Silmara Marques Allegretti

Phytol, a Diterpene Alcohol from Chlorophyll, as a Drug against Neglected Tropical Disease Schistosomiasis Mansoni

Background Schistosomiasis is a major endemic disease that affects hundreds of millions worldwide. Since the treatment and control of this parasitic disease rely on a single drug, praziquantel, it is imperative that new effective drugs are developed. Here, we report that phytol, a diterpene alcohol from chlorophyll widely used as a food additive and in medicinal fields, possesses promising antischistosomal properties in vitro and in a mouse model of schistosomiasis mansoni. Methods and findings In vitro, phytol reduced the motor activity of worms, caused their death and confocal laser scanning microscopy analysis showed extensive tegumental alterations in a concentration-dependent manner (50 to 100 µg/mL). Additionally, phytol at sublethal doses (25 µg/mL) reduced the number of Schistosoma mansoni eggs. In vivo, a single dose of phytol (40 mg/kg) administered orally to mice infected with adult S. mansoni resulted in total and female worm burden reductions of 51.2% and 70.3%, respectively. Moreover, phytol reduced the number of eggs in faeces (76.6%) and the frequency of immature eggs (oogram pattern) was significantly reduced. The oogram also showed increases in the proportion of dead eggs. Confocal microcopy studies revealed tegumental damage in adult S. mansoni recovered from mice, especially in female worms. Conclusions The significant reduction in parasite burden by this chlorophyll molecule validates phytol as a promising drug and offers the potential of a new direction for chemotherapy of human schistosomiasis. Phytol is a common food additive and nonmutagenic, with satisfactory safety. Thus, phytol has potential as a safe and cost-effective addition to antischistosomal therapy.

Molecular characterization of picobirnaviruses from new hosts.

Picobirnaviruses (PBVs) have recently been classified into the Picobirnaviridae family. They are small, non-enveloped viruses with bisegmented, double-stranded (ds) RNA genomes. Although they are found in the feces of a broad range of hosts, information regarding their genomes is limited to viruses detected from humans, rabbits, and porcine. Identification of PBVs has been done using PAGE and reverse transcription PCR (RT-PCR). In this study, we present a phylogenetic analysis of PBVs detected in the feces of dogs, snakes, and rats. In addition, we compare these strains to those from human and porcine hosts. To do so, 487 fecal specimens from dogs, snakes and rats were analyzed by PAGE. The positive specimens for PBV were tested by RT-PCR using primers for genogroup I of the PBVs. From the 11 genogroup I PBV samples, at least one from each host was sequenced and submitted for phylogenetic analysis. All of the sequences showed high homology with the human and porcine genogroup I PBV sequences. In this study we report the first detection of PBVs in snakes (8.5%). We also report a phylogenetic analysis that goes beyond humans and pigs to include dogs, rats, and snakes. However, more hosts must be included in the analysis so that we may reach better conclusions regarding the spread of these viruses.

Molecular detection of Hepatozoon spp. in Brazilian and exotic wild carnivores

Hepatozoon spp. are apicomplexan parasites that infect a wide variety of animals. The infection occurs through the ingestion of a hematophagous arthropod definitive host. Herein, we assessed the presence of Hepatozoon spp. in 165 captive wild felids and 100 captive wild canids using molecular techniques. We found that 6 felids (4 little spotted cats, 1 jaguarondi, and 1 puma) and 5 canids (2 bush dogs, 1 fox, 1 crab-eating fox, and 1 maned wolf) were positive for Hepatozoon spp. Hepatozoon spp. may be a potential pathogen and an opportunistic parasite in immunocompromised animals or if occurring in concomitant infections. Because most Brazilian wild felids and canids are endangered, knowing whether Hepatozoon infection represents a threat for these animals is crucial.

Schistosoma mansoni: in vitro schistosomicidal activity of essential oil of Baccharis trimera (less) DC.

Schistosomiasis is a chronic parasitic disease caused by the trematode species Schistosoma mansoni. Chemotherapy is the only immediate recourse to minimize the prevalence and incidence of this disease worldwide. At present, praziquantel (PZQ) is the drug of choice for the treatment of all forms of schistosomiasis. However, dependence on a single drug is concern because some strains can become resistant. In this context, medicinal plants become potential candidates as sources of new drug prototypes. This study provides findings on the schistosomicidal activity of the essential oil of Baccharis trimera in in vitro assays. During the assays parameters such as motility of adult worms, oviposition, morphological changes on the tegument and especially the mortality rate of adult worms of the BH strain were evaluated. The assays, which were carried out with four concentrations - 24, 48, 91 and 130 μg/mL - of the essential oil, have shown a promising activity regarding the parameters under study. It was possible to notice a significant decline in the motility of the worms and a mortality rate of 100% 30 h after they had been exposed to the essential oil in the concentration of 130 μg/mL. Male worms were more susceptible, producing a dose-response effect within a smaller exposition period than female worms. In what refers to morphological changes, the essential oil of B. trimera induced a peeling on the tegument surface, as well as the destruction of tubercles and spines, which resulted in smooth areas on the body surface. The essential oil also caused tegument destruction in female worms, in addition to destruction of the oral and acetabular suckers. It is the first time that the schistosomicidal activity has been reported for essential oil of B. trimera (less) DC.

Molecular Detection of Cytauxzoon spp. in Asymptomatic Brazilian Wild Captive Felids

Cytauxzoon spp. DNA was detected for the first time in blood samples from asymptomatic Brazilian wild captive felids. In 2006, 72 EDTA blood samples from seven wild felids species: Puma concolor (puma), Leopardus pardalis (ocelot), Puma yagouaroundi (jaguarundi), Leopardus wiedii (margay), Leopardus tigrinus (little spotted cat), Oncifelis colocolo (pampas cat) and Panthera onca (jaguar) were analyzed using polymerase chain reaction to amplify the 18S rRNA gene segment in order to verify the presence of Cytauxzoon spp. DNA. Nine samples were positive: six ocelots, two pumas, and one jaguar. In Brazil, wild felids may be natural reservoirs for Cytauxzoon spp.

Molecular detection of tick-borne bacterial agents in Brazilian and exotic captive carnivores

The present study aims to detect and characterize by molecular techniques, the presence of tick-borne pathogens in wild captive carnivore blood samples from Brazil. Blood was collected from 76 Brazilian felids, 23 exotic felids, 3 European wolves (Canis lupus), and 97 Brazilian canids maintained in captivity in zoos located in São Paulo and Mato Grosso states, Brazil. DNA of each sample was used in PCR reactions for Ehrlichia, Anaplasma, and Rickettsia identification. The blood from 10/100 (10%) of canids (1 European wolf, 3 bush dogs, and 6 crab-eating foxes) and from 21/99 (21%) felids (4 pumas, 6 little spotted cats, 4 ocelots, 3 jaguarundis, 1 tiger, and 3 lions) contained fragments of 16S rRNA gene of Ehrlichia spp. Fragments of Anaplasma spp. groESL and 16S rRNA genes were detected in the blood of 1/100 (1%) canids (1 bush dog) and in 4/99 (3%) felids (4 little spotted cats), respectively. Rickettsia species infections were not identified. The present work showed that new strains of Ehrlichia and Anaplasma spp. circulate among wild carnivores in Brazil.

Molecular and Serologic Detection of Ehrlichia spp. in Endangered Brazilian Wild Captive Felids

Ehrlichiosis, an emergent tick-borne disease that affects both humans and animals, may represent a threat to the survival and preservation of wild felids in Brazil. There are few studies of ehrlichiosis in wild felids in Brazil, but Ehrlichia spp. are present in domestic cats. Antibodies to Ehrlichia canis have been reported in a puma (Puma concolor). In this study we assessed the presence of these hemoparasites in the blood of Brazilian wild captive felids. Of the 72 animals tested, 5 (7%) were seropositive for the E. canis antigen, and 11 (15%) were positive for E. canis DNA sequences. We also performed sequence alignment to establish the identity of the parasite species infecting these animals using 16S rRNA and omp-1 genes. Sequences based on 16S rRNA were similar to those found in dogs and cats from Thailand, Brazil, China, and Taiwan and with E. canis obtained from a single individual (human) in Venezuela. Ehrlichia sp. sequence from sampled felines based on omp-1 gene was similar to the p28 and p30 multigene family of E. canis. To our knowledge, this is the first study of molecular detection of Ehrlichia sp. in Brazilian wild feline species.

In vitro evaluation of permeation, toxicity and effect of praziquantel-loaded solid lipid nanoparticles against Schistosoma mansoni as a strategy to improve efficacy of the schistosomiasis treatment.

Solid lipid nanoparticles (SLN) are a promising drug delivery system for oral administration of poorly-water soluble drugs because of their capacity to increase the solubility of drug molecules when loaded in their lipid matrices, with the resulting improvement of the drug bioavailability. In the present work, we have developed praziquantel (PZQ)-loaded SLN and explored the biological applications of this system for intestinal permeation of PZQ. The effect in vitro on Schistosoma mansoni culture and the cytotoxicity in HepG2 line cell were also evaluated. The results showed a significant decrease in the intestinal absorption of PZQ loaded in SLN compared to free PZQ, suggesting that the SLN matrix could act as reservoir system. In culture of S. mansoni, we observed that PZQ-loaded SLN were more effective than free PZQ, leading the death of the parasites in less time. The result was proportional to doses of PZQ (25 and 50 μg mL⁻¹) and lipid concentration. Regarding cytotoxicity, the encapsulation of PZQ into SLN decreased the toxicity in HepG2 cells in comparison to the free PZQ. From the obtained results, PZQ-loaded SLN could be a new drug delivery system for the schistosomiasis treatment especially in marginalized communities, improving the therapeutic efficacy and reducing the toxic effects of PZQ.

Hypoxia, hypoxia-inducible factor-1α and vascular endothelial growth factor in a murine model of Schistosoma mansoni infection

Schistosomiasis mansoni is a chronic parasitic disease where much of the symptomatology is attributed to granuloma formation, an immunopathological reaction against Schistosoma eggs. To more clearly understand the immunopathology of schistosomiasis, the tissue microenvironment generated by S. mansoni infected mice was investigated. Using the hypoxia marker pimonidazole, we provide immunohistochemical evidence that hypoxia occurred in inflammatory cells infiltrated around the eggs and cells surrounding granulomas in the liver, intestine, spleen and lungs of infected mice. Hypoxia-inducible factor-1α (HIF-1α) was mainly expressed in inflammatory cells surrounding the eggs and in hepatocytes surrounding cellular and fibrocellular granulomas in infected mouse liver. HIF-1α expression was also verified in granulomas in the other tissues tested (intestine, spleen and lungs). Vascular endothelial growth factor (VEGF) expression was observed in the extracellular space surrounding inflammatory cells in liver granuloma. The VEGF expression pattern verified in infected mouse liver was very similar to that observed in the other tissues tested. A strong positive correlation occurred between pimonidazole binding and HIF-1α and VEGF expression in the tissues tested, except for lung. This work is the first evidence that infection by a helminth parasite, S. mansoni, produces a hypoxic tissue microenvironment and induces HIF-1α and VEGF expression.

Anthelmintic Activity In Vivo of Epiisopiloturine against Juvenile and Adult Worms of Schistosoma mansoni

Schistosomiasis is a serious disease currently estimated to affect more that 207 million people worldwide. Due to the intensive use of praziquantel, there is increasing concern about the development of drug-resistant strains. Therefore, it is necessary to search for and investigate new potential schistosomicidal compounds. This work reports the in vivo effect of the alkaloid epiisopiloturine (EPI) against adults and juvenile worms of Schistosoma mansoni. EPI was first purified its thermal behavior and theoretical solubility parameters charaterised. In the experiment, mice were treated with EPI over the 21 days post-infection with the doses of 40 and 200 mg/kg, and 45 days post-infection with single doses of 40, 100 and 300 mg/kg. The treatment with EPI at 40 mg/kg was more effective in adult worms when compared with doses of 100 and 300 mg/kg. The treatment with 40 mg/kg in adult worms reduced parasite burden significantly, lead to reduction in hepatosplenomegaly, reduced the egg burden in faeces, and decreased granuloma diameter. Scanning electron microscopy revealed morphological changes to the parasite tegument after treatment, including the loss of important features. Additionally, the in vivo treatment against juvenile with 40 mg/kg showed a reduction of the total worm burden of 50.2%. Histopathological studies were performed on liver, spleen, lung, kidney and brain and EPI was shown to have a DL50 of 8000 mg/kg. Therefore EPI shows potential to be used in schistosomiasis treatment. This is the first time that schistosomicidal in vivo activity of EPI has been reported.