Esterase profile in a pyrethroid-resistant Brazilian strain of the cattle tick Boophilus microplus (Acari, Ixodidae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Esterase enzymes involved in pyrethroid and organophosphate resistance in a Brazilian population of Riphicephallus (Boophilus) microplus (Acari, Ixodidae)

Esterases are a group of enzymes that are reportedly associated with acaricide resistance in Riphicephallus (Boophilus) microplus. A comparative analysis was made of the esterase patterns in malathion and deltamethrin-sensitive, tolerant and resistant tick groups, using non-denaturing polyacrylamide gel electrophoresis. Electrophoretical profiles revealed four bands of esterase activity against alpha-naphthyl acetate; which were dubbed EST-1 to EST-4. The EST-3 and EST-4 were detected in all strains and were classified as carboxylesterases (CaEs). The EST-2, classified as an acetylcholinesterase (AChE), was detected in all groups, but its staining intensity increased from susceptible to resistant groups, indicating an altered production according to the degree of resistance. EST-1, which was also classified as an AChE, was detected exclusively in tolerant and resistant groups to both acaricides, but displayed greater activity in the malathion-resistant group. These data suggest that these AChEs may represent an important detoxification strategy developed to overcome the effects of acaricides. (C) 2008 Elsevier B.V. All rights reserved.

In vitro efficacy of plant extracts and synthesized substances on Rhipicephalus (Boophilus) Microplus (Acari: Ixodidae)

Herbal drugs have been widely evaluated as an alternative method of parasite control, aiming to slow development of resistance and obtain low-cost biodegradable parasiticides. This study evaluated the in vitro efficacy on Rhipicephalus (Boophilus) microplus of extracts from Carapa guianensis seed oil, Cymbopogon martinii and Cymbopogon schoenanthus leaf essential oil, and Piper tuberculatum leaf crude extract and similar synthesized substances. In the immersion test, engorged females were evaluated in five dilutions ranging from 10% to 0.030625% concentration. In the larval test on impregnated filter paper, the concentration ranged from 10% to 0.02%. The treatments and controls were done in three replicates. Chemical analysis of the oils was performed by gas chromatography. The main compounds were oleic acid (46.8%) for C. guianensis and geraniol for C. martinii (81.4%), and C. schoenanthus (62.5%). The isolated and synthesized substances showed no significant effect on larvae and adult. C. martinii and P. tuberculatum showed the best efficacy on the engorged females. The LC50 and LC90 were 2.93% and 6.66% and 3.76% and 25.03%, respectively. In the larval test, the LC50 and LC90 obtained for C. martinii, P. tuberculatum, and C. schoenanthus were 0.47% and 0.63%, 0.41% and 0.79%, 0.57% and 0.96%, respectively. The fact that geraniol is present in greater quantities in C. martinii explains its higher activity in relation to C. shoenanthus. It is necessary to validate the in vivo use of safe and effective phytoparasiticidal substances. Efforts should be focused on developing formulations that enhance the efficacy in vivo and lengthen the residual period.

In vitro activity of Artemisia annua L (Asteraceae) extracts against Rhipicephalus (Boophilus) microplus

A atividade de extratos vegetais sobre parasitas pode indicar grupos de substâncias de uso potencial no controle de Rhipicephalus (Boophilus) microplus. O objetivo do presente estudo foi investigar a ação in vitro de extratos de Artemisia annua sobre esta espécie. A concentração das lactonas sesquiterpênicas artemisinina e deoxiartemisinina presentes nos extratos vegetais, foi quantificada via cromatografia líquida de alta eficiência. Quatro extratos produzidos a partir do extrato bruto concentrado (EBC) foram avaliados sobre larvas pela metodologia do papel impregnado, com leitura após 24 horas de incubação. As fêmeas ingurgitadas foram imersas por cinco minutos no EBC e nos seus quatro extratos derivados, e incubadas para posterior análise dos parâmetros biológicos. Os extratos não tiveram eficácia sobre as larvas nas concentrações avaliadas (de 3,1 a 50 mg.mL-1). O EBC apresentou melhor eficácia sobre as fêmeas ingurgitadas (CE 50 de 130,6 mg.mL-1 e CE 90 de 302,9 mg.mL-1) que os extratos derivados. Esses resultados tendem a confirmar que a ação da artemisinina sobre as fêmeas ingurgitadas de R. (B.) microplus estaria condicionada à sua ingestão através do sangue. Nesse caso, os métodos in vitro seriam inadequados para a efetiva avaliação da ação de A. annua R.(B.) microplus.

Análise de fatores genéticos e ambientais que afetam a infestação de fêmeas bovinas da raça Caracu por carrapatos (Boophilus microplus)

Este trabalho foi conduzido com o objetivo de avaliar o efeito de fatores de meio sobre a infestação de bovinos Caracu pelo carrapato Boophilus microplus (Canestrini, 1887) e estimar parâmetros genéticos do grau de infestação por esse ectoparasita. Foram realizadas contagens em fêmeas de dois rebanhos, nas quatro estações, por dois anos consecutivos (setembro/1998 a julho/2000). Contou-se o número de carrapatos (NC) em um dos lados do animal e atribuiu-se escore visual (EC) de acordo com a quantidade de carrapatos no animal. Foram feitas de uma a oito avaliações, totalizando-se 4.079 e 3.994 observações de NC e EC, respectivamente, em 718 animais. Os dados foram analisados pelo método dos quadrados mínimos com um modelo que incluiu efeitos de rebanho (R), cor do animal (C), R x C, animal dentro de R x C como erro a, ano e estação da avaliação, espessura de pelame e idade do animal como covariável. As estimativas dos componentes de variância foram obtidas pelo método da máxima verossimilhança restrita livre de derivadas, utilizando-se um modelo que incluiu os efeitos fixos de grupo de contemporâneos (fazenda-ano-época), espessura do pelame e idade do animal como covariável e os efeitos aleatórios aditivos diretos e de ambiente permanente. Antes das análises, a variável NC foi transformada para log10 (n + 1) e EC para (x + 0,5)½, em que n é o número de carrapatos contados no animal e x, o escore (0 a 4). A incidência de carrapatos foi maior no verão e, quanto maior a espessura do pelame, maior o nível de infestação. As estimativas de herdabilidade e repetibilidade foram, respectivamente, 0,22 e 0,29 para NC e 0,15 e 0,21 para EC; a correlação genética entre NC e EC foi igual a 1,00. Os resultados sugerem que é possível obter progresso genético para resistência a carrapato pela seleção.

Tests to determine LC50 and discriminating doses for macrocyclic lactones against the cattle tick, Boophilus microplus

Laboratory tests were carried out on larvae and adults of the cattle tick Boophilus microplus to determine the toxicity of macrocyclic lactone acaricides (MLs). Technical and commercial MLs were used in larval packet test (LPT), larval immersion test (LIT) and adult immersion test (AIT). Tn LIT and AIT the toxicity of MLs was much higher than for LPT. In the AIT, diluting the injectable formulation of MLs in water was as effective as dilution in ethanol + Triton X-100. LC50, LC99.9 and 95% confidence limits were determined so that a discriminating dose (DD) could be set for larval and adult tests in order to diagnose potential resistance to MLs in field samples of the tick. These DDs are for Australian strains of B. microplus and may not be suitable for other strains until further work is carried out.The value of these diagnostic tests can only be verified if or when resistance to MLs emerges in ticks. (C) 2001 Elsevier B.V. B.V. All rights reserved.

Dermal mast cell counts in F2 Holstein x Gir crossbred cattle artificially infested with the tick Boophilus microplus (Acari : Ixodidae)

The role of dermal mast cells (DMC) in the host resistance to ticks has been studied but it is not totally explained yet. Studies have proposed that zebuine cattle breeds, known as highly resistant to ticks, have more DMC than taurine breeds. In the present study, we compared the number of adult female ticks Boophilus microplus and the mast cells' countings in the skin of F-2 crossbred Gir x Holstein cattle, before and after tick infestation. F-2 crossbred cattle (n = 148) were divided into seven groups and artificially infested with 1.0 x 10(4) B. nticroplus larvae and, 21 days afterwards, adult female-fed ticks attached to the skin were counted. Skin biopsies were taken and examined under light microscopy with a square-lined ocular reticulum in a total area of 0.0625 mm(2) in both the superficial and deep dermis. Results demonstrated that infested F-2 crossbred cattle acquired resistance against the cattle-tick B. microplus probably associated to an increase in the dermal mast cell number. It is concluded that the tick infestation may lead to an environmental modification in the dermis of parasitized hosts due to the massive migration of mast cells or their local proliferation.

Gene discovery in Boophilus microplus, the cattle tick - the transcriptomes of ovaries, salivary glands, and hemocytes

The quest for new control strategies for ticks can profit from high throughput genomics. In order to identify genes that are involved in oogenesis and development, in defense, and in hematophagy, the transcriptomes of ovaries, hemocytes, and salivary glands from rapidly ingurgitating females, and of salivary glands from males of Boophilus microplus were PCR amplified, and the expressed sequence tags (EST) of random clones were mass sequenced. So far, more than 1,344 EST have been generated for these tissues, with approximately 30% novelty, depending on the the tissue studied. To date approximately 760 nucleotide sequences from B. microplus are deposited in the NCBI database. Mass sequencing of partial cDNAs of parasite genes can build up this scant database and rapidly generate a large quantity of useful information about potential targets for immunobiological or chemical control.

Tracing heme in a living cell: hemoglobin degradation and heme traffic in digest cells of the cattle tick Boophilus microplus

Heme is present in all cells, acting as a cofactor in essential metabolic pathways such as respiration and photosynthesis. Moreover, both heme and its degradation products, CO, iron and biliverdin, have been ascribed important signaling roles. However, limited knowledge is available on the intracellular pathways involved in the flux of heme between different cell compartments. The cattle tick Boophilus microplus ingests 100 times its own mass in blood. The digest cells of the midgut endocytose blood components and huge amounts of heme are released during hemoglobin digestion. Most of this heme is detoxified by accumulation into a specialized organelle, the hemosome.We followed the fate of hemoglobin and albumin in primary cultures of digest cells by incubation with hemoglobin and albumin labeled with rhodamine. Uptake of hemoglobin by digest cells was inhibited by unlabeled globin, suggesting the presence of receptor-mediated endocytosis. After endocytosis, hemoglobin was observed inside large digestive vesicles. Albumin was exclusively associated with a population of small acidic vesicles, and an excess of unlabeled albumin did not inhibit its uptake. The intracellular pathway of the heme moiety of hemoglobin was specifically monitored using Palladium-mesoporphyrin IX (Pd-mP) as a fluorescent heme analog. When pulse and chase experiments were performed using digest cells incubated with Pd-mP bound to globin (Pd-mP-globin), strong yellow fluorescence was found in large digestive vesicles 4 h after the pulse. By 8 h, the emission of Pd-mP was red-shifted and more evident in the cytoplasm, and at 12 h most of the fluorescence was concentrated inside the hemosomes and had turned green. After 48 h, the Pd-mP signal was exclusively found in hemosomes. In methanol, Pd-mP showed maximal emission at 550 nm, exhibiting a red-shift to 665 nm when bound to proteins in vitro.The red emission in the cytosol and at the boundary of hemosomes suggests the presence of heme-binding proteins, probably involved in transport of heme to the hemosome. The existence of an intracellular heme shuttle from the digestive vesicle to the hemosome acting as a detoxification mechanism should be regarded as a major adaptation of ticks to a blood-feeding way of life. To our knowledge, this is the first direct observation of intracellular transport of heme in a living eukaryotic cell. A similar approach, using Pd-mP fluorescence, could be applied to study heme intracellular metabolism in other cell types.

Pathogenicity of isolates of Metarhizium anisopliae (Metsch.) Sorokin towards the cattle tick Boophilus microplus (Can.) (Acari : Ixodidae) under laboratory conditions

The pathogenic activity of Metarhizium anisopliae on Boophilus microplus females was assessed using five fungal isolates. Groups of 8 engorged ticks collected from cattle on free pasture were inoculated by immersion for 5 seconds in a spore suspension containing 2.5 x 10(8) conidia/ml. The groups were transferred to moist chambers in which they were maintained at 27 +/- 1 degreesC for about 15 days, their mortality being determined every 2, or occasionally 3, days. The isolate that produced the best results in this assay was used to inoculate female ticks with spore suspensions containing 0, 7.5 x 105, 7.5 x 10(6), 7.5 x 10(7) and 7.5 x 10(8) conidia/ml, under the same conditions as described above. The pathogenic action of M. anisopliae on engorged females was clearly demonstrated. Isolates E9 and AM were more effective, causing high tick mortality as well as reduced oviposition. The concentration of 7.5 x 10(8) conidia/ml was the most effective, with the fungus sporulating on 91.1% of the ticks. Mean percent oviposition was highest in the control treatment and lowest in the treatment with 7.5 x 10(8) conidia/ml.

Susceptibility of the cattle tick Boophilus microplus (Acari: Ixodidae) to isolates of the fungus Beauveria bassiana

The susceptibility of the tick Boophilus microplus to Beauveria bassiana was evaluated by inoculating eggs, larvae and engorged females of the tick with five fungal isolates at concentrations of 106, 107 and 108 conidia/ml. Tick eggs (0.25 g) were immersed in 1 ml of a suspension of the different conidial concentrations for 1 min. Similar exposure was performed by immersion of 2000 larvae and homogeneous groups of nine engorged females in 2 and 20 ml of conidial suspension, respectively. Treated eggs, larvae and adults were placed in an incubator at 27 ± 1 °C and relative humidity above 80% for evaluation of the fungal action. All fungal isolates applied at all conidial concentrations reduced the hatching rate of larvae from treated eggs by 1.36-65.58% and increased the mortality rate of inoculated larvae by 0.8-70.49%. In the bioassay with engorged females, oviposition period was reduced by 9.69-47.80%, egg mass weight by 4.71-53.87%, estimated reproduction by 8.3-60.62%, egg production index by 5.03-54.20%, percent larval hatching by 0.27-13.96%, and the mortality rate of treated females was increased by 96.60-100%. The reduction of the estimated reproduction obtained for the treated groups ranged from 8.37 to 64.52%. The sporulation of the pathogen on dead females ranged from 3.70 to 88.88% depending on the isolate and concentration used. Isolates AM 09, CB 7 and JAB 07 were the most effective and effectiveness increased with increasing concentrations of conidia in the suspensions.

PCR-based detection of Babesia bovis and Babesia bigemina in their natural host Boophilus microplus and cattle

PCR and nested-PCR methods were used to assess the frequency of Babesia bovis and Babesia bigemina infection in Boophilus microplus engorged females and eggs and in cattle reared in an area with endemic babesiosis. Blood and the engorged female ticks were from 27 naturally infested calves and 25 crossbred cows. The frequency of both Babesia species was similar in calves and cows (P > 0.05). Babesia bovis was detected in 23 (85.2%) calves and in 25 (100%) cows and B. bigemina was detected in 25 (92.6%) calves and in 21 (84%) cows. Mixed infections with the both Babesia species were identified in 42 animals, 21 in each age category. Of female ticks engorged on calves, 34.9% were negative and single species infection with B. bigemina (56.2%) was significantly more frequent (P < 0.01) than with B. bovis (4.7%). Most of the females (60.8%) engorged on cows did not show Babesia spp. infection and the frequency of single B. bovis infection (17.6%) was similar (P > 0.05) to the frequency of single B. bigemina infection (15.9%). Mixed Babesia infection was lower (P < 0.01) than single species infection in female ticks engorged either in cows (5.7%) or in calves (4.3%). An egg sample from each female was analysed for the presence of Babesia species. Of the egg samples from female ticks infected with B. bovis, 26 (47.3%) were infected while from those from female ticks infected with B. bigemina 141 (76.6%) were infected (P < 0.01). The results showed that although the frequency of both species of Babesia was similar in calves and cows, the infectivity of B. bigemina was higher to ticks fed on calves while to those ticks fed on cows the infectivity of both Babesia species was similar. © 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.