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This study was carried out aiming to evaluate the repellent and acaricidal activity of major ingredient compounds from coconut oil including their methyl ester derivatives and catnip oil against nymphs and larvae of Amblyomma sculptum. Repellent candidates, coconut oil free fatty acids (coconut FFA mainly C12, C10 and C8 acid); lauric acid (C12 acid); capric acid (C10 acid); methyl laurate; methyl caprate and 10 % each of C12, C10 and C8 acid (1:1:1) in lavender oil formulation (CFA in lavender formula) and catnip oil (Nepeta cataria), were screened using a Petri dish bioassay to assess repellency. Catnip oil, methyl caprate, methyl laurate, and CFA in lavender formulation repelled ticks strongly (P

The present study aimed to evaluate the behaviour of larvae of Rhipicephalus microplus exposed to different stimuli. A Y-olfactometer was positioned vertically and R. microplus larvae were exposed to environmental air, CO2 alone, N,N-diethyl-3-methylbenzamide (DEET) alone, and CO2 combined with the repellents DEET and (E)-2-octenal. Tests were also conducted with the olfactometer positioned horizontally; in this case, however, only CO2 was tested. In all tests conducted with the Y-olfactometer positioned vertically, CO2 activated R. microplus larvae even in the presence of DEET and (E)-2-octenal, although activation was lower when these repellents were used. In the absence of CO2 , larval behaviour against DEET was similar to that of the larvae in the control group. In the tests performed with the olfactometer positioned horizontally, the larvae had no significant response to the presence of CO2 . The larvae were not attracted to or repelled by any compound tested in either the vertical or horizontal position of the olfactometer. The lack of horizontal displacement, attraction or repellence may have been a result of the ambush behaviour of this tick species. However, when larvae were exposed to stimuli and the olfactometer was positioned vertically, the interference of attractant and repellent stimuli in larval behaviour was assessed.

The present study aimed to develop a microemulsion formulation containing thymol and eugenol for field control of Rhipicephalus sanguineus sensu lato on dogs, as well to evaluate its safety and the physical characteristics of the formulation. The microemulsion using thymol and eugenol (5.0 + 5.0 mg/mL) had as vehicles water, propylene glycol, polysorbate 80 and canola oil. On the next day the preparation (formulation freshly prepared) and after 24 months, the size of the microemulsion droplets, polydispersion index (PdI), organoleptic properties (color, viscosity), and presence of precipitate in the microemulsion were evaluated. For the field assay, on day -1, 10 English Cocker Spaniel dogs were experimentally infested with 200 larvae, 100 nymphs and 30 adults of R. sanguineus s.l. On day 0, after tick counts, the animals were divided into two groups: treated with the freshly prepared microemulsion (10 mL/kg), and control, which received the vehicle (10 mL/kg). Tick counts on dogs were performed daily for three more days. Engorged females were recovered from the dogs and their biological and reproductive parameters were monitored. The dogs' clinical parameters (temperature, mucosa color, and general physical condition) were evaluated daily. In addition, blood samples were collected before infestation to verify hematological (packed cell volume) and biochemical parameters (total serum protein, albumin, globulins, creatinine, urea, alanine transaminase, aspartate aminotransferase, and alkaline phosphatase). Freshly prepared and 24-month aged microemulsions had droplets with mean sizes of 30.94 nm and 27.93 nm, and PdI values of 0.214 and 0.161, respectively. In addition, no difference in the organoleptic properties and no precipitation formation were observed, indicating physical stability. Treatment with the microemulsion resulted in reduction of larvae (p0.05) parasitizing the dogs on day 1 while the number of nymphs and adults was not reduced (P0.05). In the evaluation of the reproductive biology of engorged females, the larval hatchability (%) was compromised (p0.05), and the microemulsion had control rate of 85.5 %. The microemulsion and its vehicles did not change the clinical, hematological and biochemical parameters of the dogs. We concluded that the microemulsion was efficient against R. sanguineus s.l. by reducing the number of larvae and affecting the reproductive parameters of engorged females, safe for dogs, and stable (physical stability) during a two-year interval.

Benzaldehyde and 2-hexanone are allomones produced by beagle dogs that reduce infestation by Rhipicephalus sanguineus sensu lato on these animals and on susceptible dogs which artificially release these repellents. These observations were obtained in previous laboratory tests or artificial infestations of susceptible dogs under controlled conditions. Here we evaluated the efficacy of collars delivering these repellents for suppressing the loads of R. sanguineus s.l. on naturally infested mixed-breed dogs under field conditions. Thirty dogs naturally infested with R. sanguineus s.l. were separated into two groups with 15 dogs each. The dogs from the treatment group received the collars with slow-release formulations of the allomones, and the dogs from the control group received collars without such compounds. Collar effectiveness tests were carried out over 30 days. All ticks found were removed from each dog, identified, and counted every collection day. The density of the different life stages of R. sanguineus s.l. varied greatly between groups and across evaluation times. Adult ticks were the stage most abundant and prevalent on infested dogs. The counts for larvae, nymphs and adults were similar between the control and the treatment groups within each tick collection day. Conversely, when we considered the total number of ticks over 30 days and gathered all life stages collected in the same animal, the dogs from the control group had significantly higher loads (35.3 ± 4.27) of ticks than the dogs from the treatment group (21.8 ± 2.96) (P0.01). In addition, the efficacy of treatment with repellent collars was high for adult ticks (30.8%) but was extremely low for reducing larval or nymphal infestation (0 or 2.6%, respectively). The present study demonstrates, for the first time, the efficacy of these volatile compounds on naturally infested dogs under field conditions. The development of a technologically enhanced device for slow release of the allomones tested here can be an advantageous alternative for reducing R. sanguineus s.l. infestation on dogs in residential environments.

The present study evaluated over two years the seasonal dynamics of Rhipicephalus microplus in a Cerrado biome of midwestern Brazil (Goiânia, Goiás) and correlated the current climatic conditions (environmental temperature, insolation, rainfall, relative humitidy and saturation deficit of the atmosphere) of this site with 30 years ago. In addition, the data collected in the present research were compared with climatic conditions data and R. microplus population dynamics conducted by our group in other regions (Formiga, Minas Gerais and Jaboticabal, São Paulo) within the same biome. For the parasitic phase dynamics, R. microplus females (4.5-8.0 mm) counts were performed on tick natural infested cows kept in Panicum maximum paddocks. To verify the larvae dynamics on pasture the flannel dragg technique on natural infested pasture was performed, and the height of the grass was measured. Five peaks of engorged R. microplus females on animals and larvae infestations on pastures were observed. Being three and two peaks during the rainy and dry season of the year, respectively. Insolation (r = 0.8758; P = 0.00009; R² = 0.7670), rainfall (r = -0.8572; P = 0.0002; R² = 0.7348), maximum environmental temperature in summer (r = 0.9999; P 0.0087; R² = 0.9988) and the saturation deficit of the atmosphere in autumn (r = -0.9789; P = 0.0211; R² = 0.9582) influenced the seasonal dynamics of R. microplus larvae on pastures. While the forage height did not influence the number of larvae found on pasture (r = 0.1545; P = 0.7682; R² = 0.0239). The comparison between the climatic data of the last 30 years with the current climatological data (2012 to 2019) in the state of Goiás, and the comparison with R. microplus population dynamics from other two locations in the same biome indicated that the increase of the environmental temperature due to the global warming possibly is a determining factor which increase the number of one or up two tick annual peaks. In addition, this climatic variable was responsible for increase the population density of ticks on pastures and animals observed in July/winter of the present study, in comparison to other regions with the same biome, as discussed in this work.

Rhipicephalus sanguineus sensu lato and Amblyomma sculptum can parasite humans and domestic animals and are vectors of pathogens, including zoonoses. Repellents are an important tool of tick control. This study aimed to compare the efficacy of N,N-diethyl- 3-methylbenzamide (DEET), a standard repellent, versus β-citronellol in a Petri dish bioassay. A semicircle of filter paper (31.8cm2) was treated with 87μl of one of four concentrations (0.200, 0.100, 0.050 and 0.025mg/cm2) of β-citronellol, DEET or solvent (ethanol). A head-to-head test was developed treating one side with increasing concentrations of β-citronellol as above mentioned, against the highest concentration of DEET. Besides that a blank assay was performed. Three males and three females were placed in the middle of the plate and their location was evaluated 5, 10 and 30min after the test was initiated. As a result, the time had no significant effect on repellency response of the ticks exposed to both compounds and their concentrations. The repellency response raised according with the increase of concentration. Additionally, our findings indicate that the tick A. sculptum was more sensitive to the compounds tested and β-citronellol showed a higher efficacy than DEET. In addition, β-citronellol could be formulated to protect humans and other animals from R. sanguineus s. l. and A. sculptum infestation, as well as the diseases transmitted by these species.

Amblyomma sculptum is a tick affecting animal and human health across Argentina, Bolivia, Paraguay and Brazil. Donkeys, Equus asinus, are known to be resistant to A. sculptum, suggesting that they can produce non-host tick semiochemicals (allomones), as already demonstrated for some other vertebrate host/pest interactions, whereas horses, Equus caballus, are considered as susceptible hosts. In this study, we tested the hypothesis that donkeys produce natural repellents against A. sculptum, by collecting sebum from donkeys and horses, collecting the odour from sebum extracts, and identifying donkey-specific volatile compounds by gas chromatography (GC) and coupled GC-mass spectrometry (GC–MS). From the complex collected blends, five main compounds were identified in both species. Hexanal, heptanal and (E)-2-decenal were found predominantly in donkey extracts, whilst ethyl octanoate and ethyl decanoate were found predominantly in horse extracts. One compound, (E)-2-octenal, was detected exclusively in donkey extracts. In Y-tube olfactometer bioassays 36 different A. sculptum nymphs were tested for each extract, compound and concentration. The dry sebum extracts and the compounds identified in both species induced neither attraction nor repellency. Only (E)-2-octenal, the donkey-specific compound, displayed repellency, with more nymphs preferring the arm containing the solvent control when the compound was presented in the test arm across four concentrations tested (p

The present study was conducted to elucidate the neuronal pathways between peripheral olfactory and taste sensilla and the synganglion in an Ixodidae tick species. The tarsus of the front legs (olfactory nerves) and the fourth palpal segment (gustatory nerves) of unfed Amblyomma americanum males and females were excised. A neuronal tracer, dextran tetramethylrhodamine, was used for filling of the sensory neurons. The synganglion preparations were examined using a confocal microscope. Neuronal arborizations from the Haller’s organ were confined to the olfactory lobes and the first pedal ganglion. The estimated number of olfactory glomeruli ranged from 16 to 22 per olfactory lobe in the females. The number of glomeruli was not counted in males because they were densely packed. Sensory neurons associated with sensilla at the distal end of the palpal organ projected into the palpal ganglion in the synganglion through the palpal nerve. Gustatory sensory neurons associated with palpal sensilla projected into a commissure with several bulges, which are confined in the palpal ganglion. The findings of distinct projection patterns of sensory neurons associated with the Haller’s organ and palpal organ in the lone star tick from this study advanced our knowledge on mechanisms of sensory information processing in ticks. Resumo O presente estudo foi conduzido para elucidar a trajetória neuronal, entre as sensilas periféricas olfativas e gustativas e o singânglio, em uma espécie de carrapato Ixodidae. O tarso da primeira pata (nervos olfativos) e o quarto segmento palpal (nervos gustativos) de machos e fêmeas não alimentados de Amblyomma americanum foram excisados. Um traçador neuronal, dextran tetrametilrodamina, foi usado para preenchimento dos neurônios sensoriais. Os singânglios foram examinados através de microscopia confocal. Arborizações neuronais do órgão de Haller foram confinadas nos lobos olfativos e primeiro gânglio pedal. O número estimado de glomérulos olfativos variou de 16 a 22 por lobo olfativo nas fêmeas. Em machos, o número de glomérulos não foi contado, pois eles estavam densamente compactados. Os neurônios sensoriais associados com as sensilas, na porção distal do órgão palpal, projetaram-se no gânglio palpal do singânglio através do nervo palpal. Neurônios sensoriais gustativos associados com a sensila palpal projetaram-se numa comissura onde havia vários bulbos. Os resultados obtidos neste estudo de padrões de projeção distintos de neurônios sensoriais associados com os órgãos de Haller e palpal no carrapato A. americanum avançam nosso conhecimento sobre os mecanismos de processamento da informação sensorial em carrapatos.

We evaluated the effects of 3β-O-tigloylmelianol from Guarea kunthiana A. Juss (Meliaceae) on oogenesis, as a larvicide and on ecdysis of the larvae and the nymphs of the cattle tick Rhipicephalus (Boophilus) microplus (Canestrini) (Acari: Ixodidae). On the oogenesis' test, 48 engorged females were divided into three groups, evaluated at 24, 48 and 72 h post-treatment. Half of the females were treated with 0.01% 3β-O-tigloylmelianol diluted in distilled water and 5% dimethyl sulfoxide (DMSO), while the other half (controls) were exposed to distilled water and 5% DMSO. After treatment, the ovaries were weighed in order to measure the gonadosomatic index (GSI) and were also subjected to standard histological technical tests. On the larvicide and ecdysis' tests, 3β-O-tigloylmelianol was tested at concentrations of 0.01, 0.005, 0.0025 and 0.00125%. Compared with the controls, there was a reduction of GSI of approximately 50% on the treated group, which started at 48 h post treatment. Overall, the protolimonoid 3β-O-tigloylmelianol has caused a significant reduction in the number of oocytes. It has also caused alteration of the cytoplasmic and germinal vesicle diameters. Morphological changes, such as vacuolization, chorion irregularity which has modified the oocytes' morphology as well as alterations on the yolk's granules were also observed. The compound was not larvicide, however, interfered in the ecdysis of the larvae and the nymphs. This study shows that the protolimonoid 3β-O-tigloylmelianol from G. kunthiana acts on oogenesis and ecdysis of R. (B.) microplus, but not as larvicide, indicating that it acts on the endocrine system of the tick.

The aim of this study was to investigate whether the combination of thymol with eugenol has a synergistic effect on the immature life stages of Rhipicephalus sanguineus sensu lato (s.l.), to evaluate the cost-benefit ratio of using these compounds in combination, and to develop a formulation combining thymol with eugenol with activity on immature stages of R. sanguineus s.l. To evaluate synergism, thymol and eugenol, combined (ratio 1:1) or not, were tested at concentrations of 2.5, 5.0, 10.0, 15.0 and 20.0 mg/mL on unfed larvae and nymphs using a larval packet test, and 0.625, 1.25, 2.5, 5.0 and 10 mg/mL on engorged larvae and nymphs using an immersion test. A cost estimate was calculated to produce 1 L of a solution containing a concentration of thymol and eugenol, combined or not, that could cause a tick mortality rate greater than 95 %. Finally, a formulation was developed, consisting of a micellar dispersion containing polymers (MDP), with thymol + eugenol (1:1), at concentrations of 2.5, 5.0, 10.0, 15.0 and 20.0 mg/mL, and the activity was evaluated on unfed and engorged larvae and nymphs. For unfed larvae and nymphs, concentrations of 2.5 and 5.0 mg/mL and 2.5, 5.0 and 10.0 mg/mL, respectively, presented synergistic effects. In tests with engorged larvae and nymphs, respective concentrations of 0.625, 1.25 and 2.5 mg/mL and 2.5 and 5.0 mg/mL had synergistic effects. The estimated costs for producing a solution of 1 L with efficacy greater than 95 % was $5.97 using only thymol (15 mg/mL), $ 5.93 using only eugenol (15 mg/mL), and $ 3.97 using thymol + eugenol (1:1 - 5,0 mg/mL). In tests with MDP, the combination of thymol + eugenol resulted in a mortality rate higher than 95 % at concentration of 10 mg/mL for unfed and engorged larvae and nymphs. Thus, the combination of thymol + eugenol, depending on the concentration, has synergistic effects and this combination lowers the cost for the active ingredients thymol and eugenol. The combination of thymol + eugenol in MDP had acaricidal activity against immature life stages of R. sanguineus s.l.

Lipids extracted from the surface of the lone star tick, Amblyomma americanum, were analyzed by high resolution mass spectrometry. Prior to lipid extraction, the adult ticks were either unfed or fed on cattle, and the fed ticks were in groups either containing males and females together, or containing only males or females. Cholesteryl esters were found on the surfaces of fed females, and they may provide a more complete description of the composition of the mounting sex pheromone. Dihydrocholesteryl esters were detected on the surfaces of unfed males and females, suggesting a possible role in survival during host-seeking. Dehydrodeoxyecdysone, found on fed females, could be a component of the genital sex pheromone. The most abundant polar surface lipids detected were acylglycerides. High levels of sphingolipids and glycerophospholipids on males fed separately might be derived, in part, from sperm development. A high level of a 20:4 fatty acid, presumably arachidonic acid, was found on the surface of fed females, indicating that it may be a component of the genital sex pheromone. A high level of docosenamide was found on the surface of fed females. Wax esters were found on the surfaces of fed ticks but not on unfed ticks. These esters could be involved in elasticity of the cuticle of engorged females or in wax coating of eggs. N-acylethanolamines were found on the surfaces of male and female ticks fed together, and on male ticks fed separately, but were absent or at low levels on females fed separately and on unfed ticks. This pattern suggests a possible role as a metabolic coordination primer pheromone.

Beagles are less susceptible to Rhipicephalus sanguineus sensu lato tick due to the production of the allomones benzaldehyde and 2-hexanone. Our previous published work showed that these compounds can reduce tick burden on susceptible dogs. Here we tested the hypothesis that an increase in repellent dose and release rate could increase repellent efficacy and persistence. Slow-release formulations of these compounds, with higher doses and release rates, were tested on artificially-infested dogs. Ten dogs were randomly assigned to two groups with five dogs each. The treated group received collars with slow-release formulations of the compounds attached, while the control group received collars with clean formulations attached. Five environmental infestations were performed, with the number of ticks (at all stages) on the dogs being counted once a day for 40 days. No significant increase in repellent efficacy was observed with the higher doses and release rates, whereas a greater persistence in repellent activity was observed. Treatment with the formulations resulted in a two-to-three-fold reduction in the number of immature stage ticks for up to three weeks. However, the number of adults was similar in both groups. Loss of repellent activity after the third week of testing coincided with a marked change in the relative release rates for the two compounds. It is hypothesized that relative amounts, rather than absolute amounts, of repellent release from slow-release formulations are important for repellent activity. We also hypothesize that the avoidance of less-preferred hosts by ticks relies on olfactory-mediated perception of specific blends of volatile cues from less preferred hosts. Resumo Beagles são menos suscetíveis ao carrapato Rhipicephalus sanguineus sensu lato devido à produção de benzaldeído e 2-hexanona. Nosso trabalho anterior já publicado mostrou que esses compostos podem reduzir a carga de carrapatos em cães suscetíveis. Aqui testamos a hipótese de que um aumento na dose destes repelentes e na taxa de liberação poderia aumentar a eficácia e a persistência do efeito repelente. As formulações de liberação lenta destes compostos, com doses e taxas de liberação mais elevadas foram testadas em cães infestados artificialmente. Dez cães foram distribuídos aleatoriamente em dois grupos com cinco cães cada. O grupo tratado recebeu colares contendo formulações de liberação lenta dos compostos, enquanto o grupo controle recebeu colares com formulações limpas. Cinco infestações ambientais foram realizadas, com o número de carrapatos (em todas as fases) nos cães sendo contados, uma vez ao dia, por 40 dias. Não se observou aumento significativo na eficácia do repelente com doses e taxas de liberação mais elevadas e, enquanto observou-se maior persistência na atividade repelente. O tratamento com as formulações resultou em uma redução de duas a três vezes no número de carrapatos dos estágios imaturos, por até três semanas. No entanto, o número de adultos foi semelhante em ambos os grupos. A perda de atividade repelente após a terceira semana de teste coincidiu com uma mudança nas taxas de liberação relativa para os dois compostos. A hipótese é que as quantidades relativas, ao invés das quantidades absolutas de liberação lenta, são importantes para a atividade repelente. Então, a hipótese é de que a repelência apresentada por hospedeiros menos susceptíveis aos carrapatos depende da percepção pelos carrapatos de misturas específicas de voláteis liberados por estes hospedeiros.

The control of Rhipicephalus microplus (Ixodida: Ixodidae) is achieved using synthetic acaricides. However, resistant tick populations are widespread around the world. Plant essential oils can act as repellents, keeping ticks away from hosts and decreasing the selection pressure on synthetic acaricides. The aim of this study was to evaluate the in vitro repellent effect of Lippia alba essential oil on R. microplus larvae. Leaves from two L. alba genotypes maintained under the same agronomic and environmental conditions were collected. Essential oil was extracted by hydrodistillation and analysed by gas chromatography-mass spectrometry (GC-MS). The major monoterpenes detected in the chemical analysis were commercially acquired and tested. For the repellency test, a glass rod was vertically fixed to measure active climbing of approximately 30 R. microplus larvae aged 14-21 days in response to essential oils and monoterpenes. Repellency was evaluated at 1 h, 3 h and 5 h after treatment. Variation in repellent action was detected between the genotypes. The major monoterpenes identified in the essential oils (limonene and carvone) showed low repellent effects in comparison with intact essential oils. Thus, the present results showed that L. alba essential oil contains bioactive compounds with great repellent activity against ticks that varies according to the plant genotype.

Rhipicephalus microplus is considered the most economically important ectoparasite of cattle worldwide. It is known that zebuine breeds of cattle are less susceptible to tick infestation than taurine breeds. Contact chemoreceptors in the cheliceral pit sensilla of ticks respond selectively to phagostimulant compounds, however their role in blood feeding relative to host susceptibility to infestation remains to be fully understood. We addressed this topic by conducting taste electrophysiology experiments with cheliceral pit sensilla preparations of R. microplus females. Solutions of five known ixodid tick phagostimulants were tested at different concentrations: sodium (NaCl), and potassium chloride (KCl) (10(-3)-10(-1)M); glucose (10(-4)-10(-1)M); adenosine triphosphate (ATP) (10(-6)-10(-2)M); and reduced l-glutathione (GSH) (10(-6)-10(-2)M). Serum samples from six susceptible animals of the Girolando breed (5/8 Bos indicus×3/8 B. taurus) and six resistant Nelore bovines (pure B. indicus) were also tested. A dose-dependent response of gustatory neurons associated with the chelicerae sensillum to NaCl, glucose, GSH, and ATP were observed. Responses by the cheliceral inner digit pit sensilla of R. microplus to KCl and glucose were also observed and they are reported here for the first time. In addition to an electrophysiological response to known phagostimulants, chemoreceptors in the chelicera of R. microplus responded differently to serum from cattle susceptible and resistant to infestation. The cheliceral pit neurons were more responsive to serum of R. microplus resistant bovines with a higher mean spike frequency (53.5±2spikess(-1)) than to serum samples from susceptible cattle (40.3±2spikess(-1)). The implications of chemosensation during tick blood feeding are discussed.

In the present study, host-related and tick pheromone-related chemical compounds were tested by means of the tip-recording technique in order to obtain electrophysiological responses in olfactory sensilla of non-fed Amblyomma cajennense ticks. The following chemicals were tested on the multiporose sensilla DI.1, located anterior to Haller's organ, and the sensillum DII.1, in the anterior pit of this organ: isobutyric acid, butyric acid, valeric acid, trans-2-heptenal, heptanal, benzaldehyde, salicylaldehyde, nonanal, m-, o- and p-tolualdehyde, 2-furaldehyde, 3-pentanone, γ-valerolactone and 1-octen-3-ol (which are all vertebrate-associated volatiles); and 2,6-dichlorophenol (2,6-DCP), 2-nitrophenol, methyl salicylate and nonanoic acid (tick pheromone components). These were used at 10−3 M and 10−2 M on at least 10 ticks per substance, and the chemicals that were found to be active at these concentrations were then tested as a series from 10−6 M to 10−2 M, in decadic steps, on at least 15 ticks per substance. 2,6-DCP was active on both sensilla, with detection thresholds of 10−6 M on the DI.1 sensillum and 10−4 M on the DII.1 sensillum. The olfactory neurons of this sensillum also responded to nonanal at the highest concentration used (10−2 M), while those of DII.1 responded not only to 2.6 DCP but also to 2-nitrophenol (to the same extent as to 2,6-DCP) and to 1-octen-3-ol. These results confirm the importance of 2,6-DCP in the chemical ecology of A. cajennense and indicate other compounds that may interfere with the behavior of this tick and which should be investigated.

Domestic dog breeds are hosts for the brown dog tick, Rhipicephalus sanguineus sensu lato, but infestation levels vary among breeds. Beagles are less susceptible to tick infestations than English cocker spaniels due to enhanced production of 2-hexanone and benzaldehyde that act as volatile tick repellents. We report the use of prototype slow-release formulations of these compounds to reduce the burden of R. sanguineus s. l. on English cocker spaniel dogs. Twelve dogs were randomly assigned to two groups with six dogs each. The treated group received collars with slow-release formulations of the compounds attached, while the control group received collars with clean formulations attached. Five environmental infestations were performed, with the number of ticks (at all stages) on the dogs being counted twice a day for 45 days. The counts on the number of tick stages found per dog were individually fitted to linear mixed effects models with repeated measures and normal distribution for errors. The mean tick infestation in the treated group was significantly lower than in the control group. For larvae and nymphs, a decrease in tick infestation was observed at the fifth count, and for adults, lower average counts were observed in all counts. The compounds did not interfere with the distribution of the ticks on the body of the dogs, as a similar percentage of ticks was found on the anterior half of the dogs (54.5% for the control group and 56.2% for the treated group). The biological and reproductive parameters of the ticks were not affected by the repellents. This study highlights for the first time the potential use of a novel allomone (repellent)-based formulation for reduction of tick infestation on susceptible dogs.

The evolution of resistance of Rhipicephalus (Boophilus) microplus to synthetic acaricides has given rise to the need for new scientific investigations on alternative ways to control this tick. In this regard, various studies on plants have been developed in an attempt to find extracts with acaricidal properties. Evaluations on plant extracts for controlling R. (B.) microplus have grown intensely over the last decade. There are many advantages from using plant extracts: for example, they can be used in organic cattle farming or even replace synthetic acaricides and they are associated with lower environmental and food contamination, slower development of resistance and lower toxicity to animals and humans. In vitro studies on plant extracts have shown promising results, but most of these extracts have not been tested on animals to validate their use. Difficulties in preparing proper formulations, differences in the chemical composition of plants of the same species due to extrinsic and intrinsic factors and sparse information on active acaricide compounds are hindrances that need to be addressed in order to enable progress within this scientific field. A evolução da resistência do Rhipicephalus (Boophilus) microplus aos acaricidas sintéticos tem impulsionado novas investigações científicas sobre métodos alternativos para controlar este carrapato. Considerando isso, vários estudos com plantas têm sido desenvolvidos numa tentativa de encontrar extratos com propriedades acaricidas. Avalições de extratos de plantas para o controle de R. (B.) microplus tem sido intensificadas nesta última década. Existem muitas vantagens com o uso de extratos de plantas no controle deste carrapato, como: eles podem ser utilizados na produção orgânica de bovinos, ou mesmo substituir os acaricidas sintéticos, além do mais, estão associados com baixa contaminação ambiental e dos alimentos, desenvolvimento mais lento de resistência e baixa toxicidade para animais e seres humanos. Estudos in vitro de extratos de plantas têm apresentado resultados promissores, mas a maioria destes extratos não têm sido testada em animais para validar estes resultados. Dificuldades para preparar formulações apropriadas, diferenças na composição química de plantas de uma mesma espécie devido a fatores intrínsecos e extrínsecos e informações esparsas.sobre os princípios ativos são entraves que precisam ser solucionados visando o desenvolvimento deste campo de pesquisa.

The use of a concentrate emulsion of Melia azedarach green fruits and a suspension of the fungus Beauveria bassiana was evaluated in the control of Rhipicephalus microplus on artificially infested cattle. The evaluation was conducted following the protocol established by the Brazilian Agriculture Ministry. Five groups of 4 or 5 animals were allocated to one of the following treatments: emulsion concentrate of M. azedarach at 0.25% (T AZED 0.25%), emulsion concentrate of M. azedarach at 0.5% (T AZED 0.5%), B. bassiana at 2.4 × 10 8 conidia (T BASS), association of the concentrate of M. azedarach at 0.25% with B. bassiana at 2.4 × 10 8 conidia (T AZED 0.25% + BASS), and control (untreated). The association of the two compounds provided better results than any one isolated treatment, indicating compatibility or perhaps a synergy between M. azedarach and B. bassiana . This treatment resulted in fewer engorged females (129 ± 70) than in the control group (233 ± 82), showing high performance against all developmental stages of the tick. Results revealed an apparent synergistic effect of M. azedarach and B. bassiana in the control of R. microplus that should be further investigated.

Neosporosis is considered to be one of the main causes of abortions in bovines. In this study we evaluated the congenital transmission and occurrence of abortions by Neospora caninum in an embryo transfer center in Neropolis, Goiás. Serological samples from 101 recipients, 61 donors, and 90 calves were analyzed. Among these animals, 32.67% of the recipients, 22.22% of the donors, and 6.66% of the calves were positive for N. caninum. The rate of vertical transmission was 24%. There was a statistically significant difference between antibody titer of the recipient cows and the serological status of the calves. There was no statistically significant relationship between N. caninum serological status and reproductive rates such as estrum repetition and embryo production. Eight abortions occurred, six (75%) in positive females and two (25%) in negative females. These results indicate that neosporosis may be an important cause of failing reproduction and that in embryo transfer centers recipients should be previously examined, and those that are seronegative for Neospora should be chosen to reduce abortion and the birth of seropositive calves.

This study was carried out with the objective of evaluating the efficacy of a 2,6-dichlorophenol (2,6-DCP) lure to control Dermacentor nitens (Acari: Ixodidae). Slow-release formulations of the pheromone formulated with and without cypermethrin were prepared. Olfactometer bioassays were used to define the best dose of the pheromone and to evaluate the effect of cypermethrin with 2,6-DCP attractiveness. Sexually active males were released 15 cm from 2 cmx1 cm pieces of polypropylene treated with different odors: 2,6-DCP in a liposphere system (1.5, 30 and 300 microg--without cypermethrin and 30 microg--with cypermethrin) and 2,6-DCP in hexane (30 microg). The tests were conducted 7 and 15 days after the preparation of the odor sources. The percentages of males that oriented, or showed directional movement toward the stimulus, and their tracks were recorded. Lures (10 cmx2 cm pieces of polypropylene) treated with the best dose of the liposphere formulation (30 microg) were prepared. The lures were evaluated in horses that had been artificially infested with D. nitens. The horses' ears were infested with 3000 D. nitens larvae per ear, once weekly for 4 weeks. The animals were divided into three groups: control, 2,6-DCP and 2,6-DCP+cypermethrin. On day 0, the lures of their respective treatments were attached to the horses' napes. From days 6 to 20 after attachment, female ticks of 4 mm or over in length were counted on the ears, every 2 or 3 days. Olfactometer analysis showed higher orientation to 30 microg dose and more prolonged release of the pheromone in the liposphere formulation than in hexane; cypermethrin did not interfere with the attractive effect of the pheromone. The lures were efficient in the first 10 days after attachment, when the mean number of females was higher in the control group (24.9) than in 2,6-DCP and cypermethrin (5.4), and 2,6-DCP (9.2) groups. After that period the number of females was similar in the control and treated groups. These results indicate that 2,6-DCP lures used in a liposphere formulation can control D. nitens for at least 10 days by preventing its copulation. However, further evaluation of longer-term pheromone release under natural conditions is needed in order to validate this kind of control. In addition, the use of extra lures on the horse's tail may help to control populations on the hindquarters.

Studies have shown that the brown dog tick, Rhipicephalus sanguineus sensu lato, when fed on the beagle breed of dog, Canis lupus familiaris, development negatively affected in comparison with tick development after feeding on the English cocker spaniel breed. Thus leading to the suggestion that beagle dogs are be tick-resistant dogs. Behavioural studies have demonstrated that more ticks are attracted by extracts from cocker spaniels than from beagles and that the odour of beagles is a repellent. To test the hypothesis that resistant hosts produce repellent compounds, we undertook comparative chemical analysis on beagle odour and cocker spaniel extracts using coupled high-resolution gas chromatography-mass spectrometry (GC-MS) and also used Petri-dish and olfactometer behavioural assays to assess the response of ticks to identified non-host compounds. The beagle odour extracts contained almost three times as many chemical compounds as cocker spaniel samples. Several non-host compounds were identified, i.e. 2-hexanone, benzaldehyde, nonane, decane and undecane. In Petri-dish assays, 2-hexanone was repellent at 30 min at concentrations of 0.200 and 0.050 mg cm(-2), whilst at 10 min, the 0.100 mg cm(-2) concentration was repellent. Benzaldehyde repelled ticks at 30 min (0.200 mg cm(-2)) and at 5 min (0.050 mg cm(-2)). Undecane was repellent for R. sanguineus s.l. ticks for the first 5 min at the highest concentration tested. Nonane and decane did not show any significant repellency at any concentration or time evaluated. When 2-hexanone and benzaldehyde were combined, an increase in the repellency rate was observed, with activity comparable or better than N,N-diethyl-3-methylbenzamide (DEET). In olfactometer bioassays, a 1:1 mixture of 2-hexanone:benzaldehyde and DEET were repellent for R. sanguineus s.l. adults at the concentration of 0.200 mg cm(-2). This study identified non-host semiochemicals that mediate avoidance of the beagle dog breed by R. sanguineus s.l. This finding may enable development of new approaches to control this tick.

The population dynamics of all stages of the Cayenne tick, Amblyomma cajennense (Fabricius) (Acari: Ixodidae) on horses was evaluated over a period of 2 years in the district of Pedro Leopoldo, State of Minas Gerais, Brazil. Every 14 days, the left side of 20 horses was brushed for collection of immature stages; counts of adults were also undertaken. Infestation by larvae was detected from April to August, whereas nymphs were observed from June to October. Infestation by adults was detected throughout the year, and the highest population density occurred from September to March. The number of males was always higher than the number of females, but with considerable reduction in the male : female ratio between April and July. It was observed that 25% of the horses carried 41% of the infesting ticks, and 20% carried only 10% of the ticks during the entire period of the study.

The efficacy of ripe fruit extracts of Melia azedarach L. (Rutales: Meliaceae) was evaluated against the tick, Boophilus microplus (Canestrini) (Acari: Ixodidae). Ripe fruits of M. azedarach dried and powdered were extracted by Soxhlet apparatus successively using hexane, CHCl3 and 96% aqueous ethanol. Larvae and engorged females were immersed in decreasing concentrations from 0.25% to 0.015% of each extract. The mortality of larvae was evaluated 24, 72 and 168 h after treatment. The effectiveness of treatment against engorged females was assessed by measuring egg production. All tested extracts caused mortality of B. microplus larvae, with higher mortality rates observed in CHCl3 (100%) and hexanic extract (98%) than in ethanolic extract (50%) 168 h after treatment. The mortality was dependent on concentration and on time after treatment. Similarly hexanic and CHCl3 extracts showed higher effectiveness (varying from 14% to 100%) against B. microplus engorged females than ethanolic extract (varying from 0% to 46%). Melia azedarach extracts did not kill the adult females, but inhibited partially or totally egg production and embryogenesis. These results show that the less polar the extract of M. azedarach ripe fruits the more its effectiveness against larvae and engorged females of B. microplus. This plant may therefore be useful in the control of resistant B. microplus populations.

The objective of this study was to investigate some aspects of horse resistance to natural infestations of Anocentor nitens and Amblyomma cajennense over a 2-year period. Free-living stages of A. nitens were used to evaluate the influence of season of the year on horse resistance. Every 2 weeks, 10 selected engorged females, weighing 150–329 mg, were removed from naturally infested horses and were individually placed into glass tubes closed with a cotton stopper, and maintained in an incubator. The biological parameters evaluated varied according to the season of the year, i.e., reproductive efficiency, maximum larval survival (MLS) and egg-hatching rates were significantly lower in spring/summer than in fall/winter. At 14-day intervals, females of A. nitens and A. cajennense were counted on each side of the body of five mares. A comparison between the animal with the lowest infestation and the animal with the highest infestation showed greater differences in animals infested with A. nitens than those infested with A. cajennense. Ten animals were found to carry 64.9% of the total parasite burden of A. cajennense and 88.6% of the highest infestations of A. nitens females. A seasonal variation on intensity of horse resistance to A. nitens was observed during the study. However, this was not observed for A. cajennense. Cross-resistance was observed in animals with the highest total number of A. cajennense and the highest number of A. cajennense adults (r=0.817), larvae (r=0.756), and A. nitens females (r=0.799). These results led to the conclusion that changes in horse resistance to A. nitens occur seasonally. As far as the total population of A. cajennense and the female stage of A. nitens are concerned, horses develop cross-resistance.

A study on the population behavior of Amblyomma cajennense larvae, nymphs and adult free-living stages was carried out in Pedro Leopoldo County, Minas Gerais State, Brazil, from July 1995 to July 1997. Larvae were collected from pasture from April to October using the technique of drag sampling with flannelette. The maximal larval population was observed during autumn (May) representing, respectively, 48 and 82% of the total numbers recovered during the first and the second years of the experiment. Nymphs and adults were captured throughout the year by the use of traps with carbon dioxide as chemical attractant. The highest population of nymphs occurred during the winter (July), representing 39.5% of the total collected specimens. Adults had the highest population density on pasture during spring and summer, i.e., from August to May, with peaks in January and February, representing 46 and 38.3% of the total collected specimens during the first and the second year of study, respectively.

The aim of this study was to investigate whether there are sex pheromones in the tropical horse tick, Anocentor nitens (Neumann). When virgin males and females, fed on rabbits for 3-7 d, were tested reciprocally, there was a direct correlation between attractiveness and the period of feeding; attraction was greater (and with the fastest responses) in males and females fed for at least 6 d (sexually active males and confirmed attractive females). The capacity of sexually active males to orient topward confirmed attractive females decreased as the emission distance increased; the maximum distance that evoked a response by males was 6 cm. Confirmed attractive females lost their attractiveness approximately 2 h after being separated from the host, or after being killed by ethanol injection, although some females continued to attract sexually active males up to 4 h after death. An extract was produced by immersing confirmed attractive females in hexane and submitting them to ultrasound. Sexually active males were more attracted to and spent more time on glass bead dummies treated with this extract when compared with those treated with the hexane alone. Our results indicate the presence of at least 2 sex pheromones (attractant and mounting) in attractive females of A. nitens.

Phagostimulants are chemical compounds that stimulate feeding. Some tick species are known to have pores in their inner cheliceral digits, called pit sensillum (ps), which are involved in taste perception. This study investigated the existence of ps in chelicerae of Rhipicephalus sanguineus ticks by scanning electron microscopy (SEM) and their involvement in the perception of phagostimulatory substances, such as the salts potassium chloride and sodium chloride (KCl and NaCl), sugars (glucose, sucrose and fructose), purines (guanine and hypoxanthine), the nucleotide adenosine triphosphate (ATP) and the tripeptide reduced glutathione (GSH), as well as their combinations at different concentrations. By means of scanning electron microscopy, a ps was observed at the expected location. Using a single-sensillum recording technique, strong activity by R. sanguineus cheliceral sensilla in response to glucose, ATP, GSH and high concentrations of salts was recorded. The responses to ATP and to KCl at 1 M were multicellular, while the responses to the other stimulant compounds were monocellular. Glucose and GSH stimulated different neurons. The taste response of R. sanguineus chelicerae seemed to be selective, given that substances that were not expected to participate in this tick's biology were not perceived. (C) 2013 Elsevier B.V. All rights reserved.

Made available in DSpace on 2013-09-27T14:53:05Z (GMT). No. of bitstreams: 1 WOS000300129500002.pdf: 340641 bytes, checksum: e37008783ca84a0d8598cb946bac8d78 (MD5) Previous issue date: 2012-02-01 Made available in DSpace on 2013-09-30T19:24:37Z (GMT). No. of bitstreams: 1 WOS000300129500002.pdf: 340641 bytes, checksum: e37008783ca84a0d8598cb946bac8d78 (MD5) Previous issue date: 2012-02-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T15:34:42Z No. of bitstreams: 1 WOS000300129500002.pdf: 340641 bytes, checksum: e37008783ca84a0d8598cb946bac8d78 (MD5) Made available in DSpace on 2014-05-20T15:34:42Z (GMT). No. of bitstreams: 1 WOS000300129500002.pdf: 340641 bytes, checksum: e37008783ca84a0d8598cb946bac8d78 (MD5) Previous issue date: 2012-02-01 Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) The control of parasitic diseases in small ruminants is mainly done with the use of synthetic anthelmintics. However, incorrect and indiscriminate use of these products has caused the emergence of parasite resistance. Plants with anthelmintic activity are used in folk veterinary medicine, but it is necessary to investigate and scientifically validate low-cost phytotherapeutic alternatives for future use to control gastrointestinal nematodes in small ruminants by family farmers. Thus, the aim of this study was to evaluate the in vitro anthelmintic effect of plant extracts from Melia azedarach and Trichilia claussenii by the egg hatch test (EHT) and larval development test (LDT) against sheep gastrointestinal nematodes. The hexane extract of M. azedarach fruits was extracted through cold percolation and the methanol extract of T. claussenii leaves was obtained by extraction at room temperature in solvents in order of increasing polarity. The efficacy results were analyzed using the Probit program of SAS. The M. azedarach extract showed a LC50 of 572.2 mu g/mL and LC99 of 1137.8 mu g/mL in the EHT, and LC50 of 0.7 mu g/mL and LC99 of 60.81 mu g/mL in the LDT. In turn, the T. claussenii extract presented a LC50 of 263.8 mu g/mL and LC99 of 522.5 mu g/mL in the EHTand LC50 of 1.11 mu g/mL and LC99 of 26.4 mu g/mL in the LDT. Comparing the extracts of the species from the Meliaceae family, T. claussenii showed greater anti-parasite potential in vitro than M. azedarach. However, studies on the isolated compounds, toxicity and administration forms to animals are also needed to validate low-cost alternative herbal remedies for use to control gastrointestinal nematodes by family farmers. (C) 2011 Elsevier B.V. All rights reserved. Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) Pecuaria Sudeste, BR-13560970 São Paulo, Brazil Inst Invest Agr Mocambique, Direcao Ciencias Anim, Maputo, Mozambique Ctr Univ Cent Paulista, São Paulo, Brazil Universidade Federal de Goiás (UFG), Goiania, Go, Brazil Univ Estadual Paulista, São Paulo, Brazil Univ Estadual Paulista, São Paulo, Brazil EMBRAPA: MP2 02.07.06.009.00.00

In order to clarify the role of 2,6-dichlorophenol (2,6-DCP) in the courtship of Amblyomma cajennense, sexually mature males that had previously fed on rabbits were tested in bioassays. The males were released onto dummies treated with whole female extract or synthetic 2,6-DCP at a concentration of two female equivalents, or with hexane (control), and their responses were observed. In the presence of both the extract and 2,6-DCP, excitation was observed among the males, expressed in the form of touching and probing the dummy, and mounting occurred readily. The percentages of mounting (73%) and tipping over (60%) were equal in the two treatments and higher than in the control group (27 and 20%, respectively). Relatively short durations of mounting were recorded, and these were statistically similar in all treatments. Almost all instances of mounting resulted in tipping-over behavior. A few isolated cases of males that went directly to ventral positioning without mounting were observed. It was confirmed that 2,6-DCP alone is capable of mediation of mounting behavior in A. cajennense.

This study was developed to evaluate the repellent activity of N,N-diethyl-3-methylbenzamide (DEET) against Amblyomma cajennense nymphs. Two repellent bioassays were compared and the effective concentration and repellent time were calculated. The fingertip test was accomplished to evaluate in vivo four concentrations of the compound (0.200; 0.100; 0.050 and 0.025 mg.cm-2) and the filter-paper bioassay to evaluate in vitro the two highest concentrations. The compound provided repellence higher than 90% in all concentrations and at least 95% repellency in the highest concentration over 5 hours. The effective concentration against 50% of tested nymphs (EC50) was 0.006 mg.cm-2 and the EC99 was 0.036 mg.cm-2. Those concentrations were lower than the ones obtained against other tick species, denoting the effectiveness of DEET against A. cajennense. The repellency time against 50% of the ticks (RT50) was 4.8 hours and the RT90 was 2.7 hours. Both bioassays were adequate to evaluate A. cajennense repellency and provided similar results; however the in vivo test is more appropriate to estimate the effective concentration and repellency time. Este estudo foi conduzido com o objetivo de avaliar a atividade repelente do N,N-diethyl-3-methylbenzamide (DEET) sobre ninfas de Amblyomma cajennense. Dois bioensaios para a avaliação de repelência foram comparados e cálculos da concentração eficaz e do tempo de repelência foram realizados. Foram empregados o bioensaio da ponta do dedo, para avaliação in vivo de quatro concentações do químico (0,200; 0,100; 0,050 e 0,025 mg.cm-2) e o bioensaio do papel filtro, para a avaliação in vitro das duas concentrações mais altas. O composto conferiu mais de 90% de repelência em todas as concentrações utilizadas e 95% de repelência por mais de cinco horas na maior concentração. A concentração do composto efetiva contra 50% das ninfas testadas (CE50) foi de 0,006 mg.cm-2 e a CE99 foi de 0,036 mg.cm-2. Estas concentrações são mais baixas do que as observadas em outras espécies de carrapatos, denotando a efetividade do princípio contra A. cajennense. O tempo de repelência de 50% dos carrapatos (TR50) foi de 4,8 horas e o TR90 de 2,7 horas. Os dois bioensaios avaliados permitiram a observação de percentuais de repelência igualmente altos e se mostraram adequados para tal avaliação, sendo que o teste in vivo é mais indicado para cálculo da concentração eficaz e da duração da repelência.

Repellence responses of Amblyomma cajennense nymphs to callicarpenal, intermedeol, Hyptis suaveolens essential oil, extract of Melia azedarach, Cymbopogon nardus, Spiranthera odoratissima, Chenopodium ambrosioides, Ageratum conyzoides, Mentha pulegium, Ruta graveolens, and Memora nodosa were studied. Among these the extract of C. nardus stood out because of the long-lasting repellence, maintaining, in the highest concentration, 35 h of protection against 90% of the nymphs. The essential oil of H. suaveolens and the extracts of C. ambrosioides and A. conyzoides showed good repellence index (66%) when applied in high concentrations. However, greater protection could be obtained at higher concentrations but with a shorter repellence time. Callicarpenal, intermedeol, extract of M. Pulegium, and M. nodosa leaves showed moderate repellence in high concentrations. Extracts from M. azedarach, R. graveolens, S. odoratissima, and M. nodosa roots showed little or no repellent effect. These results show that some plant extracts may represent a promising alternative in the control of infestations by A. cajennense.

Extratos hexânicos obtidos dos frutos de Melia azedarach foram testados sobre fêmeas ingurgitadas e larvas de Rhipicephalus (Boophilus) microplus. Foram produzidos quatro extratos: um de frutos verdes e três com frutos maduros, sendo um com os frutos imediatamente processados, outro com frutos armazenados por cinco meses e um testado após armazenamento do extrato por 2 (dois) anos a 4ºC. Os testes foram feitos em triplicata, em diluições de 0,25% a 0,0156%. A eficácia dos extratos foi avaliada através da comparação da Reprodução Estimada dos lotes "controle e tratado" para fêmeas, da mortalidade para as larvas, e do cálculo das DL50 e da potência relativa para larvas e fêmeas. O extrato de frutos verdes apresentou uma eficácia variando de 3,6% a 100% para fêmeas, e o de frutos maduros variou de 5,2% a 99,7%. Os dois extratos causaram uma mortalidade de 100% das larvas nas maiores concentrações. O extrato de frutos armazenados apresentou eficácia de 14% a 83% sobre fêmeas, enquanto o extrato armazenado em geladeira apresentou eficácia de 8,4% a 100% e ambos não apresentaram efeito larvicida. Os resultados obtidos demonstram uma superioridade do extrato verde, com uma menor DL50 e potência 1,497 vezes superior ao extrato de frutos maduros. Hexanic extracts gotten of the fruits of cinamomo (Melia azedarach) were tested on engourged females and larvae of Rhipicephalus (Boophilus) microplus. Four extracts were produced: one of unripe fruits and three of ripe fruits, being one with the fruits immediately processed, another one with fruits stored for five months and one tested after storage of the extract for 2 (two) years at 4ºC. The tests were made in triplicate, in 0.25%- 0.0156% dilutions. The effectiveness of extracts was evaluated through the comparison of the Reproduction Estimate of the groups "controlled and treated" for females, of mortality for the larvae, and of the calculation of the DL50 and the relative power for larvae and females. The extract of unripe fruits presented the effectiveness varying the 3.6% - 100% for females, and of ripe fruits it varied of 5.2% - 99.7%. The two extracts caused a mortality of 100% of the larvae in the highest concentrations. The extract of stored fruits presented effectiveness of 14% - 83% on females, while the extract stored in refrigerator presented effectiveness of 8.4% 100% and both did not present larvicidal effect. The results demonstrate a superiority of the unripe extract, with a minor DL50 and superior power 1.497 times to the extract of ripe fruits.

Canine babesiosis is an infectious disease caused by either Babesia gibsoni or Babesia canis protozoans. The latter is also classified under three different phylogenetic groups, referred to as subspecies B. canis canis, B. canis vogeli and B. canis rossi. The objective of the present study was to validate and standardize a PCR assay to discriminate the organisms at the subspecies level. First, the reference sequences of the 18S rRNA, 5.8S rRNA and 28S rRNA genes, including the internal transcribed spacer 1 (ITS1) and 2 (ITS2) of the most common species and subspecies of the genus Babesia were retrieved from the GenBank database. Subspecies-specific primers (BAB3, BAB4 and BAB5) and one genus-specific primer were designed from the alignment of the sequences. The PCR assays were evaluated in three different combinations of primer pairs in order to assure complete specificity for each reaction. The results of the tests had demonstrated effectiveness of the novel primer pairs BAB1/BAB3, BAB1/BAB4 and BAB1/BAB5 for the amplification of the subspecies-specific target fragments of 746 bp (B. c. canis), 546 bp (B. c. vogeli) and 342 bp (B. c. rossi) by PCR. The original enzymatic amplification assays with novel primers reported in this paper were confirmed to be a reliable tool for the specific discrimination among B. canis subspecies by single-step PCR assays.

Populational flux of the adult phase of Stomoxys calcitrans was observed in the municipal district of Pedro Leopoldo, Minas Gerais, Brazil. Three biocenoses were selected for the study: stable agrobiocenosis, pastural agrobiocenosis and eubiocenosis. The occurrence and the populational flux of the insects, using the Magoon trap for their capture, were established. For each trap located in different biocenoses, a crossbred calf (Bos taurus × Bos indicus) approximately 6-month-old was used as “live bait,” exposed weekly for 48 h in the traps. Of the three agrobiocenoses studied, the stable agrobiocenosis contributed the greatest number of specimens of. S. calcitrans captured, corresponding to 96.9% of the total flies of this species collected. S. calcitrans shows seasonal behavior for approximately 6 months (spring and summer being the rainiest months of the year). The population peaked during the months of November and December. During the months of July and August, there was no capture of flies.

Anocentor nitens is a widespread ectoparasite of horses in Brazil. A study of the seasonal dynamics of the parasitic phase of this tick was made by regular census of engorging females (≥4 mm) on free-ranging horses, fortnightly from July 1995 to July 1997, at Pedro Leopoldo, Minas Gerais. Twenty horses were divided into four groups of five animals per group, each having a similar mix of resistant and less resistant animals, based on initial infestation. Every 2 weeks, one of the groups, in sequence, was treated with an acaricide to prevent excessive infestations, and female ticks were censused 38 days later. The acaricide dips did not interfere with the seasonal dynamics of the tick population, which produced three major peaks of activity over a 12-month period. The first two peaks were in the hot, wet season and the third in the cold dry season. Tick numbers were similar for the two 12-month periods, decreasing from the spring and summer to the autumn and winter. Infestation rates were not equal among different horses. Twenty percent of the horses were responsible for 48% of the total parasite load, while 30% accounted for only 2.5% of the total parasite load. Principal predilection sites were; the ear (61%), mane (21%), and muzzle and face (6%). Measures for the strategic control of A. nitens ticks are discussed.

An understanding of seasonal dynamics in the free-living phase of the tropical horse tick, Anocentor nitens, is vital for effective control measures. This study examines seasonal changes in the oviposition and incubation periods, eclosion rates, reproductive success, and larval survival in the free-living phase during the period June 1995–July 1997. Ten engorged female ticks, placed individually in wire-gauze tubes, were exposed in a field of Brachiaria decumbens fortnightly each month, and monitored to measure the duration of the various reproductive parameters, and success. Larval abundance was estimated indirectly, through the percentage of females with viable progeny, and directly by using a flagging method. Duration of the free-living cycle ranged from 12.3 weeks (October and December) to 23.5 weeks (March). The cycle was prolonged in cold and dry months. Larval survival was longer from February to July, and reduced from August to February. Oviposition was recorded in all months, and the egg production index ranged from 26.7 to 47.1%. The eclosion rate ranged from 0.1 to 76.6% in different months. Monthly estimates of larval abundance in the pasture were similar for the two methods used, being higher in months with higher temperatures, relative humidity and rainfall.

We report on an evaluation of the systematic position of the tick Anocentor nitens (Neumann), examining particularly whether Anocentor should be regarded as a subgenus of Dermacentor or as a separate genus. Twelve species of Ixodidae were analyzed phenetically by using 24 characters of adult ticks. A phenogram indicated 3 clusters, with Anocentor more closely related to Rhipicephalus than to Dermacentor. The results of this study endorse the validity of the monotypic genus Anocentor.

This study aimed to analyze ovary histology and quantify total protein in the hemolymph of Rhipicephalus (Boophilus)microplus females treated with hexane extracts from green fruits of Melia azedarach. Eight engorged females were immersed in the extract at 0.25% concentration, and eight in water containing 5% acetone (control). The females were dissected 72 hours after treatment, and the ovaries were weighed and subjected to standard histological techniques. The total protein concentration was measured in the hemolymph of 200 females, of which 100 were treated as described above and 100 served as a control. In the treated group, ovary weight reduction and predominance of immature oocytes were observed. In addition, there were decreases in the diameters of the cytoplasm and germ vesicle of the oocytes in the treated group, compared with the controls. The protein concentration in the hemolymph was higher in the treated group than in the controls. The morphological changes observed in the treated ovaries included: presence of vacuolization; alteration of oocyte morphology, which changed from rounded to elongated; deformation of the chorion; and disorganization of the yolk granules. These results demonstrate the action ofM. azedarach fruit extracts on R.(B.) microplus oogenesis. Este estudo foi desenvolvido, visando analisar a histologia do ovário e quantificar as proteínas totais na hemolinfa de fêmeas deRhipicephalus (Boophilus)microplus tratadas com extrato hexânico de frutos verdes deMelia azedarach. Oito fêmeas ingurgitadas foram tratadas por imersão com o extrato na concentração de 0,25%, e oito com água contendo 5% de acetona (controle). As fêmeas foram dissecadas 72 horas após o tratamento e os ovários foram pesados e submetidos a técnicas histológicas padrões. A concentração total de proteína foi mensurada na hemolinfa de 200 fêmeas, sendo 100 tratadas como descrito anteriormente e 100 como controle. Foi observada redução do peso dos ovários, predomínio de ovócitos imaturos e houve diminuição nos diâmetros do citoplasma e da vesícula germinal dos ovócitos do grupo tratado em comparação ao controle. A concentração de proteína na hemolinfa foi mais alta no grupo tratado que no controle. As alterações morfológicas observadas nos ovários tratados foram a presença de vacuolizações, alteração da morfologia dos ovócitos que mudaram de redondos para alongados, deformação do córion e desorganização dos grânulos de vitelo. Estes resultados demonstram a ação do extrato de M. azedarach na ovogênese de R.(B.) microplus.

It is already known that the beagle breed of domestic dogs produces semiochemicals capable of repelling the brown dog tick, Rhipicephalus sanguineus sensu lato (s.l.). With a view to discovering new non-host semiochemicals as tick repellents, we compared the semiochemicals produced by a putative tick-resistant breed of dog, miniature pinscher, with known tick-resistant (beagle) and tick-susceptible (English cocker spaniel) breeds. Two non-host compounds produced by beagles, i.e. 2-hexanone and benzaldehyde, were shown to be present in samples collected from all three breeds. Furthermore, two compounds, 6-methyl-5-hepten-2-one and 1,2,4-trimethylbenzene, were found in higher amounts in samples collected from miniature pinscher dogs. The mean amounts of benzaldehyde, 2-hexanone and 1,2,4-trimethylbenzene were similar for beagles and miniature pinschers (P > 0.05) and higher than the means observed for cocker spaniels (P < 0.05), whereas the mean amount of 6-methyl-5-hepten-2-one produced by miniature pinschers was significantly higher (P < 0.05) than for the other breeds of dogs. In Petri-dish assays with adult R. sanguineus s.l., 6-methyl-5-hepten-2-one was repellent for all observation periods evaluated for the two highest concentrations (0.100 and 0.200 mg.cm-2, P < 0.01). The obtained results support our hypothesis that miniature pinschers are a tick-resistant dog breed and agree with previous observations of miniature pinschers being the breed least parasitized by ticks. Furthermore, the non-host semiochemical 6-methyl-5-hepten-2-one has potential to be developed for use as a repellent for the protection of susceptible dogs from R. sanguineus s.l. ticks.