boi) é um fator crucial à maior rentabilidade da pecuária brasileira, porém ainda constitui-se um desafio. Um maior conhecimento das práticas adotadas no combate destes parasitos faz-se necessário para que se possam estruturar estratégias de controle mais próximas da realidade do produtor rural e mais fáceis de serem executadas. Este estudo caracterizou, através de entrevistas, as práticas adotadas no controle desses ectoparasitos em 23 propriedades da mesorregião do Triângulo Mineiro e Alto Paranaíba, Minas Gerais. A maioria dos entrevistados reconheceu a importância desses parasitos para a atividade pecuária, entretanto, diversos problemas relativos ao controle parasitário foram observados. O controle da mosca-dos-chifres e do carrapato-do-boi era uma prática rotineira em, respectivamente, 17,4% e 95,7% das propriedades, geralmente realizado em função de elevado grau de infestação dos animais. Mais de seis aplicações de ectoparasiticidas eram realizadas por ano em todas as propriedades que empregavam tratamentos para o controle da mosca e em 76,5% daquelas que combatiam o carrapato. Os produtos eram aplicados principalmente com bombas costais manuais (63,6%) e sem a contenção dos animais em 45,5% das propriedades. A diluição dos produtos segundo recomendações dos fabricantes era realizada por 45,4% dos entrevistados, porém, 63,6% aplicavam um volume de solução por animal menor do que o tecnicamente recomendado. Utilizava-se principalmente a associação de piretróides e organofosforados para o controle de ambos os parasitos. Apesar de utilizar rotineiramente ectoparasiticidas, o uso de equipamento de proteção individual (EPI) não era comum entre os entrevistados. A maioria dos entrevistados conhecia algumas características epidemiológicas dos parasitos, entretanto, o controle parasitário adotado na maior parte das propriedades tende a comprometer não apenas a eficácia dos tratamentos, mas a suscetibilidade dos parasitos e a sustentabilidade do controle.

Assessing bovine babesiosis in Rhipicephalus (Boophilus) microplus ticks and 3 to 9-month-old cattle in the middle Magdalena region, Colombia

Babesia sp. is a protozoan hemoparasite that affects livestock worldwide. The Colombian Middle Magdalena is an enzootic region for babesiosis, but there is no previous research providing detail on its transmission cycle. This study aims to assess some Babesia sp. infection indicators in cattle and ticks from the area, by using direct microscopic and molecular techniques to detect the infection. In the cattle, 59.9% and 3.4 % positivity values for B. bigemina and mixed infection (B. bovis + B. bigemina) were found respectively. In ticks, the positivity of B. bigemina reached 79.2% and 9.4% for the mixed infection. The degree of infestation in the region was 3.2 ticks per bovine. There was positive correlation between tick control acaricide frequencies and infestation in bovines. This leads us to infer that control periodicity greater than 90 days, in stable zones, is an abiotic factor that benefits the acquisition of protective immunity in calves, the natural control of the infection and eventual disease absence. It is necessary to monitor the disease by applying new entomological and parasitological indicators showing the complexity of this phenomenon.

Resposta de linfonodos em bovinos inoculados a campo com a vacina recombinante rSBm7462 anti Rhipicephalus (Boophilus) microplus)

A dependência exclusiva de compostos químicos para o controle de Rhipicephalus (Boophilus) microplus tornou-se uma das maiores preocupações científicas e econômicas dos últimos anos, e como consequência, estão sendo realizadas pesquisas para o desenvolvimento de vacinas. O objetivo deste trabalho foi avaliar a resposta de linfonodos de bovinos imunizados a campo com o peptídeo rSBm7462 anti R. (B.) microplus. Foram utilizados 14 bovinos mestiços (Bos taurus x Bos indicus), com idades entre 4-10 meses, mantidos em duas propriedades rurais do norte do estado de Minas Gerais. Os animais receberam três imunizações do peptídeo rSBm7462, aplicados por via subcutânea, com intervalo de 30 dias. Após 15 dias de cada imunização, os linfonodos pré-escapulares foram coletados e fixados por 18 horas em formol. Posteriormente, foram incluídos em Paraplast e as amostras foram coradas pela técnica hematoxilina-eosina (HE) para a observação de eventos celulares. Para a identificação do antígeno nos linfonodos dos animais imunizados, foi realizada a técnica de imuno-histoquímica (IHQ) com o método peroxidase-anti-peroxidase (PAP). A resposta de linfonodos dos bovinos inoculados foi avaliada pelas análises de formação de centros germinais (CG), hiperplasia de cordões medulares (CM) e a presença do antígeno rSBm7462 em células PAP+, demonstrando que o peptídeo recombinante rSBm7462 induz uma resposta imune adaptativa T-dependente, caracterizada nos tecidos linfóides secundários pela formação de estruturas que conferem afinidade e memória imunológica.

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.

Structure and Mode of Action of Microplusin, a Copper II-chelating Antimicrobial Peptide from the Cattle Tick Rhipicephalus (Boophilus) microplus

Microplusin, a Rhipicephalus (Boophilus) microplus antimicrobial peptide (AMP) is the first fully characterized member of a new family of cysteine-rich AMPs with histidine-rich regions at the N and C termini. In the tick, microplusin belongs to the arsenal of innate defense molecules active against bacteria and fungi. Here we describe the NMR solution structure of microplusin and demonstrate that the protein binds copper II and iron II. Structured as a single alpha-helical globular domain, microplusin consists of five alpha-helices: alpha 1 (residues Gly-9 to Arg-21), alpha 2 (residues Glu-27 to Asn-40), alpha 3 (residues Arg-44 to Thr-54), alpha 4 (residues Leu-57 to Tyr-64), and alpha 5 (residues Asn-67 to Cys-80). The N and C termini are disordered. This structure is unlike any other AMP structures described to date. We also used NMR spectroscopy to map the copper binding region on microplusin. Finally, using the Gram-positive bacteria Micrococcus luteus as a model, we studied of mode of action of microplusin. Microplusin has a bacteriostatic effect and does not permeabilize the bacterial membrane. Because microplusin binds metals, we tested whether this was related to its antimicrobial activity. We found that the bacteriostatic effect of microplusin was fully reversed by supplementation of culture media with copper II but not iron II. We also demonstrated that microplusin affects M. luteus respiration, a copper-dependent process. Thus, we conclude that the antibacterial effect of microplusin is due to its ability to bind and sequester copper II.

(1)H, (15)N and (13)C assignments of the Rhipicephalus (Boophilus) microplus anti-microbial peptide microplusin

Microplusin, a Rhipicephalus (Boophilus) microplus anti-microbial peptide (AMP) is the first member of a new family of cysteine-rich AMPs with histidine-rich regions at the N- and C-termini, which is being fully characterized by biophysical and biochemical methods. Here we report the NMR resonance assignments for (1)H, (15)N, and (13)C nuclei in the backbone and side chains of the microplusin as basis for further studies of structure, backbone dynamics and interactions mapping.

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.

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.

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.

Resistance to cypermethrin, deltamethrin and chlorpyriphos in populations of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) from small farms of the State of Sao Paulo, Brazil

A field survey of resistance was conducted based on the larval packet test technique with synthetic pyrethroids (cypermethrin and deltamethrin) and organophosphates (chlorpyriphos) in Rhipicephalus (Boophilus) microplus field populations from six different regions of the State of Sao Paulo (Brazil). 82.6% of the populations showed resistance to cypermethrin, 86.36% to deltamethrin and 65.25% to chlorpyriphos, with 50% presenting resistance to both SP and OP acaricide. According to the questionnaires completed by the producers, OP + SP mixtures followed by SP-only formulations were the products most commonly used for controlling the cattle tick in the surveyed areas. The present study showed high occurrence of resistance to SP and OP in the State of Sao Paulo, Brazil and revealed the type of strategy adopted by small dairy farms in this state. This information is fundamental in order to establish the monitoring of resistance on each farm individually, contributing to the rational use of acaricides for the control of R. (B.) microplus. (C) 2011 Elsevier B.V. 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.

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.

In vitro tests to establish LC(50) and discriminating concentrations for fipronil against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) and their standardization

Laboratory test was carried out on larvae and adults of the cattle tick, Rhipicephalus (Boophilus) microplus, to determine fipronil toxicity. Adult immersion test (AIT, N = 26), larval immersion test (LIT, N = 71) and larval packet test (LPT, N = 41) were standardized using susceptible strain (Mozo). Dose-response curves were compared with a fipronil resistant strain. Four variables were analyzed from AIT results: mortality, weight of eggs on day 7 and on day 14, index of fertility, and index of fecundity. For larval test, dose mortality curves were analyzed. In spite of the high LC(50) variability, all variables determined for AIT were appropriate to discriminate both strains. AIT and LIT had more sensitivity than LPT, with larger resistance factors. It was used two times LC(99.9) as discriminating doses (DCs) following FAO suggestion. For mortality by AIT, LIT and LPT the DCs were estimated: 4.98 ppm, 7.64 ppm and 2365.8 ppm, respectively, for Mozo strain. DCs mortality values estimated for resistant strain by AIT, LIT and LPT were: 6.96 x 10(5) ppm, 343.26 ppm and 5.7 x 10(3) ppm, respectively and their respective resistant factors were: 202.4, 5.36 and 1.52. Protocols for AIT, LIT and LPT have been presented in this paper. (C) 2009 Elsevier B.V. All rights reserved.

Determination of the susceptibility of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) to ivermectin and fipronil by Larval Immersion Test (LIT) in Uruguay

Rhipicephalus (Boophilus) microplus is an important cattle pest in Uruguay, and the law regulates its control. It is resistant to organophosphates, synthetic pyrethroids and, as recently discovered, to fipronil. Resistance to macrocyclic lactones (MLs) and amitraz have not been documented; however, veterinarians and farmers have reported treatment failures. The objective of the present work was to study the susceptibility of cattle tick strains from different Uruguayan counties to ivermectin (IVM) and fipronil by using the Larval Immersion Test (LIT). The Mozo strain was used as the susceptible reference strain. From 2007 to 2009, twenty-eight tick populations were collected from different cattle farms with and without history of IVM or fipronil use. A probit analysis estimated dose-mortality regressions, lethal concentrations (LC), and confidence intervals. The resistance ratio (RR) was determined at the LC(50) and LC(90) estimates. To classify a tick population in relation to resistance, three categories based on a statistical analysis of LC and RR between field populations and Mozo strains were defined: susceptible (no differences), incipient resistance (differences and RR(50) < 2) and resistant (differences and RR(50) >= 2). Eighteen field populations were tested with IVM and five of them presented a RR(50) range between 1.35 and 1.98 and the LC(50/90), which is statistically different from the Mozo strain (incipient resistance). However, the RR(90) increases >= 2 in four of the populations, confirming that tick resistance to IVM is emergent. The low RR values obtained could be a result of a low frequency of treatments with IVM. Twenty-seven tick populations were tested with fipronil and six were diagnosed as resistant according to the LIT. Cross-resistance was not observed between fipronil and IVM on these tick populations. The current study presents different R. (B.) microplus populations with an incipient resistance to IVM, and indicates that the fipronil tick resistance is restricted to certain areas in Uruguay. (c) 2011 Elsevier B.V. All rights reserved.

Cell Death in Salivary Glands of Rhipicephalus (Boophilus) microplus (Canestrini, 1887) (Acari: Ixodidae) Females at Semi-engorged Feeding Stage

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