Skin test and tick immune status in susceptible and resistant cattle in Brazil

A cutaneous hypersensitivity test (CHT) was used to correlate host resistance to ticks and type of reaction elicited to unfed larval extract-ULE of the cattle tick Boophilus microplus in European and Indian cattle. Twenty calves were separated into four groups of five animals each: naïve or preinfested Indian or European cattle. CHT was induced by intradermal inoculation of 0.1 ml of ULE cattle tick B. microplus (50 μg protein) in the calf ear. Ear thickness was measured using calipers before and 10 min, 1, 2, 6, 18, 24, 48, 72, 96, and 144 h postinoculation (PI). Preinfested European calves showed only an immediate type reaction with maximum response (75% increase in ear thickness) at 10 min PI. On the other hand, preinfested Indian calves presented an immediate response with maximum reaction (70% increase in ear thickness) between 10 min and one hour PI, and a delayed type reaction at 72 h PI (60% increase in ear thickness). These results point out the crucial role of the cellular immune response of cattle in the expression of resistance to cattle tick B. microplus. Skin test might be useful in the ranking of cattle according to the susceptibility/resistance to ticks.

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.

Structural and cytochemical changes in the salivary glands of the Rhipicephalus (Boophilus) microplus (CANESTRINI, 1887) (Acari : Ixodidae) tick female during feeding

This study describes the morphology of salivary glands of Rhipicephalus (Boophilus) microplus female ticks at beginning of feeding (24-48 h of attachment) and semi-engorged (4-5 days of attachment) to verify the degenerative characteristics of these organs and the secretory phase in which the process begins. At the beginning of feeding, secretion granules had been observed only in the cytoplasm of cells b, c(1), c(2), c(4) (type II acinus) and d (type III acinus), as well as large nuclei with regular and preserved morphology. In the semi-engorged females the acini presented few normal cells, few partially preserved ones, and the remaining ones in several stages of degeneration, that is, with retraction and cytoplasmic vacuolization, and nuclei with chromatin in several stages of condensation, picnotic and/or in fragmentation. In type I acinus and in the excretory ducts of the studied glands, at both feeding stages, no degenerative characteristic was observed. In females of R. (B.) microplus, the salivary glands degenerate asynchronically and precociously when compared with those of others tick's species. (c) 2006 Elsevier B.V. All rights reserved.

Genetic parameters and investigation of genotype x environment interactions in Nellore x Hereford crossbred for resistance to cattle ticks in different regions of Brazil

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

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.

Tristeza Parasitária em bovinos na região de Botucatu – SP: estudo retrospectivo de 1986 – 2007

A retrospective study of Cattle Tick Fever was made with animals up to one year old, which occurred from 1986-2007 in Botucatu-SP in the influence area of the Veterinary Hospital of the School of Veterinary Medicine and Animal Science - UNESP. There were 232 cases and 57 deaths. The main etiological agent causing the disease on this region is Anaplasma marginale identified as a single agent in 31.5% of the cases. Most of the cases occurred on autumn in mixed-blood animals two to six months old. Predominant clinical signs included apathy, hyporexia or anorexia, dehydration, weight loss and pale mucosa. The high incidence and mortality observed justify prophylactic actions to have the balance between host, agent, vector, and environment, thus avoiding the clinical form of disease and death of livestock herds in the region.

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.

Esterase profile in the postembryonic development of Rhipicephalusmicroplus

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

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.

Rhipicephalus (Boophilus) microplus (Canestrini, 1887) (Acari: Ixodidae): Acid phosphatase and ATPase activities localization in salivary glands of females during the feeding period

This study investigates the presence and the localization of acid phosphatase and ATPase in the salivary glands of Rhipicephalus (Boophilus) microplus female ticks during feeding. Semi-engorged females showed a larger amount of acid phosphatase compared to those at beginning of feeding, localized mainly in the apical portion of the secretory cells, and in the basal labyrinth of the interstitial cells. Ultrastructural observations also demonstrated its presence in secretion granules and inside some nuclei of secretory cells at beginning of feeding. Acid phosphatase in a free form probably has a hemolymph and/or ribosomal origin and participates in salivary gland secretion control. ATPase was detected in basal membrane of all types of acini and/or in the cytoplasm of the secretory cells at both feeding stages. The enzyme activities found strongly suggests that cell death by apoptosis occurs during the degenerative process. © 2006 Elsevier Inc. All rights reserved.

In vitro activity of pineapple extracts (Ananas comosus, Bromeliaceae) on Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

Measures to control the cattle tick, Rhipicephalus (Boophilus) microplus, based only on chemical products are becoming unsustainable, mainly because of the development of resistance. The objective of this study was to test the effect of the aqueous extract of pineapple skin (AEPS) and bromelain extracted from the stem (Sigma-Aldrich®, B4882) on engorged females and larvae of R. (B.) microplus in vitro. These substances were diluted in water and evaluated at eight concentrations. Engorged females were collected and distributed in groups of 10, with three repetitions for each treatment. After immersion in the solutions, the females were placed in an incubator for observation of survival, oviposition and larval hatching. The larval packet method was used, also with three repetitions with about 100 larvae each. The packets were incubated and the readings were performed after 24h. The estimated reproduction and efficacy of the solutions were calculated. The LC50 and LC90 were estimated using the Probit procedure of the SAS program. The eight concentrations were compared within each treatment by the Tukey test. For the experiment with engorged females, the most effective concentrations were 125, 250 and 500mg/mL: 33%, 48% and 59% for the AEPS and 27%, 51% and 55% for the bromelain. The LC50 and LC90 values were, respectively, 276 and 8691mg/mL for AEPS and 373 and 5172mg/mL for bromelain. None of the dilutions tested was effective against the larvae of R. (B.) microplus. This is the first report of the action of pineapple extracts or their constituents on cattle ticks. The results demonstrate that further studies regarding composition of tick cuticle, with evaluation of other solvents and formulations, should be conducted seeking to enhance the effect of pineapple extracts and compounds against this ectoparasite. © 2013 Elsevier Inc.

O papel das imunidades nas relações parasito-hospedeiro: o carrapato Rhipicephalus (Boophilus ) microplus e bovinos resistentes ou susceptíveis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Aplicação do fungo Metarhizium anisopliae em pastagem visando o controle do carrapato Boophilus microplus em bovinos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Aspectos ecológicos de Boophilus microplus (CANESTRINI, 1887) (ACARINA: IXODIDAE) no município de Franca, nordeste de São Paulo

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Efficacy of fipronil (1.0 mg/kg) against Rhipicephalus (Boophilus) microplus strains resistant to ivermectin (0.63 mg/kg)

The present study aimed to evaluate the efficacy of fipronil (1 mg/kg) against three strains of ivermectin-resistant Rhipicephalus (Boophilus) microplus (R. (B.) microplus), naturally infesting cattle from different states of Brazil. Three rural properties with a history of macrocyclic lactones ineffectiveness against the cattle tick, and low frequency use of fipronil in the herd, were selected for the study. The animals were randomized according to the mean tick counts, performed on days 3, 2 and I, into three groups with 10 animals each: T01, control (saline solution); T02, subcutaneous ivermectin (0.63 mg/kg) and T03, topical fipronil (1 mg/kg). Treatment was performed on day 0. Counts of partially engorged female ticks were performed on days 3,7 and 14 post-treatment (DPT), and then every 7 days until the 49th DPT. In all three experiments, it was possible to diagnose resistance of R. (B.) microplus to ivermectin (0.63 mg/kg). The maximum efficacy (arithmetic mean) obtained for ivermectin was 64% in experiment II. On the other hand, the formulation containing fipronil (1 mg/kg) reached high efficacy values (>= 97%) in all three experiments. The results from all experiments in this study demonstrate the high efficacy of 1 mg/kg fipronil, administered pour-on in naturally infested cattle, against strains of R. (B.) microplus that are resistant to 630 mcg/kg ivermectin. (C) 2014 Elsevier B.V. All rights reserved.