Ecdysone modulates both ultrastructural arrangement of hindgut and attachment of Trypanosoma cruzi DM 28c to the rectum cuticle of Rhodnius prolixus fifth-instar nymph.

Studies on the effects of azadirachtin treatment, ecdysone supplementation and ecdysone therapy on both the ultrastructural organization of the rectum in 5th-instar nymph of Rhodnius prolixus and the ex vivo attachment behavior of Trypanosoma cruzi under these experimental conditions were carried out. Control insects had a typical and significant organization of the rectum cuticle consisted of four main layers (procuticle, inner epicuticle, outer epicuticle, and wax layer) during the entire period of the experiment. Both azadirachtin treatment and ecdysone supplementation avoid the development of both outer epicuticle and wax layer. Oral therapy with ecdysone partially reversed the altered organization and induce the development of the four main rectal cuticle layers. In the same way, the ex vivo attachment of T. cruzi to rectal cuticle was blocked by azadirachtin treatment but ecdysone therapy also partially recovered the parasite adhesion rates to almost those detected in control insects. These results point out that ecdysone may be a factor responsible - directly or indirectly - by the modulation of rectum ultrastructural arrangement providing a superficial wax layer to the attachment followed by metacyclogenesis of T. cruzi in the rectum of its invertebrate hosts.

Acaricidal Properties of Bio-Oil Derived From Slow Pyrolysis of Crambe abyssinica Fruit Against the Cattle Tick Rhipicephalus microplus (Acari: Ixodidae).

Slow pyrolysis is a process for the thermochemical conversion of biomasses into bio-oils that may contain a rich chemical composition with biotechnological potential. Bio-oil produced from crambe fruits was investigated as to their acaricidal effect. Slow pyrolysis of crambe fruits was performed in a batch reactor at 400°C and chemical composition was analyzed by gas chromatography-mass spectrometry (GC-MS). The bio-oil collected was used in bioassays with larvae and engorged females of the cattle tick Rhipicephalus microplus. Biological assays were performed using the larval packet test (LPT) and adult immersion test. The GC-MS of crambe fruit bio-oil revealed mainly hydrocarbons such as alkanes and alkenes, phenols, and aldehydes. The bio-oil in the LPT exhibited an LC90 of 14.4%. In addition, crambe bio-oil caused female mortality of 91.1% at a concentration of 15% and a high egg-laying inhibition. After ovary dissection of treated females, a significant reduction in gonadosomatic index was observed, indicating that bio-oil interfered in tick oogenesis. Considering these results, it may be concluded that slow pyrolysis of crambe fruit affords a sustainable and eco-friendly product for the control of cattle tick R. microplus.

Biotechnological Potential of Eugenol and Thymol Derivatives Against Staphylococcus aureus from Bovine Mastitis.

Bovine mastitis is an infectious disease that affects the mammary gland of dairy cattle with considerable economic losses. Staphylococcus aureus is the main microorganism involved in this highly contagious process, and the treatment is only using antibiotics. Currently, the search for new treatment and/or compounds is still in need due to microbial resistance. In this work, we evaluated the potential of eugenol and thymol derivatives against S. aureus strains from bovine mastitis. On that purpose, nine derivatives were synthesized from eugenol and thymol (1-9), and tested against 15 strains of S. aureus from subclinical bovine mastitis. Initially, the strains were evaluated for the biofilm production profile, and those with strong adherence were selected to the antimicrobial sensitivity determination in the Minimum Inhibitory Concentration (MIC) assays. Herein the compounds toxicity was also evaluated by in silico analysis using Osiris DataWarrior® software. The results showed that 60% of the strains were considered strongly adherent and three strains (S. aureus 4271, 4745 and 4746) were selected for the MIC tests. Among the nine eugenol and thymol derivatives tested, four were active against the evaluated strains (MIC = 32 µg mL-1) within CLSI standard values. In silico analysis showed that all derivatives had cLopP < 5, cLogS > - 4 and TPSA < 140 Å2, and similar theoretical toxicity parameters to some antibiotics currently on the market. These molecules also showed negative drug-likeness values, pointing to the originality of these structures and theoretical feasibility on escaping of resistance mechanism and act against resistant strains. Thus, these eugenol derivatives may be considered as promising for the development of new treatments against bovine mastitis and future exploring on this purpose.

Evaluation of essential oils as an ecological alternative in the search for control Rhipicephalus microplus (Acari: Ixodidae).

The cattle tick Rhipicephalus microplus is a significant problem for livestock, causing losses of billions of dollars per year. This work aimed to determine the chemical composition of essential oils obtained from Laurus nobilis and Copaifera officinalis and evaluate activity against engorged R. microplus females. Chemical composition analyzed by GC-MS revealed the presence of 39 components accounting for 95.38% of the oil in L. nobilis, the most abundant being 1,8-cineol (25.7%), trans-sabinene-hydrate (20.8%), and α-terpinil acetate (15.0%). Chemical analysis of C. officinalis oil identified 25 components corresponding to 80.5% of the total constituents, where the major compounds were ß-caryophyllene (21.1%), caryophyllene oxide (10.7%), and α-trans-bergamotene (9.3%). Adult immersion test (AIT) showed that L. nobilis essential oil at 5% or 10% caused 80.5% mortality of engorged females after 24 h and reached 96.9% and 100% mortality on the third day after treatment, respectively. While the essential oil from C. officinalis caused 84.7% mortality after six days at 10% and at 5%, achieved approximately 100% mortality rate at the end of the experiment (day 15). Both essential oils and the combination significantly inhibited egg-laying; however, the combination treatment showed higher effectiveness than the isolated oils at 2.5%. A possible synergic action of L. nobilis and C. officinalis against the cattle tick R. microplus is therefore suggested. The present work introduces a potential alternative for the development of a formulation environment-friendly (green pesticide) used to control cattle tick infestations.

First report of meticillin-resistant Staphylococcus aureus harboring mecC gene in milk samples from cows with mastitis in southeastern Brazil.

MRSA infection and colonization have been reported in both companion and food-chain animals, highlighting MRSA as an important veterinary and zoonotic pathogen. Another mec allele, the mecC gene, also confers beta-lactam resistance in Staphylococcus aureus and shows 69% nucleotide identity to mecA. The main aim of this study was to investigate the genotypic and clonal profile of methicillin-resistant S. aureus (MRSA) from cows with mastitis in dairy herds. Thirty-five samples suggestive of bovine subclinical mastitis were evaluated, and S. aureus were detected in all of them using both phenotypic and molecular approaches. According to the multilocus sequence typing (MLST), the S. aureus isolates were assigned in five different STs, 21 (60%) showed ST 742, 6 (17%) ST97, 4 (11%) ST1, 2 (6%) ST30, and 2 (6%) ST126. The presence of mecA was not observed in any of these isolates whereas mecC was detected in nine of them (9/35; 26%). The Panton-Valentine leukocidin (PVL) genes were detected in a total of 4 isolates. Among the 35 isolates analyzed, 26 showed resistance to penicillin. Changes in the S. aureus epidemiology due to the detection of MRSA in milk samples from cows presenting with bovine subclinical mastitis may have consequences for public health in Brazil, challenging the empirical therapy and animal management, with potential medical and social outcomes. To the best of our knowledge, this is the first report describing mecC MRSA in Southeastern Brazil.

Chemical composition and acaricidal activity of Ocotea notata (Lauraceae), an endemic species from Brazil, against the cattle tick Rhipicephalus microplus / Composición química y actividad acaricida de Ocotea notata (Lauraceae), una especie endémica de Brasil, contra la garrapata del ganado Rhipicephalus microplus

Rhipicephalus microplus, is one parasites that cause severe productivity losses in the cattle industry of Brazil and shows increasing resistance to conventional pesticides. This research aims to study the chemical composition, and acaricidal activity of the essential oil from Ocotea notata leaves, a brazilian endemic species, against R. microplus. The effect on R. microplus engorged adult females was evaluated using the immersion test. The oil reduced the survival by 90% after incubation for 15 days and there was 100% reduction for posture inhibition and reproductive capacity. These results suggest that the O. notata essential oil has activity on the R. microplus.

Rhipicephalus microplus, es un parásito que causa graves pérdidas de productividad en la industria ganadera de Brasil y muestra una creciente resistencia a los pesticidas convencionales. Esta investigación tiene como objetivo estudiar la composición química y la actividad acaricida del aceite esencial de las hojas de Ocotea notata, una especie endémica brasileña, contra R. microplus. El efecto sobre las hembras adultas engordadas de R. microplus se evaluó mediante la prueba de inmersión. El aceite redujo la supervivencia en 90% después de la incubación durante 15 días y hubo una reducción del 100% para la inhibición de la postura y la capacidad reproductiva. Estos resultados sugieren que el aceite esencial de O. notata tiene actividad contra R. microplus.

Exploring the Insecticide and Acaricide Potential of Development Regulators obtained from Restinga vegetation from Brazil.

As a part of our continuing search for insect and arthropod development regulators from Brazilian restinga vegetation of the Rio de Janeiro State, crude extracts, purified fractions and essential oils were submitted to screening tests seeking for biological activities on the development of the insects Rhodnius prolixus, Dysdercus peruvianus, Oncopeltus fasciatus and Rhipicephalus (Boophilus) microplus. Up to now, 102 secondary metabolites have been detected in the fractions, among them monoterpenes, sesquiterpenes and two triterpenes which were obtained from the species, Eugenia sulcata, Pilocarpus spicatus, Manilkara subsericea, Myrciaria floribunda and Zanthoxylum caribaeum. These secondary plant metabolites are considered of interest for the use of studies related to arthropod endocrinology, vector-parasite interaction system, and population control of vector insect and agricultural pest. The observed biological activities were surprisingly high, involving increased mortality, molting and metamorphosis inhibition, paralysis, corporeal deformities, apparition of permanent nymphs, of adultoids and juvenoids, partial or total inhibition of oviposition and egg hatching. These compounds are now being studied further to determine if they may or may not be useful in controlling insect populations and/or interfere with the life cycle and vector transmission of parasites to animal and human populations.

A new lipid carrier protein in the cattle tick Rhipicephalus microplus.

Tick infestation in cattle reflects the main cause of economic loss to cattle producers. This is due to several reasons but mainly to their ability to feed on blood and generate a huge amount of eggs. Lipid transport in arthropods is achieved by highly specialized hemolymphatic lipoproteins, which resemble those described in vertebrate blood. Such lipoproteins continuously deliver lipids through the blood to growing eggs. The injection of radioactive [3H] palmitic acid into tick hemocoel showed that the gut, ovary, fat body and Gene's organ were the main organs of incorporation of this labeled fatty acid. The rate of [3H] palmitic acid incorporation by the organs was high up to 30 min after injection. The [3H] palmitic acid incorporated by these organs was later found in phospholipids and neutral lipids. Here, we describe the purification and characterization of a key player of lipid dynamics in tick hemolymph. The Rhipicephalus microplus lipid-apolipoprotein complex (RmLCP) is a new high-density lipoprotein (1.18 g/mL), which accounts for over 90% of [3H] palmitic acid present in the hemolymph. It has a native molecular weight of 420 kDa and is composed of one subunit of 122 kDa. Protein identification analysis of RmLPC subunit showed two better hits: vitellogenin 2 (23% protein coverage) and vitellogenin 5 (29% protein coverage), respectively and similarities with hemolymphatic apolipoproteins of arachnids such as the tick Ixodes scapularis (80%), the mite Galendromus occidentalis (44%) and the spider Parasteatoda tepidariorum (43%) and also for the insects Locusta migratoria (45%), Drosophila melanogaster (42%) and Manduca sexta (47%) to vitellogenin 2 and tick Ixodes scapularis (83%), the crab Limulus polyphemus (55%) and the oyster Crassostrea gigas (55%) to vitellogenin 5. Furthermore, it shows a distinct lipid composition from most arthropod lipoproteins, being composed of 40% free cholesterol, 27% phospholipids, 20% triacylglycerol and 15% hydrocarbons. In addition to binding most hemolymphatic fatty acids, this lipoprotein also binds and transports free cholesterol. In conclusion, the present study provides insight into the macromolecules involved in arachnid metabolism, which have significant potential for future use for the biological control of ticks.

Lipophorin Drives Lipid Incorporation and Metabolism in Insect Trypanosomatids.

Insect trypanosomatids are inhabitants of the insect digestive tract. These parasites can be either monoxenous or dixenous. Plant trypanosomatids are known as insect trypanosomatids once they and are transmitted by phytophagous insects. Such parasites can be found in latex, phloem, fruits and seeds of many plant families. Infections caused by these pathogens are a major cause of serious economic losses. Studies by independent groups have demonstrated the metabolic flow of lipids from the vertebrate host to trypanosomatids. This mechanism is usually present when parasites possess an incomplete de novo lipid biosynthesis pathway. Here, we show that both insect trypanosomatids Phytomonas françai and Leptomonas wallacei incorporate (3)H-palmitic acid and inorganic phosphate. These molecules are used for lipid biosynthesis. Moreover, we have isolated the main hemolymphatic lipoprotein, Lipophorin (Lp) from Oncopeltus fasciatus, the natural insect vector of such parasites. Both parasites were able to incorporate Lp to be utilized both as a lipid and protein source for their metabolism. Also, we have observed the presence of Lp binding sites in the membrane of a parasite. In conclusion, we believe that the elucidation of trypanosomatid metabolic pathways will lead to a better understanding of parasite-host interactions and the identification of novel potential chemotherapy targets.

Acaricidal properties of the essential oil from Zanthoxylum caribaeum against Rhipicephalus microplus.

Zanthoxylum caribaeum Lamarck (Rutaceae) is plant species with a variety of medical applications, including insecticidal activity. This study determined the bioacaricidal activity of the essential oil from Z. caribaeum leaves against engorged Rhipicephalus (Boophilus) microplus (Canestrini, 1887) females using the adult immersion test. For this purpose, three serial concentrations (5.0, 2.5, and 1.25%, vol:vol, in 1% dimetilsulfoxide) of the essential oil were used. Essential oil 5% caused 65% mortality on the first day after treatment, 85% on the second day, and 100% mortality by the fifth day. To our knowledge, this is the first demonstration of the acaricidal activity of the essential oil from Z. caribaeum leaves against cattle ticks.

Acaricidal properties of the formulations based on essential oils from Cymbopogon winterianus and Syzygium aromaticum plants.

The cattle tick, Rhipicephalus (Boophilus) microplus, has caused serious harm to livestock raising in Brazil, considering the costs of controlling it, loss of revenue due to smaller production of milk and meat, and damage to leather, in addition to transmitting diseases. The use of medicinal plants is considered an alternative to the recurring resistance to chemicals. Due to the need for efficient alternatives with less environmental impact, this study aimed to develop contact formulations with essential oils from the Java citronella (Cymbopogon winterianus) and clove (Syzygium aromaticum) plants and to assess in vitro the effects in different stages of the tick cycle. In the present study, concentrations from 0.5-15.0% of the essential oils incorporated in the formulations were used. The ticks from different geographical areas were treated with those formulations, and their effects on the production levels of eggs, on the larvae hatching, and their efficiency on ticks were assessed. The obtained results were compared with other commercial acaricidal products. After the 20th day of treatment, the formulations with citronella essential oil had 2.09-55.51% efficiency, depending on the concentration of the oil incorporated. The efficiency of the treatment with formulations containing clove essential oil was higher, from 92.47-100%. The results showed the acaricidal effects of the formulations tested when compared to commercial chemical products. In vivo studies should be performed in order to assess the efficiency of those formulations in the fields, aiming to use these products as an alternative for controlling cattle ticks.

Glycoinositolphospholipids from Trypanosomatids subvert nitric oxide production in Rhodnius prolixus salivary glands.

BACKGROUND: Rhodnius prolixus is a blood-sucking bug vector of Trypanosoma cruzi and T. rangeli. T. cruzi is transmitted by vector feces deposited close to the wound produced by insect mouthparts, whereas T. rangeli invades salivary glands and is inoculated into the host skin. Bug saliva contains a set of nitric oxide-binding proteins, called nitrophorins, which deliver NO to host vessels and ensure vasodilation and blood feeding. NO is generated by nitric oxide synthases (NOS) present in the epithelium of bug salivary glands. Thus, T. rangeli is in close contact with NO while in the salivary glands. METHODOLOGY/PRINCIPAL FINDINGS: Here we show by immunohistochemical, biochemical and molecular techniques that inositolphosphate-containing glycolipids from trypanosomatids downregulate NO synthesis in the salivary glands of R. prolixus. Injecting insects with T. rangeli-derived glycoinositolphospholipids (Tr GIPL) or T. cruzi-derived glycoinositolphospholipids (Tc GIPL) specifically decreased NO production. Salivary gland treatment with Tc GIPL blocks NO production without greatly affecting NOS mRNA levels. NOS protein is virtually absent from either Tr GIPL- or Tc GIPL-treated salivary glands. Evaluation of NO synthesis by using a fluorescent NO probe showed that T. rangeli-infected or Tc GIPL-treated glands do not show extensive labeling. The same effect is readily obtained by treatment of salivary glands with the classical protein tyrosine phosphatase (PTP) inhibitor, sodium orthovanadate (SO). This suggests that parasite GIPLs induce the inhibition of a salivary gland PTP. GIPLs specifically suppressed NO production and did not affect other anti-hemostatic properties of saliva, such as the anti-clotting and anti-platelet activities. CONCLUSIONS/SIGNIFICANCE: Taken together, these data suggest that trypanosomatids have overcome NO generation using their surface GIPLs. Therefore, these molecules ensure parasite survival and may ultimately enhance parasite transmission.

LDL uptake by Leishmania amazonensis: involvement of membrane lipid microdomains.

Leishmania amazonensis lacks a de novo mechanism for cholesterol synthesis and therefore must scavenge this lipid from the host environment. In this study we show that the L. amazonensis takes up and metabolizes human LDL(1) particles in both a time and dose-dependent manner. This mechanism implies the presence of a true LDL receptor because the uptake is blocked by both low temperature and by the excess of non-labelled LDL. This receptor is probably associated with specific microdomains in the membrane of the parasite, such as rafts, because this process is blocked by methyl-ß-cyclodextrin (MCBD). Cholesteryl ester fluorescently-labeled LDL (BODIPY-cholesteryl-LDL) was used to follow the intracellular distribution of this lipid. After uptake it was localized in large compartments along the parasite body. The accumulation of LDL was analyzed by flow cytometry using FITC-labeled LDL particles. Together these data show for the first time that L. amazonensis is able to compensate for its lack of lipid synthesis through the use of a lipid importing machinery largely based on the uptake of LDL particles from the host. Understanding the details of the molecular events involved in this mechanism may lead to the identification of novel targets to block Leishmania infection in human hosts.

Effects of platelet-activating factor on the interaction of Trypanosoma cruzi with Rhodnius prolixus.

We investigated the effects of platelet-activating factor (PAF) on the interaction of Trypanosoma cruzi with Rhodnius prolixus. The parasites (epimastigotes) were treated with PAF and/or WEB 2086 (PAF antagonist) for 1 h prior to the interaction experiments. PAF stimulated both in vivo and ex vivo interactions between T. cruzi and R. prolixus while WEB 2086 abrogated these effects. PAF-treated epimastigotes also showed an increase in surface negativity and in the amount of surface sialic acid. Neither of these effects was observed when the epimastigotes were treated with neuraminidase following PAF treatment. In the ex vivo interaction experiments, the number of epimastigotes bound to the midguts of the insects was reduced when the epimastigotes had been treated with neuraminidase. We conclude that PAF modulates the interaction of T. cruzi with R. prolixus by altering the amount of sialyl residues at the surface of the parasite.

Trypanosoma rangeli uptakes the main lipoprotein from the hemolymph of its invertebrate host.

During its life cycle Trypanosoma rangeli crosses the hemolymph of its invertebrate host. In the present study, we demonstrate for the first time the uptake of lipophorin (Lp), the main lipid-transporting particle of insect hemolymph. We observed that living T. rangeli parasites uptake lipids from both 32P- and 3H-, or 125I-labeled Lp. However, the parasites do not uptake any other hemolymphatic protein such as 32P-labeled vitellogenin. The presence of a specific receptor to Lp in the parasite surface is suggested based on experiments using 125I-Lp. We also investigated the intracellular fate of lipids using Texas Red-labeled phosphatidylethanolamine-Lp. Parasites were observed under confocal microscope and displayed fluorescent-labeled lipids close to the flagellar pocket and in vesicles at the posterior region. In conclusion, this study raises a novel set of molecular events which takes place during vector-parasite interaction.