Márcia Cristina de Sena Oliveira

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

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

Abiotic factors controlling the establishment and abundance of the invasive golden mussel Limnoperna fortunei

Limnoperna fortunei (Bivalvia, Mytilidae), a freshwater bivalve native to Southern Asia, has been an invasive species in South America since 1991. It spread upstream in the La Plata basin reaching the Paraguay River in the vicinity of the Pantanal wetland, Brazil, around 1998. The role of abiotic factors in controlling establishment and abundance of this species is not well known, making projections of its risk of further spread difficult. This study evaluates the importance of abiotic factors to L. fortunei populations established in rivers of the Pantanal, focusing on larval and juvenile densities and taking advantage of a wide range of seasonal variability in water temperature, flow, dissolved oxygen, and suspended material. Temperature, river stage (influencing several water characteristics) and water velocity are the main variables related to the larval and juvenile densities. In the Pantanal, environmental variables vary over a broader range compared with other South American locations, subjecting L. fortunei to oxygen depletion, low calcium, low pH, and high water velocity and suspended solids, associated with low chlorophyll a concentrations. The combined effect of several of these conditions may explain the relatively low densities in some Pantanal sites. However, they probably will not prevent the persistence of populations in the Pantanal and the eventual establishment of viable populations in upriver systems connected to the Pantanal. These results are pertinent not only to this species but also to other aquatic invasive invertebrates whose expansion may be limited by thermal extremes, episodic oxygen depletion, and waters that are too dilute or acidic for optimal biocalcification.

Artificial infestation of Boophilus microplus in beef cattle heifers of four genetic groups

Resistance of beef cattle heifers to the cattle tick Boophilus microplus was evaluated by artificial infestation of 66 beef cattle heifers of the following genetic groups: 16 Nelore (NE), 18 Canchim x Nelore (CN), 16 Angus x Nelore (AN) and 16 Simmental x Nelore (SN). The animals, with a mean age of 16.5 months, were maintained with no chemical tick control in a Brachiaria decumbens pasture. Four artificial infestations with 20,000 B. microplus larvae were carried out 14 days apart and from day 18 to day 22 of each infestation the number of engorged female ticks (> 4.5 mm) was counted on the left side of each heifer. Data were analyzed as the percentage of return (PR = percentage of ticks counted relative to the number infested), transformed to (PR)1/4, and as log10 (Cij + 1), in which Cij is the number of ticks in each infestation, using the least squares method with a model that included the effects of genetic group (GG), animal within GG (error a), infestation number (I), GG x I and the residual (error b). Results indicated a significant GG x I interaction, because AN and SN heifers had a higher percentage of return than CN and NE heifers, while CN heifers showed a higher percentage of return than the NE heifers only in infestations 3 and 4. Transformed percentages of return were NE = 0.35 ± 0.06, AN = 0.89 ± 0.06, CN = 0.54 ± 0.05 and SN = 0.85 ± 0.06.

Oxygen depletion events control the invasive golden mussel (Limnoperna fortunei) in a tropical floodplain.

Over the past 10xa0years the golden mussel (Limnoperna fortunei), an exotic species native to eastern Asia, has become widespread and abundant in the Pantanal Wetland, Brazil. Oxygen concentrations are often low in rivers and floodplain waters of the Pantanal and oxygen depletion events can last for several weeks during the rising water phase. Although mortality of L. fortunei has been documented during oxygen depletion events, its tolerance to hypoxic and anoxic conditions is poorly understood, in part because changes in oxygen availability are accompanied by other changes such as decreased pH. We analyzed interannual variation in densities of adults and juveniles in relation to varying oxygen conditions, and tested the tolerance of L. fortunei to oxygen depletion events in a floodplain lake and in the laboratory. Mussels died after 5xa0days of an oxygen depletion event in a floodplain lake, and a population established there in 2005 was extirpated in 2006 owing to hypoxic conditions. Laboratory tests confirmed that mussels died more quickly in water from the oxygen depletion event. Annual oxygen depletion events must control the density of the invasive golden mussel in the Pantanal, maintaining low densities and periodically extirpating them from some habitats.

Infestação natural de fêmeas bovinas de corte por ectoparasitas na Região Sudeste do Brasil

It was evaluated in this work, the degree of natural infestation by external parasites in beef cattle females from three genetic groups. Countings of cattle tick (Rhipicephalus (Boophilus) microplus), horn fly (Haematobia irritans) and beef-worm (Dermatobia hominis) were performed from July 2003 to December 2004 in females from the genetic groups Nellore (NE), Angus x Nellore (AN), Canchim x Nellore (CN), and Simmental x Nellore (SN) naturally infested. It was realized from 6 to 10 countings in each animal, totalizing 5,384 observations in females at seven physiological stages (calf; empty heifers, pregnant heifers, primiparous cows with or without calf; and pluriparous cows, with or without calf). Data, which were transformed by log10 (n + 1), were analyzed by the least square method with a statistical model that included effects of the female genetic group, of the animal within a genetic group (error a), year-season counting, physiological state, and year-season x genetic group interaction. Differences among genetic groups depended on year-season counting; however, in general, Nellore females were the least infected by ticks, and Angus x Nellore females were the most infected by horn flies and by beef-worms. There was a physiological stage effect of the female in all the three studied characteristics and the differences changed accordingly to the trait. Despite of the counting year-season x genetic group, there are differences among Nellore, Canchim x Nelore, Angus x Nellore and Simmental x Nelore genetic groups regarded to the degree of natural infestation by cattle tick, horn fly and beef-worm.

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

The activity of plant extracts on parasites may indicate groups of substances that are potentially useful for controlling Rhipicephalus (Boophilus) microplus. The aim of the present study was to investigate the in vitro action of Artemisia annua extracts on this tick. The concentrations of the sesquiterpene lactones artemisinin and deoxyartemisinin present in plant extracts were quantified via high-performance liquid chromatography. Four extracts produced from the concentrated crude extract (CCE) were evaluated on larvae using the impregnated paper method, with readings after 24 hours of incubation. The engorged females were immersed in the CCE and in its four derived extracts for five minutes, with incubation for subsequent analysis of biological parameters. The extracts were not effective on the larvae at the concentrations tested (3.1 to 50 mg.mL(-1)). The CCE showed greater efficacy on engorged females (EC(50) of 130.6 mg.mL(-1) and EC(90) of 302.9 mg.mL(-1)) than did the derived extracts. These results tend to confirm that the action of artemisinin on engorged females of R. (B.) microplus is conditional to their blood intake. In this case, in vitro methods would be inadequate for effective evaluation of the action of A. annua on R. (B.) microplus.

DNA Barcoding Green Microalgae Isolated from Neotropical Inland Waters

This study evaluated the feasibility of using the Ribulose Bisphosphate Carboxylase Large subunit gene (rbcL) and the Internal Transcribed Spacers 1 and 2 of the nuclear rDNA (nuITS1 and nuITS2) markers for identifying a very diverse, albeit poorly known group, of green microalgae from neotropical inland waters. Fifty-one freshwater green microalgae strains isolated from Brazil, the largest biodiversity reservoir in the neotropics, were submitted to DNA barcoding. Currently available universal primers for ITS1-5.8S-ITS2 region amplification were sufficient to successfully amplify and sequence 47 (92%) of the samples. On the other hand, new sets of primers had to be designed for rbcL, which allowed 96% of the samples to be sequenced. Thirty-five percent of the strains could be unambiguously identified to the species level based either on nuITS1 or nuITS2 sequences’ using barcode gap calculations. nuITS2 Compensatory Base Change (CBC) and ITS1-5.8S-ITS2 region phylogenetic analysis, together with morphological inspection, confirmed the identification accuracy. In contrast, only 6% of the strains could be assigned to the correct species based solely on rbcL sequences. In conclusion, the data presented here indicates that either nuITS1 or nuITS2 are useful markers for DNA barcoding of freshwater green microalgae, with advantage for nuITS2 due to the larger availability of analytical tools and reference barcodes deposited at databases for this marker.

In vitro and in vivo evaluation of the activity of pineapple (Ananas comosus) on Haemonchus contortus in Santa Inês sheep.

The development of resistance to anthelmintics has prompted research into alternative methods of controlling intestinal nematodes in ruminants. This study aimed to assess the activity of Ananas comosus on Haemonchus contortus in Santa Inês sheep. The aqueous extract of pineapple skin (AEPS), bromelain from pineapple stems (B4882) and residue from pineapple processing was evaluated in in vitro and in vivo tests. The enzymatic activity of substances was analyzed by the azocasein method. The egg hatch test (EHT) and larval development test (LDT) were performed using the Embrapa2010 isolate of H. contortus. In the in vivo test, 36 sheep artificially infected with H. contortus were divided into six groups: G1: 2 g/kg BW of the aqueous extract administered for three days; G2: 2 g/kg BW of the industrial pineapple residue for 60 days; G3: 180 mg/animal of bromelain in a single dose; G4: negative control I; G5: positive control (levamisole phosphate); and G6: negative control II. The eggs per gram (EPG) in the feces were counted till 28 days after treatment. LC₅₀ and LC₉₀ were obtained by the probit procedure, while the in vivo test results were analyzed by GLM. The aqueous extract in the in vitro and in vivo test, the bromelain and industrial residue presented 0.102, 0.157, 1.864 and 0.048 enzyme units/mL, respectively. In the egg hatch test, the LC₅₀ and LC₉₀ were respectively 31 and 81 mg/mL for the aqueous extract and 0.50 and 2 mg/mL for bromelain. In the larval development test, the LC₅₀ and LC₉₀ were respectively 1.7 and 7.3 mg/mL for the aqueous extract and 0.019 and 0.086 mg/mL for bromelain. In the in vivo test, the general efficacies of the treatments in relation to the negative control were 22.6%, 42.2%, 3.65% and 89% for the aqueous extract, industrial pineapple residue, bromelain and positive control respectively. The transformed EPG values were 3.19 ± 0.59, 3.32 ± 0.25, 2.85 ± 0.66, 3.44 ± 0.50, 2.28 ± 0.93 and 2.75 ± 0.94 for the aqueous extract, industrial residue, bromelain, negative control I, positive control and negative control II respectively. The results for all the treated groups differed significantly (p<0.05) from the positive control, and although the residue presented efficacy of 42.2%, there was no statistical difference (p>0.05) in relation to the negative control. Therefore, both the aqueous extract and bromelain were effective in vitro, but showed reduced anthelmintic efficacy in vivo. For the pineapple residue, the 42.2% in vivo efficacy in reducing the EPG and the possibility of reducing environmental contamination through reuse of industrial residue indicate it can also be useful for control of this parasite.

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

Measures to control the cattle tick, Rhipicephalus (Boophilus) microplus, based only on chemical products are becoming unsustainable, mainly because of the development of resistance. The objective of this study was to test the effect of the aqueous extract of pineapple skin (AEPS) and bromelain extracted from the stem (Sigma-Aldrich®, B4882) on engorged females and larvae of R. (B.) microplus in vitro. These substances were diluted in water and evaluated at eight concentrations. Engorged females were collected and distributed in groups of 10, with three repetitions for each treatment. After immersion in the solutions, the females were placed in an incubator for observation of survival, oviposition and larval hatching. The larval packet method was used, also with three repetitions with about 100 larvae each. The packets were incubated and the readings were performed after 24 h. The estimated reproduction and efficacy of the solutions were calculated. The LC(50) and LC(90) were estimated using the Probit procedure of the SAS program. The eight concentrations were compared within each treatment by the Tukey test. For the experiment with engorged females, the most effective concentrations were 125, 250 and 500 mg/mL: 33%, 48% and 59% for the AEPS and 27%, 51% and 55% for the bromelain. The LC(50) and LC(90) values were, respectively, 276 and 8691 mg/mL for AEPS and 373 and 5172 mg/mL for bromelain. None of the dilutions tested was effective against the larvae of R. (B.) microplus. This is the first report of the action of pineapple extracts or their constituents on cattle ticks. The results demonstrate that further studies regarding composition of tick cuticle, with evaluation of other solvents and formulations, should be conducted seeking to enhance the effect of pineapple extracts and compounds against this ectoparasite.

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.