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.

Molecular characterization of Hepatozoon canis in dogs from Colombia

Hepatozoonosis is a tick-borne disease whose transmission to dogs occurs by ingestion of oocysts infected ticks or feeding on preys infested by infected ticks. Until now, there is no previous report of molecular characterization of Hepatozoon sp. in dogs from Colombia. EDTA blood samples were collected from 91 dogs from central-western region of Colombia (Bogota, Bucaramanga, and Villavicencio cities) and submitted to 18S rRNA Hepatozoon sp. PCR and blood smears confection. Phylogenetic analysis was used to access the identity of Hepatozoon species found in sampled dogs. From 91 sampled dogs, 29 (31.8%) were positive to Hepatozoon sp. (25 dogs were only positive in PCR, 1 was positive only in blood smears, and 3 were positive in both blood smears and PCR). After sequencing, the found Hepatozoon sp. DNA showed 100% of identity with Hepatozoon canis DNA isolates. The phylogenetic tree supported the identity of the found Hepatozoon sp. DNA, showing that the isolates from Colombia were placed in the same clade than other H. canis isolates from Venezuela, Spain, and Taiwan. This is the first molecular detection of H. canis in dogs from Colombia.

Endectocide activity of a new long-action formulation containing 2.25% ivermectin+1.25% abamectin in cattle

The present work aimed to evaluate the endectocide activity of a new injectable long-action formulation, containing ivermectin (IVM) and abamectin (ABA). In each one of the four experiments performed, the following groups were formed: group I: 2.25% IVM (450 mu g/kg) + 1.25% ABA (250 mu g/kg), group II: 3.15% IVM (630 mu g/kg) and group III: control. Eighteen bovine naturally infected by gastrointestinal nematoda were selected for anthelmintic evaluation and necropsied on posttreatment day (PTD) 14 to estimate the total parasitic burden. For the Rhipicephalus (Boophilus) microplus field trial, 30 bovine were selected by means of counts of semi-engorged R. (B.) microplus and the therapeutic and residual efficacy evaluated by tick counts on PTDs 1, 3, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84 and 91. In the stall test, 15 calves were artificially infested with 5000 R. (B.) microplus (Mozzo strain) larvae three times a week and daily collections of all the engorged female ticks detached from each calf were performed until the PTD 80. Forty bovine naturally infected with Dermatobia hominis larvae were selected and the number of larvae was counted by visual and tactile inspection on PTDs 3, 7, 14, 28, 35, 49, 63, 77, 91 and 105. In this trial, a formulation containing 1% doramectin (200 mu g/kg) was also used. IVM + ABA formulation and 3.15% IVM eliminated four of the eight species of nematode identified. The anthelmintic efficacy of the avermectins association against Haemonchus placei, Cooperia spatulata and C. punctata was 89.64%, 98.84% and 97.69%, while 3.15% IVM achieved 30.98%, 84.79% and 75.56%, respectively. The two formulations evaluated showed reduced acaricide action on the PTD I and 3, reaching high efficacy percentages from PTD 14 onward. The IVM + ABA showed efficacy above 95% in the period between PTDs 21 and 49. In the stall test, it observed no difference (P > 0.05) between the two formulations regarding the R. (B.) microplus counts during the entire evaluation period. IVM + ABA reduced the number of ticks from the PTD 1 to 77 (P < 0.05) and 3.15% IVM reduced (P < 0.05) the tick number from PTD 4 up to PTD 80. The three endectocides showed no difference (P > 0.05) regarding the number of D. hominis larvae and prevented this parasite reestablishment until PTD 105. These results indicate that the IVM + ABA association showed higher anthelmintic activity and similar efficacy against arthropods to the formulation containing 3.15% IVM. (C) 2008 Elsevier B.V. All rights reserved.

Potential role for dog fleas in the cycle of Leishmania spp.

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

An enzyme-linked immunosorbent assay (elisa) for the detection of antibodies against babesia boris in cattle

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Quantification of sex pheromone from Anocentor nitens by gas chromatography-mass spectrometry-selected ion monitoring

Two highly sensitive and selective methods based on gas chromatography coupled to mass spectrometry (GC-MS) in the selected ion monitoring (SIM) mode have been developed for the quantification of 2,6-dichlorophenol (2,6-DCP), a sex pheromone of the tick females of Anocentor nitens. Standard addition method and calibration curve techniques using 5-bromine-4-hydroxy-3- methoxybenzaldehyde (5-BrV) as internal standard (IS) afforded detection limit of 0.1ngml-1. The calibration curve was linear over the concentration range from 0.5 to 500ngml-1 for 2,6-DCP. Results show that the concentration range of sex pheromone in the extracts samples was 1.08-10.35ngml-1. The methods developed provided reliable procedures to determine amounts of 2,6-DCP present in ticks. © 2003 Elsevier B.V. All rights reserved.

Demodicose bovina no estado da Paraíba

Cases of bovine demodicosis caused by Demodex bovis were reported in a Sindhi herd from December 1989 to January 1992. Both localized and generalized forms were diagnosed. This is the first report of the generalized form in Brazil. In the first two years, demodicosis was diagnosed only in cattle < 2 years old, whereas animals of all ages were positive in the last two years. Prevalence varied from 20.4% (11/54) to 53.1% (26/49) and 13.2% (12/91) to 14.8% (9/61) for cattle < 2 years old and > 2 years old, respectively. Clinical signs varied from a few small nodules to a thickened skin with soft large nodules in the localized and generalized forms, respectively. Main microscopic features of the nodules in the generalized form consisted of acanthosis with hyperqueratosis, chronic sebaceous adenitis, subcutaneous muscular necrosis, focal cellular degeneration of the epidermis basal layer and presence of large number of mites inside the lumen of dilated hair follicles. In addition, a chronic perifoliculitis was observed, characterized by lymphoplasmocytic infiltrate which also contained macrophages and neutrophils. It is suggested that poor nutrition and stress due to prolonged drought probably contributed to the increase of susceptibility of the herd to mite infestation.

A new species of Pterodectes Robin, 1877 (Proctophyllodidae: Pterodectinae) from the pale-breasted thrush, Turdus leucomelas (Passeriformes: Turdidae)

A new species of feather mite, Pterodectes fissuratus sp. n., is described from the pale-breasted thrush, Turdus leucomelas Veillot (Passeriformes: Turdidae), in Brazil. This species is easily distinguished by having numerous well-pronounced lacunae and a longitudinal median furrow on prodorsal and hysteronotal shields in both sexes. Copyright © 2005 Magnolia Press.

Éster de sacarose no controle do Varroa estructor em abelhas africanizadas

This study aimed to determine the effect of sucrose ester on the control of Varroa destructor mite infestation in Africanized honeybees. For the in vitro experiments, the product was tested in bees and mites at five concentrations obtained through dilution in water (T0: 100% distilled water; T1: 0.5%; T2: 1%; T3: 2%; T4: 5%; and T5: 10% sucrose ester).For the field studies, the experimental design was completely randomized, with four treatments and seven replicates, totaling 28 colonies, from which seven were the controls, seven were treated with 0.1% sucrose ester, seven with 0.2% sucrose ester, and seven hives with 0.5% sucrose ester diluted in water. In the in vitro study, the sucrose ester at 0.5% concentration caused mite and bee mortality. In the field tests, the product at 0.2% concentration reduced Varroa destructor infestation in Africanized honeybees and, therefore, may be used as a tool to control this pest. At 0.1, 0.2, and 0.5% concentrations, sucrose ester did not impair the establishment of open and capped brood areas, as well as stored food areas in the hive, suggesting it is not toxic to Africanized honeybees.

Flower mites decrease nectar availability in the rain-forest bromeliad Neoregelia johannis

Nectarivorous flower mites can reduce the volume of nectar available to pollinators. The effects of the flower mite Proctolaelaps sp. on nectar availability in flowers of a melittophilous bromeliad Neoregelia johannis (Bromeliaceae) was evaluated in a coastal rain forest in south-eastern Brazil. In a randomized block experiment utilizing 18 flower pairs, one per bromeliad ramet, pollinators (Bombus morio) and mites were excluded, and then nectar volume, sugar concentration and sugar mass were quantified over the anthesis period. Mites significantly reduced nectar volume early in the morning (6h00-8h00), but not later (10h00-12h00). Mites decreased total volume of nectar available up to 22%. Sugar concentration in nectar was higher earlier in the morning, and decreased between 10h00-12h00. The pronounced consumption of nectar by mites during the period of higher sugar concentration reduced the total amount of sugar available to pollinators by 31%. This is the first study showing that flower mites decrease nectar rewards in a melittophilous plant. Because nectar volume by itself incompletely describes nectar production rates and the effects of nectar removal by flower mites on the availability of sugar, our study highlights the inclusion of sugar content in future studies assessing the effects of thieves on nectar production rates. Copyright © 2010 Cambridge University Press.

Effect of fungicides on the mite fauna of prunus persica L. cultivars in Presidente Prudente, SP, Brazil

The objective of this study was to characterize the diversity of phytophagous and predatory mites on peach cultivars either with or without fungicide treatment, in the city of Presidente Prudente, State of São Paulo, Brazil. In order to evaluate the effect of fungicides, leaf samples were colleted at random from treated and untreated plants of the Tropical, Aurora 1 and Aurora 2 cultivars, from June 2004 to February 2006. From the results obtained it can be concluded that: plants with or without treatment showed high populations of predatory mites, indicating that the treatments were innocuous on the mite populations. A diversified composition of the mite community was observed. The Phytoseiidae family had the highest richness in numbers and species of mites. Euseius citrifolius and E. concordis were the most abundant species. Plants with or without treatment had high abundances of predatory mites, with a predominance of E. citrifolius.

Primeiro relato de atividade patogênica de Hirsutella thompsonii (Fischer) sobre o ácaro-da-erinose-da-lichia Aceria litchii (Keifer)

The litchi erineum mite, Aceria litchii (Keifer) is found in all producing regions attacking leaves and flowers of litchi plants. The mite attack young leaves and causes the erinea on leaf surface, which later become brown galls with velvety appearance. Severe attacks can cause leaf drop and destruction of branches end directly production affecting. In 2009 year it was registered a heavy infestation of the pest on litchi plants (Litchi chinensis Sonn.) in the municipality of Casa Branca, São Paulo, Brazil (2127'O; 4702'S; 679 m altitude). In June, many galls caused by mite infestation showed a mycelium of white coloration and many eriophyid dead. The fungus was identified as Hirsutella thompsonii (Fischer). The results suggest that galls may facilitate the fungus development and its permanence on the plants. Thus, the possibility of mite biological control with H. thompsonii should be investigated.

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.

Babesia bovis infection in cattle in the southwestern Brazilian Amazon

The present study provides the first epidemiological data on infection with Babesia bovis in cattle raised in the southwestern Brazilian Amazon. Blood clot samples were filtered through nylon cloth before being submitted to DNA extraction. PCR and nested-PCR were applied to assess the frequency of infection with B. bovis in calves with ages from 4 to 12 months bred in 4 microregions each in the states of Rondônia and Acre. After the DNA was extracted from the samples, the infection in cattle was investigated by amplification of the rap1 gene from B. bovis. The DNA amplification results revealed a frequency of infection with B. bovis of 95.1% (272/286) in the samples from Rondônia and 96.1% (195/203) in those from Acre. The high frequency of B. bovis infection in the animals with ages from 4 to 12 months indicates a situation of enzootic stability in the regions studied. The infection rates are comparable to those detected by immunodiagnostic techniques in other endemic regions of Brazil. © 2012 Elsevier GmbH.