Proteolytic activity of excretory/secretory products of Cochliomyia hominivorax larvae (Diptera: Calliphoridae) / Atividade proteolítica de produtos de excreção/secreção de Cochliomyia hominivorax (Diptera: Calliphoridae)

The protein profiles and proteolytic activity of the excretory secretory products (E/SP) of the first (L1), second (L2) and third (L3) larval stages of Cochliomyia hominivorax were studied in the laboratory. Analysis on the E/SP protein profile was carried out using polyacrylamide gel containing sodium dodecyl sulfate (SDS-PAGE). The E/SP of each larval stage (L1, L2 and L3) treated with protease inhibitors, containing 30µg, 40µg and 50µg of protein, was applied to the 10% polyacrylamide gel. The proteolytic activity of the crude E/SP was analyzed in gels copolymerized with gelatin and by colorimetric assays using azocasein as a substrate, with the characterization of the proteases using synthetic inhibitors. Different protein profiles were observed for the larval instars, with L1 presenting the most complex profile. Nevertheless, various protein bands were observed that were common to all the larval instars. The E/SP of all the instars showed proteolytic activity on gelatin, evidenced by proteolysis zones, predominantly with apparently higher molecular masses in L1, while for L2 and L3 the proteolysis zones could also be observed in regions with lower masses. Tests with protease inhibitors using gelatin as substrate showed that the E/SP of larvae were mainly composed of serine proteases. Additionally, inhibition was observed in L2 E/SP treated previously with EDTA, an inhibitor of metalloproteases. The assays with azocasein revealed a gradual increase of proteolytic activity on this substrate with larval development progress, with the strongest inhibitions being observed after treatments with 3,4-dichloroisocoumarin (DCI) for E/SP of L1, L2 and L3. These results suggest that C. hominivorax larvae produce different proteases, a fact that can be related to the parasite's vital processes for survival, such as penetration into the host's tissues and nutrition during the larval stage.(AU)

Os perfis protéicos e a atividade proteolítica dos produtos de excreção/secreção (PE/S) das larvas de primeiro (L1), segundo (L2) e terceiro (L3) estágios de Cochliomyia hominivorax foram estudados em laboratório. Os perfis protéicos foram obtidos por eletroforese em géis de poliacrilamida (SDS-PAGE). Os PE/S de cada fase larval (L1, L2 e L3), tratados com inibidores de proteases, contendo 30µg, 40µg e 50µg de proteína, foram aplicados em géis de poliacrilamida a 10%. A atividade proteolítica dos PE/S na sua forma nativa, foi analisada em géis co-polimerizados com gelatina e por testes colorimétricos usando a azocaseína como substrato, com a caracterização das proteases feita por meio de inibidores sintéticos. Diferentes perfis protéicos foram observados para os instares larvais, com L1 apresentando o perfil mais complexo. Apesar disso, foram observadas várias bandas protéicas comuns a todos os estágios larvais. Os PE/S de todos os instares mostraram atividade proteolítica sobre a gelatina, evidenciada por zonas de proteólise, com predominância de massas moleculares aparentes mais altas em L1, enquanto que para L2 e L3 as zonas de proteólise puderam ser observadas também em regiões de menores massas. Os testes com inibidores de proteases usando a gelatina como substrato mostraram que os PE/S de L1, L2 e L3 eram compostos principalmente de serina-proteases. Adicionalmente, inibição foi observada nos PE/S de L2 tratada previamente com EDTA, um inibidor de metalo-proteases. Os ensaios com a zocaseína revelaram um aumento gradual da atividade proteolítica sobre este substrato com o progresso do desenvolvimento larval, com a mais forte inibição sendo observada após o tratamento com 3,4 dicloroisocumarina (DCI) para os PE/S de L1, L2 e L3. Estes resultados sugerem que as larvas de C. hominivorax produzem diferentes proteases, fato que pode estar relacionado a processos vitais para a sobrevivência do parasita, tais como a penetração nos tecidos dos hospedeiros e nutrição durante os estágios larvais.(AU)

Description of the larva of Amblyomma calcaratum Neumann, 1899 (Acari: Ixodidae) by light and scanning electron microscopy.

The larval stage of Amblyomma calcaratum Neumann is described using optical and scanning electron microscopy. Unfed larvae were obtained from a colony of A. calcaratum originating from engorged females collected on Tamandua tetradactyla in the Jaraguá Mountain (23°40'S, 45°44'W), São Paulo County, Brazil. Eleven larvae were prepared and mounted on slides and observed under a light microscope equipped with a drawing tube. Three specimens were prepared for SEM. Several morphological characters are described, including the chaetotaxy of the idiosoma, palpi, and Haller's organ, as well as morphological features of the idiosoma, gnathosoma, and legs of A. calcaratum larvae. In addition, topographical and numerical patterns of integumentary structures on the larval idiosoma are described using a recently proposed nomenclature. On the idiosoma, setaes, lyrifissures, small glands, and large wax glands were found. These structures were observed isolated or associated over the entire idiosoma, except on the scutum, which lacks large wax glands. The topographical and numerical patterns of integumentary structures of the A. calcaratum larva showed only minor differences when compared with patterns of other Amblyomma larvae; however, a few key features can be used to differentiate A. calcaratum from other members of this genus.

Babesia bovis infection in cattle in the southwestern Brazilian Amazon.

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

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.

Genotype characterization of Haematobia irritans from different Brazilian geographic regions based on randomly amplified polymorphic DNA (RAPD) analysis / Caracterização genotípica de Haematobia irritans procedentes de diferentes regiões geográficas brasileiras baseada na análise do DNA polimórfico amplificado ao acaso (RAPD-PCR)

Blood-sucking diptera are important parasites in bovine 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. An important aspect while studying the variability within a species is the study of the geographic structure of its populations and, attempting to find out the genetic flow of Brazilian populations of horn fly, the RAPD technique, which is suited for this purpose, has been used. The use of molecular markers generated from RAPD made it possible to identify the geographic origin of samples from different Brazilian geographic regions, as well as to estimate the genotypic flow among the different Brazilian populations of the horn fly.

Dípteras hematófagos são importantes parasitas dentro de sistemas de produção de bovinos, especialmente em confinamento. Haematobia irritans, a mosca-dos-chifres, é uma das espécies que maiores problemas causa em sistemas de produção de bovinos, dado ao intenso estresse que impõe aos animais. Um importante aspecto quando se estuda a variabilidade genética dentro das espécies é o estudo da estrutura geográfica destas populações. Buscando-se estimar a similaridade genotípica das diferentes populações brasileiras da mosca do chifre utilizou-se a técnica do DNA polimórfico amplificado ao acaso (RAPD-PCR), que mostrou-se eficiente para tal propósito. A utilização dos marcadores moleculares gerados através da técnica de RAPD-PCR tornou possível a identificação da origem geográfica das amostras das diferentes regiões geográficas brasileiras, assim como, estimar o fluxo genotípico entre as diferentes populações brasileiras da mosca-dos-chifres.

[Reproductive behavior and longevity of isolated and grouped couples of Lucilia cuprina, under controlled conditions]. / Comportamento reprodutivo e longevidade de casais isolados e agrupados se Lucilia cuprina, sob condições controladas.

The Lucilia cuprina laboratory breeding depends, fundamentally, on reproductive behavior and longevity information. In the present study it was verified that, due to the species isolation, there was a remarkable increase in the number of eggs/female (1344.18), when compared to the number of grouped eggs/female (732.78). The eggs viability was reduced due to the isolation of L. cuprina female. The longevity rate was estimated in 29.41 and 31.20 days for grouped males and females, respectively. For isolated males and females the longevity rate was 25.20 and 33.89, respectively.