Luciana G. Brito

The anthelmintic effect of plant extracts on Haemonchus contortus and Strongyloides venezuelensis

The indiscriminate use of anthelmintics has resulted in the establishment of parasite resistance. Thus, this study aimed to evaluate the in vitro antiparasitic effect of plant extracts on Haemonchus contortus in sheep and the in vivo effect on Strongyloides venezuelensis in Rattus norvegicus. The plant extracts from Piper tuberculatum, Lippia sidoides, Mentha piperita, Hura crepitans and Carapa guianensis, produced at different research institutions, were chemically analyzed and evaluated through the egg hatch test (EHT) and larval development test (LDT) in H. contortus. P. tuberculatum (150 and 250 mg kg(-1) of body weight) was evaluated for its anthelmintic action on R. norvegicus experimentally infected with S. venezuelensis. In the EHT, the LC(50) and LC(90) of the extracts were respectively as follows: 0.031 and 0.09 mg mL(-1) for P. tuberculatum, 0.04 and 0.13 mg mL(-1) for L. sidoides, 0.037 and 0.10 mg mL(-1) for M. piperita, 2.16 and 17.13 mg mL(-1) for H. crepitans and 2.03 × 10(-6) and 1.22 × 10(-12) mg mL(-1) for C. guianensis. In the LDT, the LC(50) and LC(90) were respectively: 0.02 and 0.031 mg mL(-1) for P. tuberculatum, 0.002 and 0.04 mg mL(-1) for L. sidoides, 0.018 and 0.03 mg mL(-1) for M. piperita, 0.36 and 0.91 mg mL(-1) for H. crepitans and 17.65 and 1890 mg mL(-1) for C. guianensis. The extract of P. tuberculatum showed the following substances: piperamides as (Z)-piplartine, (E)-piplartine, 8,9-dihydropiplartine, piperine, 10,11-dihydropiperine, 5,6 dihydropiperlongumine and pellitorine. The major compounds of the oils were thymol (76.6%) for L. sidoides, menthol (27.5%) for M. piperita and oleic acid (46.8%) for C. guianensis. Regarding the in vivo test, neither dose of P. tuberculatum caused any significant reduction (P>0.05) in worm burden and fecal egg counts compared with the control group. We conclude that the extracts of P. tuberculatum, L. sidoides and M. piperita have effective activity when tested in vitro, but the doses of the extract of P. tuberculatum have no effect when employed in in vivo tests.

Evaluation of the Efficacy of Acaricides Used to Control the Cattle Tick, Rhipicephalus microplus, in Dairy Herds Raised in the Brazilian Southwestern Amazon.

The adult immersion test (AIT) was used to evaluate the efficacy of acaricide molecules used for control of Rhipicephalus microplus on 106 populations collected in five municipalities in the state of Rondônia in the Brazilian South Occidental Amazon region. The analysis of the data showed that the acaricide formulations had different efficacies on the tick populations surveyed. The synthetic pyrethroids (SPs) acaricides were the least effective (48.35–76.84%), followed by SP + organophosphate (OP) associations (68.91–81.47%) and amidine (51.35–100%). For the macrocyclic lactones (MLs), the milbemycin (94.84–100%) was the most effective, followed by spinosad (93.21–100%) and the avermectins (81.34–100%). The phenylpyrazole (PZ) group had similar efficacy (99.90%) to the MLs. Therefore, SP acaricides, including associations with OP, and formulations based on amidine presented low in vitro efficacy to control the R. microplus populations surveyed.

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 microplus. 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.

Description of the larva of Amblyomma ovale Koch, 1844 (Acari: Ixodidae) by light and scanning electron microscopy

Abstract The larval stage of Amblyomma ovale Koch is described using optical and scanning electron microscopy. Unfed larvae were obtained from a colony of A. ovale originating from engorged females collected on domestic dogs from Monte Negro County, state of Rondônia, Western Amazon, Brazil. Fifteen larvae were prepared and mounted on slides, and observed under a light microscope equipped with a drawing tube. Five specimens were prepared for scanning electron microscopy. Several morphological characters are described for the first time, including the chaetotaxy of the idiosoma, palps and Hallers organ, as well as morphological features of the idiosoma, gnathosoma and legs. In addition, topographical and numerical patterns of integumentary pores (porotaxy) on the idiosoma of larval A. ovale are described using a recently proposed nomenclature. Three types of integumentary pores were identified on the idiosoma of A. ovale larvae: lyrifissures, small glands, and large wax glands. These structures were observed isolated or associated over the entire idiosoma, except on the scutum, which lacks large wax glands. Larvae of A. ovale showed similar topographical and numerical patterns of integumentary structures, with 6 pairs of large wax glands (1 pair dorsal/5 pairs ventral), 24 pairs of lyrifissures (11 pairs dorsal/13 pairs ventral), and 54 pairs of small glands (32 pairs dorsal/22 pairs ventral). These topographical and numerical patterns of integumentary structures of larval A. ovale showed only minor differences when compared with patterns of other Amblyomma larvae; however, a few key features can be used to differentiate A. ovale from other members of its genus.

New tick records in Rondônia, Western Brazilian Amazon

In the present study, we provide new tick records from Vilhena Municipality, in the Southeast of the State of Rondônia, Northern Brazil. Ticks collected from a capybara, Hydrochoerus hydrochaeris (Linnaeus), were identified as Amblyomma romitii Tonelli-Rondelli (1 female), and Amblyomma sp. (1 larva). Ticks collected from a harpy eagle, Harpia harpyja (Linnaeus), were identified as Amblyomma cajennense (Fabricius) (16 nymphs) and Haemaphysalis juxtakochi Cooley (1 nymph). Ticks collected from a yellow-footed tortoise, Chelonoidis denticulada (Linnaeus), were identified as Amblyomma rotundatum Koch (10 females, 2 nymphs), and Amblyomma sp. (2 larvae). The present record of A. romitii is the first in the State of Rondônia, and represents the southernmost record for this tick species, indicating that its distribution area is much larger than currently recognized. Although both A. cajennense and H. juxtakochi have been reported parasitizing various bird species, we provide the first tick records on a harpy eagle. A. rotundatum is widespread in the State of Rondônia, and has been previously reported on the yellow-footed tortoise. The present records increase the tick fauna of Rondônia to 26 species.

Anaplasma marginale infection in cattle from south-western Amazonia

The present study provides the first epidemiological data regarding infection by Anaplasma marginale in cattle reared in south-western Brazilian Amazonia. One simple procedure was adapted for the extraction of DNA from blood clots collected in seven microregions of Rondonia State and two mesoregions of Acre State. PCR method was used to asses the frequency of A. marginale infections in 4 to12-month-old cattle. The cattle infection was investigated by polymerase chain reaction (PCR) using the specific primer msp5 for A. marginale. The DNA amplifications revealed that the mean frequency of A. marginale infection was 98.6% (1,627/1,650) in samples from Rondonia, and 92.87% (208/225) in samples from Acre. The high frequency of A. marginale infections in 4 to 12-month-old cattle indicate a situation of enzootic stability in the studied areas and are comparable to those detected by immunodiagnosis in different endemic regions in Brazil. The DNA extraction of clotted blood method described here can be used for epidemiological studies on anaplasmosis and other bovine hemoparasites.

Babesia bovis and Babesia bigemina infection levels estimated by qPCR in Angus cattle from an endemic area of São Paulo state, Brazil

The levels of infection by Babesia bovis and Babesia bigemina were estimated by absolute quantification through the quantitative PCR technique (qPCR). Fifty-one contemporaneous Angus cattle were evaluated on two occasions. The number of standard female Rhipicephalus microplus ticks present on the left side of the body was counted and blood samples were drawn from the tail vein into tubes containing the anticoagulant EDTA. The blood samples were submitted to DNA extraction and used to quantify the number of copies (NC) of DNA from B. bovis and B. bigemina by qPCR. The data on tick count and number of DNA copies were transformed for normalization and analyzed by a mixed model method. A multivariate model with repeated measures of the same animal, including the effects of collection, parasite species and their interaction, was used. The repeatability values were obtained from the matrix of (co)variances and were expressed for each species. The correlations between the counts of different species on the same animal, in the same collection or different collections, were also estimated. The results showed the qPCR could distinguish the two between infection by the two Babesia species. Infection levels by B. bovis and B. bigemina were detected in 100% and 98% of the animals, respectively. Significant differences were found (P<0.05) between the NC of the two Babesia species, B. bovis 1.49±0.07 vs. B. bigemina 0.82±0.06. Low repeatabilities were found for the counts of R. microplus and NC of B. bovis and B. bigemina: 0.05, 0.10 and 0.02, respectively. The correlations between R. microplus count and NC of B. bovis and B. bigemina were both very near zero. However, an association was observed between the NC of the two species, with a correlation coefficient of 0.30 for measures from the same collection. The absence of associations between the quantity of DNA from B. bovis and B. bigemina and the tick counts suggests that the variation of parasitemia by the hemoparasites did not depend on the tick infestation levels at the moment of each collection. The repeatability values estimated indicate that under the study conditions, the variations in the tick infestation levels and of parasitemia by B. bovis and B. bigemina depend more on factors related to each collection than on intrinsic factors of the animal.

Description of the larva of Amblyomma pacae Aragao, 1911 (Acari: Ixodidae) by light and scanning electron microscopy

Abstract The larval stage of Amblyomma pacae Aragão is described using optical and scanning electron microscopy. Unfed larvae were obtained from a colony of A. pacae originating from engorged females collected on Agouti paca (Linnaeus) from Monte Negro County, state of Rondônia, Western Amazon, Brazil. Several morphological characters are described for the first time, including the chaetotaxy of the idiosoma, palpi and Hallers organ, as well as morphological features of the idiosoma, gnathosoma and legs. The topographical and numerical patterns of integumentary pores (porotaxy) of the larva are described using a recently proposed nomenclature. Only a few morphological differences were observed between the A. pacae larva and other Amblyomma spp. larvae previously described from Brazil; however, some of these characters can be used to differentiate A. pacae from other species of this genus.

Genotype characterization of the Haematobia Irritans (diptera: muscidae) from Brazil, Dominican Republic and Colombia based on randomly amplified polymorphic dna (rapd) analysis

Blood-sucking flies are important parasites in animal production systems, especially regarding confinement conditions. Haematobia irritans, the horn fly, is one of the most troublesome species within bovine production systems, due to the intense stress imposed to the animals. H. irritans is one of the parasites of cattle that cause significant economic losses in many parts of the world, including South America. In the present work, Brazilian, Colombian and Dominican Republic populations of this species were studied by Random Amplified Polymorphic DNA (RAPD) to assess basically genetic variability between populations. Fifteen different decamer random primers were employed in the genomic DNA amplification, yielding 196 fragments in the three H. irritans populations. Among H. irritans samples, that from Colombia produced the smallest numbers of polymorphic bands. This high genetic homogeneity may be ascribed to its geographic origin, which causes high isolation, low gene flow, unlike the other American populations, from Brazil and Dominican Republic. Molecular marker fragments, which its produced exclusive bands, detected in every sample enabled the population origin to be characterized, but they are also potentially useful for further approaches such as the putative origin of Brazilian, Colombian and Dominican Republic populations of horn fly from South America. Similarity indices produced by chemo metric analysis showed the closest relationships between flies from Brazil and Dominican Republic, while flies from Colombia showed the greatest genotypic differentiation relative to the others populations.

Molecular quantitative assay for esterase-mediated organophosphate resistance in Rhipicephalus microplus

The use of pesticides is the main tool to control infestations of the cattle tick Rhipicephalus microplus, and organophosphate (OP) is one of the most used compounds for this purpose. Carboxylesterases (ChEs) are targets for OP pesticides in arthropods, and acetylcholinesterase 2 (AChE2) and esterase 1 (EST1) are metabolic enzymes involved in the xenobiotic detoxification process. The increase in the synthesis of these enzymes can be detected by the quantitative polymerase chain reaction (qPCR) assay, which was used to identify cattle tick populations resistant to OP pesticides. For that, two field populations of R. microplus were used, one previously identified by the larval packet test (LPT) as OP -sensitive (LC50=0.13μg/cm2) and the other OP-resistant (LC50=8.14μg/cm2). To promote the OP enzyme detoxification, groups of 10 females of the resistant strain were immersed in solutions of diazinon in technical grade at concentrations of 1.0mg/ml, 2.5mg/ml, and 5.0mg/ml. The ticks that survived diazinon exposure were submitted to qPCR assay, which enabled observing an increase in AChE2 and EST1 synthesis in the OP-resistant strain when compared to the susceptible strain. The initial results of expression analysis suggest that the qPCR assay can discriminate OP-resistant and susceptible populations. The development and improvement of molecular diagnostic tests to identify pesticide resistant R. microplus populations are priorities and in the near future it will be important to expand the molecular targets involved in OP resistance, which could be used for better selection of effective strategies to control cattle tick populations.