Schistosoma mansoni: In vivo evaluation of Phyllanthus amarus hexanic and ethanolic extracts.

Human schistosomiasis is an important neglected tropical disease caused by blood flukes of the genus Schistosoma and is responsible for more than 280,000 deaths annually. Treatment for this disease relies currently on a single drug, praziquantel (PZQ). Concerns regarding PZQ resistance and insensitivity of juvenile schistosomes have increased the interest in resorting to medicinal plants for alternative drug therapies. This study aimed to perform an in vivo schistosomicidal activity evaluation of crude hexanic (HE) and ethanolic (EE) extracts obtained from Phyllanthus amarus in mice infected with Schistosoma mansoni (BH strain). Mice were treated orally with a single dose of 100 or 250 mg/kg, on two different infection periods, 30 and 45 days post-infection (dpi). Parameters such as worm recovery, faecal egg count, intestinal tissue egg count and liver histopathology were evaluated. Treatment against young adult (30 dpi) and adult (45 dpi) worms were more effective compared to the control group treated with PZQ. At a concentration of 250 mg/kg (30 dpi) EE showed a 54.4% female reduction and a 61.2% total worm reduction whilst at a concentration of 100 mg/kg (45 dpi) HE showed a 40.6% female worm reduction and a 45.3% total worm reduction. Histopathological examination showed a granuloma decrease in both number and size for groups treated with 250 mg/kg of HE (45 dpi) or EE (30 or 45 dpi). From these results, it can be concluded that both hexanic and ethanolic extracts have antischistosomal activities, however, act differently according to the parasites age. The schistosomicidal activity results in groups treated 30 days post infection is extremely important since praziquantel does not show activity against the juvenile forms of Schistosoma.

Ameliorating the antiparasitic activity of the multifaceted drug ivermectin through a polymer nanocapsule formulation.

Ivermectin (IVM) is a potent antiparasitic widely used in human and veterinary medicine. However, the low oral bioavailability of IVM restricts its therapeutic potential in many parasitic infections, highlighting the need for novel formulation approaches. In this study, poly(ε-caprolactone) (PCL) nanocapsules containing IVM were successfully developed using the nanoprecipitation method. Pumpkin seed oil (PSO) was used as an oily core in the developed nanocapsules. Previously, PSO was chemically analyzed by headspace solid-phase microextraction coupled to gas chromatography/mass spectrometry (HS-SPME/GC-MS). The solubility of IVM in PSO was found to be 4266.5 ± 38.6 µg/mL. In addition, the partition coefficient of IVM in PSO/water presented a logP of 2.44. A number of nanocapsule batches were produced by factorial design resulting in an optimized formulation. Negatively charged nanocapsules measuring around 400 nm demonstrated unimodal size distribution, and presented regular spherical morphology under transmission electron microscopy. High encapsulation efficiency (98-100%) was determined by HPLC. IVM-loaded capsules were found to be stable in nanosuspensions at 4 °C and 25 °C, with no significant variations in particle size observed over a period of 150 days. Nanoencapsulated IVM (0.3 mM) presented reduced toxicity to J774 macrophages and L929 fibroblasts compared to free IVM. Moreover, IVM-loaded nanocapsules also demonstrated enhanced in vitro anthelmintic activity against Strongyloides venezuelensis in comparison to free IVM. Collectively, the present findings demonstrate the promising potential of PCL-PSO nanocapsules to improve the antiparasitic effects exerted by IVM.

The effect of Spondias mombin L. against Strongyloides venezuelensis: An in vitro approach.

Strongyloidiasis is a neglected tropical disease mainly caused by the nematode parasite Strongyloides stercoralis. Current treatment consists in the administration of ivermectin or, alternatively, albendazole (or analogues). Concerns regarding these drugs' irregular cure rates and side effects, raise a need for therapeutic alternatives. In this study, we tested the in vitro effect of Spondias mombin L. ethanolic extract against the laboratory model for strongyloidiasis, Strongyloides venezuelensis. The ethanolic extract was further fractionated and each fraction was also tested. Tested fractions were analyzed through thin layer chromatography and gas chromatography (GC/MS). Our results showed that S. mombin extract and fractions had a better in vitro effect than ivermectin, particularly fraction 4 which showed the better results causing 100% mortality in 4 h after exposure to an extract concentration of 400 µg/mL of RPMI medium and caused 100% mortality 12 h after exposure to an extract concentration of 50 µg/mL. Scanning electron microscopy showed that this fraction caused both wrinkling and peeling of the parasites cuticle, whilst ivermectin only caused wrinkling. GC/MS showed a high percentage of monoaromatic phenolic lipids (3-R phenol and 3-R1 phenol), which were likely responsible for the anti-Strongyloides effect. The use of polyvinylpyrrolidone reduced the efficiency, thus raising a need for alertness when using this excipient. Our results suggest that S. mombin is a potential source of compounds that could be used for stongyloidiasis treatment.

Centesimal composition and meat yield of Hoplias malabaricus: association with intestinal parasites.

Hoplias malabaricus is a non-migratory fish commonly found in the Mogi Guaçu River basin, mainly feeding on fish, small crustaceans and insects. It forms part of the diet for humans, birds and some mammals. This fish has great nutritional value, with both good quality and good quantities of essential vitamins and amino acids. Regarding parasitic fauna, this fish can host different species of helminths in its gastrointestinal tract. The aim of the present study was to investigate the possible interference of parasitism in the meat yield from H. malabaricus and the centesimal composition. For this purpose, fish specimens were collected from marginal lagoons of the Mogi Guaçu River (Pirassununga, state of São Paulo, Brazil) using hooks and fishing nets. We found that all specimens of H. malabaricus were parasitized by at least one species, including larvae of Contracaecum sp. (Nematoda: Anisakidae). Parasitism did not have any significant influence on centesimal composition, but meat yield was negatively correlated with the abundance of larvae.

Investigating Immunization With Nucleotide Enzymes of Schistosoma mansoni: Nucleoside Diphosphate Kinase and Adenylosuccinate Lyase as New Antigenic Targets Against Schistosomiasis.

Schistosomiasis, caused by Schistosoma mansoni trematode worm, affects more than 1.5 million people in Brazil. The current treatment consists in the administration of Praziquantel, the only medicine used for treatment for more than 40 years. Some of the limitations of this drug consist in its inactivity against schistosomula and parasite eggs, the appearance of resistant strains and non-prevention against reinfection. Thus, the objective of this study was to evaluate the effect of immunization with recombinant functional enzymes of the purine salvage pathway of S. mansoni, Nucleoside Diphosphate Kinase (NDPK) and Adenylosuccinate Lyase (ADSL), to evaluate the host immune response, as well as the parasite load after vaccination. For this, Balb/c mice were divided into 5 groups: control (uninfected and untreated), non-immunized/infected, NDPK infected, ADSL infected, and NDPK + ADSL infected. Immunized groups received three enzyme dosages, with a 15-day interval between each dose, and after 15 days of the last application the animals were infected with 80 cercariae of S. mansoni. On the 47th day after the infection, fecal eggs were counted and, on the 48th day after the infection, the evaluation of leukocyte response, parasite load, antibody production, cytokines quantification, and histopathological analysis were performed. The results showed that immunizations with NDPK, ADSL or NDPK + ADSL promoted a discreet reduction in eosinophil counts in lavage of peritoneal cavity. All immunized animals showed increased production and secretion of IgG1, IgG2a, and IgE antibodies. Increased production of IL-4 was observed in the group immunized with the combination of both enzymes (NDPK + ADSL). In addition, in all immunized groups there were reductions in egg counts in the liver and intestine, such as reductions in liver granulomas. Thus, we suggest that immunizations with these enzymes could contribute to the reduction of schistosomiasis transmission, besides being important in immunopathogenesis control of the disease.

Proteomic, metabolic and immunological changes in Biomphalaria glabrata infected with Schistosoma mansoni.

Mansonic schistosomiasis is a neglected disease transmitted by Biomphalaria spp. snails. Understanding what happens inside the intermediate host is important to develop more efficient ways of reducing schistosomiasis prevalence. Our purpose was to characterize metabolic and immunological changes in Biomphalaria glabrata 24 h after exposure to Schistosoma mansoni. For this purpose, proteins were extracted from snails' whole tissue with Tris-Urea buffer and digested with tripsin. Mass spectrometry was performed and analyzed with MaxQuant and Perseus software. Also, the hemolymph of five snails 24 h post exposure was collected, and the numbers of hemocytes, levels of urea, uric acid, nitric oxide, calcium, glycogen and alanine and aspartate aminotransferases activities were assessed. Snails were also dissected for measurement of glycogen content in the cephalopodal region and gonoda-digestive gland complex. Globin domain proteins were found to be up-regulated; also the number of circulating hemocytes was significantly higher after 24 h of exposure to the parasite. NO levels were higher 24 h post exposure. Several proteins associated with energy metabolism were found to be up-regulated. Glycogen analysis showed a significant decrease in the gonad-digestive gland complex glycogen content. We found several proteins which seem to be associated with the host immune response, most of which were up-regulated, however some were down-regulated, which may represent an important clue in understanding B. glabrata - S. mansoni compatibility.

Bovine fasciolisis in São Paulo state, Brazil.

Fasciolosis is a zoonotic disease with a worldwide distribution caused by Fasciola hepatica, which leads to severe economic losses in cattle such as reducing meat and milk production, livers condemnation, growth retardation and increase in mortality. From October 2008 to April 2011, condemned bovine livers in slaughterhouses of different municipalities from São Paulo state, Brazil were evaluated for the presence of Fasciola hepatica. Out of 20,635 analyzed livers, 1422 were infected with F. hepatica. These cattle came from 33 municipalities, out of which 16 showed infected animals and where 7 municipalities did not show statistical difference between each month throughout the year: Tuiuti - 276/1408 (19,6%), Atibaia - 44/257 (17,1%), Joanópolis - 116/738 (15,7%), Bragança Paulista - 318/2316 (13,3%), Piracaia - 182/1442 (12,6%), Santo Antonio de Posse - 118/1005 (11,7%), Amparo 131/2003 (6,5%). The other nine municipalities, Monte Alegre do Sul, Descalvado, Campinas, Morungaba, Pedreira, Socorro, Munhoz, Jaguariúna and Itatiba showed a positive percentage varying from 5.08% to 1.46%. Our results demonstrated the presence of F. hepatica in this region was higher than official data, bringing the need for control measures. There is also an apparent increase in fasciolosis two to three months after low to medium precipitation, however high precipitation causes a decrease in fasciolosis prevalence.

Gastrointestinal, skin and blood parasites in Didelphis spp. from urban and sylvatic areas in São Paulo state, Brazil.

Didelphis (Marsupialia, Didelphimorphia) are synanthropic mammals, whose omnivorous diet predisposes them to infections caused by endoparasites. Their higher frequency in urban areas makes them potential carriers of zoonotic protozoans and helminths, enhancing potential transmission to humans. Our purpose was to study two common species, Didelphis albiventris (54 individuals) and D. aurita (2 individuals), which were screened for blood, skin and intestinal parasites in animals captured in urban areas and in riparian forest regions associated with the Capivari River Basin, in Monte Mor's municipality, São Paulo state (SP), Brazil. Blood and tissue samples were collected for DNA extraction and PCR. Fecal samples were collected and submitted to two sedimentation and two flotation methods. 77.6% of fecal samples were positive for nematode eggs, 34.5% for trematode eggs and 32.7% for protozoans. Two D. aurita specimens were naturally infected by Trypanosoma cruzi. Molecular analysis in a D. albiventris captured on a forested rural area was positive for Leishmania sp. DNA. Several parasites were found infecting Didelphis sp., demonstrating that this group of animals can harbor important zoonotic parasites, potentially playing a role as sylvatic reservoirs for human and domestic animal pathogens.

Sesquiterpenes evaluation on Schistosoma mansoni: Survival, excretory system and membrane integrity.

The main challenge in schistosomiasis control has been the emergence of drug-resistant parasites. Since the 1970's, praziquantel (PZQ) is the single drug for treatment. This fact highlights the importance to research news chemotherapeutic agents. In the last years, S. mansoni excretory system and tegument have been major targets for drug development. The purpose of this study was to evaluate the effect of sesquiterpenes, alpha-humulene and trans-caryophyllene on S. mansoni survival, excretory system and membrane integrity, after in vitro exposure. The in vitro studies, showed that sesquiterpenes reduced egg production and motor activity of worms at sublethal concentrations, and caused death in a concentration-dependent manner (100 and 200µg/mL). Tegumental analysis by Scanning Electron Microscopy (SEM), showed tegument damage. Additionally, it was possible to observe lesions, evidenced by intense marking trough Hoechst probe, in the tegument and suckers of worms exposed to 200µg/mL. In this study, we also showed that resorufin is only capable of identifying the interaction of sesquiterpenes in males excretory system, Pgp expression and inferring that females are more tolerant to treatments. Thus, the present study results contribute to an understanding of alpha-humulene and trans-caryophyllene effect over these targets, contributing for the development of schistosomicidal drugs.