Leishmania spp. parasite isolation through inoculation of patient biopsy macerates in interferon gamma knockout mice

Isolation of Leishmania parasite and species identification are important for confirmation and to help define the epidemiology of the leishmaniasis. Mice are often used to isolate pathogens, but the most common mouse strains are resistant to infection with parasites from the Leishmania (Viannia) subgenus. In this study we tested the inoculation of interferon gamma knockout (IFNγ KO) mice with biopsy macerates from Leishmania-infected patients to increase the possibility of isolating parasites. Biopsies from twenty five patients with clinical signs of leishmaniasis were taken and tested for the presence of parasites. Immunohistochemical assay (IHC) and conventional histopathology detected the parasite in 88% and 83% of the patients, respectively. Leishmania sp. were isolated in biopsy macerates from 52% of the patients by culture in Grace's insect medium, but 13% of isolates were lost due to contamination. Inoculation of macerates in IFNγ KO mice provides isolation of parasites in 31.8% of the biopsies. Most isolates belong to L. (Viannia) subgenus, as confirmed by PCR, except one that belongs to L. (Leishmania) subgenus. Our preliminary results support the use of IFNγ KO mice to improve the possibility to isolate New World Leishmania species.

Boosting the suppressive effects of Ascaris suum components in IFN-γ-deficient mice

High molecular weight components from Ascaris suum extract suppress ovalbumin-specific immunity in mice. In IFN-γ-deficient mice, ovalbumin-specific delayed-type hypersensitivity reactions are more strongly downregulated by these suppressive components. Here, the cellularity of the delayed-type hypersensitivity reaction in IFN-γ-deficient mice and the increased downregulation induced by Ascaris suum components were analyzed. IL-12p40-dependent neutrophilic influx was predominant. Suboptimal doses of the suppressive fraction from this nematode completely inhibited the hypersensitivity reaction, thus indicating intensification of the immunosuppression under conditions of intense recruitment of IFN-γ-independent neutrophils.

The anticancer drug tamoxifen is active against Trypanosoma cruzi in vitro but ineffective in the treatment of the acute phase of Chagas disease in mice

The activity of the antineoplastic drug tamoxifen was evaluated against Trypanosoma cruzi. In vitro activity was determined against epimastigote, trypomastigote and amastigote forms of CL14, Y and Y benznidazole resistant T. cruzi strains. Regardless of the strain used, the drug was active against all life-cycle stages of the parasite with a half maximal effective concentration ranging from 0.7-17.9 µM. Two experimental models of acute Chagas disease were used to evaluate the in vivo efficacy of treatment with tamoxifen. No differences in parasitemia and mortality were observed between control mock-treated and tamoxifen-treated mice.

Antibody responses elicited in mice immunized with Bacillus subtilis vaccine strains expressing Stx2B subunit of enterohaemorragic Escherichia coli O157:H7

No effective vaccine or immunotherapy is presently available for patients with the hemolytic uremic syndrome (HUS) induced by Shiga-like toxin (Stx) producedbyenterohaemorragic Escherichia coli (EHEC) strains, such as those belonging to the O157:H7 serotype. In this work we evaluated the performance of Bacillus subtilis strains, a harmless spore former gram-positive bacterium species, as a vaccine vehicle for the expression of Stx2B subunit (Stx2B). A recombinant B. subtilis vaccine strain expressing Stx2B under the control of a stress inducible promoter was delivered to BALB/c mice via oral, nasal or subcutaneous routes using both vegetative cells and spores. Mice immunized with vegetative cells by the oral route developed low but specific anti-Stx2B serum IgG and fecal IgA responses while mice immunized with recombinant spores developed anti-Stx2B responses only after administration via the parenteral route. Nonetheless, serum anti-Stx2B antibodies raised in mice immunized with the recombinant B. subtilis strain did not inhibit the toxic effects of the native toxin, both under in vitro and in vivo conditions, suggesting that either the quantity or the quality of the induced immune response did not support an effective neutralization of Stx2 produced by EHEC strains.

Analysis of mangrove forest succession, using sediment cores: a case study in the Cananéia -Iguape coastal system, São Paulo Brazil

Sediment cores are an essential tool for the analysis of the dynamics of mangrove succession. Coring was used to correlate changes in depositional environments and lateral sedimentary facies with discrete stages of forest succession at the Cananéia-Iguape Coastal System in southeastern Brazil. A local level successional pattern was examined based on four core series T1) a sediment bank; T2) a smooth cordgrass Spartina alterniflora bank; T3) an active mangrove progradation fringe dominated by Laguncularia racemosa, and; T4) a mature mangrove forest dominated by Avicennia schaueriana. Cores were macroscopically described in terms of color, texture, sedimentary structure and organic components. The base of all cores exhibited a similar pattern suggesting common vertical progressive changes in depositional conditions and subsequent successional colonization pattern throughout the forest. The progradation zone is an exposed bank, colonized by S. alterniflora. L. racemosa, replaces S. alterniflora as progradation takes place. As the substrate consolidates A. schaueriana replaces L. racemosa and attains the greatest structural development in the mature forest. Cores collected within the A. schaueriana dominated stand contained S. alterniflora fragments near the base, confirming that a smooth cordgrass habitat characterized the establishment and early seral stages. Cores provide a reliable approach to describe local-level successional sequences in dynamic settings subject to drivers operating on multiple temporal and spatial scales where spatial heterogeneity can lead to multiple equilibria and where similar successional end-points may be reached through convergent paths.

Habitat suitability of Anopheles vector species and association with human malaria in the Atlantic Forest in south-eastern Brazil

Every year, autochthonous cases of Plasmodium vivax malaria occur in low-endemicity areas of Vale do Ribeira in the south-eastern part of the Atlantic Forest, state of São Paulo, where Anopheles cruzii and Anopheles bellator are considered the primary vectors. However, other species in the subgenus Nyssorhynchus of Anopheles (e.g., Anopheles marajoara) are abundant and may participate in the dynamics of malarial transmission in that region. The objectives of the present study were to assess the spatial distribution of An. cruzii, An. bellator and An. marajoara and to associate the presence of these species with malaria cases in the municipalities of the Vale do Ribeira. Potential habitat suitability modelling was applied to determine both the spatial distribution of An. cruzii, An. bellator and An. marajoara and to establish the density of each species. Poisson regression was utilized to associate malaria cases with estimated vector densities. As a result, An. cruzii was correlated with the forested slopes of the Serra do Mar, An. bellator with the coastal plain and An. marajoara with the deforested areas. Moreover, both An. marajoara and An. cruzii were positively associated with malaria cases. Considering that An. marajoara was demonstrated to be a primary vector of human Plasmodium in the rural areas of the state of Amapá, more attention should be given to the species in the deforested areas of the Atlantic Forest, where it might be a secondary vector.

Effect of CO2 and 1-octen-3-ol attractants for estimating species richness and the abundance of diurnal mosquitoes in the southeastern Atlantic forest, Brazil

Studies have shown that both carbon dioxide (CO2) and octenol (1-octen-3-ol) are effective attractants for mosquitoes. The objective of the present study was to evaluate the attractiveness of 1-octen-3-ol and CO2 for diurnal mosquitoes in the southeastern Atlantic forest. A Latin square experimental design was employed with four treatments: CDC-light trap (CDC-LT), CDC-LT and 1-octen-3-ol, CDC-LT and CO2 and CDC-LT with 1-octen-3-ol and CO2. Results demonstrated that both CDC-CO2 and CDC-CO2-1-octen-3-ol captured a greater number of mosquito species and specimens compared to CDC-1-octen-3-ol; CDC-LT was used as the control. Interestingly, Anopheles (Kerteszia) sp. was generally attracted to 1-octen-3-ol, whereas Aedes serratus was the most abundant species in all Latin square collections. This species was recently shown to be competent to transmit the yellow fever virus and may therefore play a role as a disease vector in rural areas of Brazil.

Observations on Haemagogus janthinomys Dyar (Diptera: Culicidae) and other mosquito populations within tree holes in a gallery forest in the northwestern region of Sao Paulo state, Brazil

In 2000, an outbreak of sylvatic yellow fever possibly occurred in gallery forests of the Grande river in the Paraná basin in the northwestern region of São Paulo state. The aim of this study was to obtain information on the bionomics of Haemagogus and other mosquitoes inside tree holes in that area. Eighteen open tree holes were sampled for immature specimens. Adults were collected twice a month in the forest in Santa Albertina county from July 2000 to June 2001. The seasonal frequency of fourth instars was obtained by the Williams geometric mean (Mw), while the adult frequency was estimated either by hourly arithmetic or the Williams' means. Cole's index was applied to evaluate larval inter-specific associations. Among the ten mosquito species identified, the most abundant was Aedes terrens Walker followed by Sabethes tridentatus Cerqueira and Haemagogus janthinomys Dyar. Larval and adult abundance of these species was higher in summer than in winter. Although larval abundance of Hg. janthinomys peaked in the rainy season, correlation with rainfall was not significant. Six groups of larval associations were distinguished, one of which the most positively stable. The Hg. janthinomys and Ae. terrens association was significant, and Limatus durhamii Theobald was the species with most negative associations.

Pathogenicity of different rabies virus isolates and protection test in vaccinated mice

This study was aimed to evaluate and compare the pathogenicity of rabies virus isolated from bats and dogs, and to verify the efficacy of a commercial rabies vaccine against these isolates. For evaluation of pathogenicity, mice were inoculated by the intramuscular route (IM) with 500MICLD50/0.03mL of the viruses. The cross-protection test was performed by vaccinating groups of mice by the subcutaneous route and challenged through the intracerebral (IC) route. Isolates were fully pathogenic when inoculated by the IC route. When inoculated intramuscularly, the pathogenicity observed showed different death rates: 60.0% for the Desmodus rotundus isolate; 50.0% for dog and Nyctinomops laticaudatus isolates; 40.0% for Artibeus lituratus isolate; 9.5% Molossus molossus isolate; and 5.2% for the Eptesicus furinalis isolate. Mice receiving two doses of the vaccine and challenged by the IC route with the isolates were fully protected. Mice receiving only one dose of vaccine were partially protected against the dog isolate. The isolates from bats were pathogenic by the IC route in mice. However, when inoculated through the intramuscular route, the same isolates were found with different degrees of pathogenicity. The results of this work suggest that a commercial vaccine protects mice from infection with bat rabies virus isolates, in addition to a canine rabies virus isolate.

Occurrence and molecular characterization of Cryptosporidium spp. isolated from domestic animals in a rural area surrounding Atlantic dry forest fragments in Teodoro Sampaio municipality, State of São Paulo, Brazil

The aim of this study was to assess the occurrence of Cryptosporidium in domestic animals in rural properties surrounding rain forest fragments within the municipality of Teodoro Sampaio, southeastern Brazil. Conventional sucrose flotation method followed by molecular characterization of the parasites by sequencing PCR products amplified from SSU rRNA gene were used. Stool samples were collected from domestic animals raised as pets and livestock in all rural properties surrounding three forest fragments. Samples from cattle (197), equine (63), pigs (25), sheep (11), and dogs (28) were collected from 98 rural properties. The frequency of occurrence of Cryptosporidium within each animal species was 3.0% (6/197) among cattle and 10.7% (3/28) among dogs. Cryptosporidium was not detected in stool samples from equine, sheep, and pigs. All sequences obtained from the six samples of calves showed molecular identity with Cryptosporidium andersoni while all sequences from dog samples were similar to C. canis. The frequency of occurrence of Cryptosporidium in these domestic animal species was low. The absence of C. parvum in the present study suggests that the zoonotic cycle of cryptosporidiosis may not be relevant in the region studied. The presence of Cryptosporidium species seldom described in humans may be, otherwise, important for the wild fauna as these animals are a source of infection and dissemination of this protozoan to other animal species. The impact and magnitude of infection by C. andersoni in wild ruminants and C. canis in wild canids have to be assessed in future studies to better understand the actual importance of these species in this region.

Antidepressant-like effects of an apolar extract and chow enriched with Nepeta cataria (catnip) in mice

Nepeta cataria (catnip) is a plant used in pet toys and to treat human diseases. Catnip has also been used in the treatment of some depressive disorders. In this paper, we studied the antidepressant, anxiogenic, and motor activity effects of acute and repeated feeding of chow enriched with 10% N. cataria leaves and the acute and repeated administration of apolar and polar extracts of N. cataria leaves in male mice. The results showed that repeated feeding and acute and repeated administration with the apolar extract reduced immobility in the behavioral despair test but did not alter elevated plus maze and open-field parameters. Acute feeding and the acute and repeated administration of the polar extracts of N. cataria leaves did not alter the behavior of mice. These data suggest that N. cataria has antidepressant properties. Moreover, this antidepressant activity was present in the apolar extract.

Effect of cyhalothrin on Ehrlich tumor growth and macrophage activity in mice

Cyhalothrin, a pyrethroid insecticide, induces stress-like symptoms, increases c-fos immunoreactivity in the paraventricular nucleus of the hypothalamus, and decreases innate immune responses in laboratory animals. Macrophages are key elements in cellular immune responses and operate at the tumor-host interface. This study investigated the relationship among cyhalothrin effects on Ehrlich tumor growth, serum corticosterone levels and peritoneal macrophage activity in mice. Three experiments were done with 10 experimental (single gavage administration of 3.0 mg/kg cyhalothrin daily for 7 days) and 10 control (single gavage administration of 1.0 mL/kg vehicle of cyhalothrin preparation daily for 7 days) isogenic BALB/c mice in each experiment. Cyhalothrin i) increased Ehrlich ascitic tumor growth after ip administration of 5.0 x 106 tumor cells, i.e., ascitic fluid volume (control = 1.97 ± 0.39 mL and experimental = 2.71 ± 0.92 mL; P < 0.05), concentration of tumor cells/mL in the ascitic fluid (control = 111.95 ± 16.73 x 106 and experimental = 144.60 ± 33.18 x 106; P < 0.05), and total number of tumor cells in the ascitic fluid (control = 226.91 ± 43.22 x 106 and experimental = 349.40 ± 106.38 x 106; P < 0.05); ii) increased serum corticosterone levels (control = 200.0 ± 48.3 ng/mL and experimental = 420.0 ± 75.5 ng/mL; P < 0.05), and iii) decreased the intensity of macrophage phagocytosis (control = 132.3 ± 19.7 and experimental = 116.2 ± 4.6; P < 0.05) and oxidative burst (control = 173.7 ± 40.8 and experimental= 99.58 ± 41.7; P < 0.05) in vitro in the presence of Staphylococcus aureus. These data provide evidence that cyhalothrin simultaneously alters host resistance to Ehrlich tumor growth, hypothalamic-pituitary-adrenocortical (HPA) axis function, and peritoneal macrophage activity. The results are discussed in terms of data suggesting a link between stress, HPA axis activation and resistance to tumor growth.

Dose-response effects of systemic anandamide administration in mice sequentially submitted to the open field and elevated plus-maze tests

The endocannabinoid system is involved in the control of many physiological functions, including the control of emotional states. In rodents, previous exposure to an open field increases the anxiety-like behavior in the elevated plus-maze. Anxiolytic-like effects of pharmacological compounds that increase endocannabinoid levels have been well documented. However, these effects are more evident in animals with high anxiety levels. Several studies have described characteristic inverted U-shaped dose-response effects of drugs that modulate the endocannabinoid levels. However, there are no studies showing the effects of different doses of exogenous anandamide, an endocannabinoid, in animal models of anxiety. Thus, in the present study, we determined the dose-response effects of exogenous anandamide at doses of 0.01, 0.1, and 1.0 mg/kg in C57BL/6 mice (N = 10/group) sequentially submitted to the open field and elevated plus-maze. Anandamide was diluted in 0.9% saline, ethyl alcohol, Emulphor® (18:1:1) and administered ip (0.1 mL/10 g body weight); control animals received the same volume of anandamide vehicle. Anandamide at the dose of 0.1 mg/kg (but not of 0.01 or 1 mg/kg) increased (P < 0.05) the time spent and the distance covered in the central zone of the open field, as well as the exploration of the open arms of the elevated plus-maze. Thus, exogenous anandamide, like pharmacological compounds that increase endocannabinoid levels, promoted a characteristic inverted U-shaped dose-response effect in animal models of anxiety. Furthermore, anandamide (0.1 mg/kg) induced an anxiolytic-like effect in the elevated plus-maze (P < 0.05) after exposing the animals to the open field test.

Remyelination in experimentally demyelinated connexin 32 KnockOut mice

The aim of this study was to evaluate the role of connexin 32 (Cx 32) during remyelination of the peripheral nervous system, through a local injection of either 0,1% ethidium bromide solution or saline in the sciatic nerve of Cx 32 knockout mice. Euthanasia was performed ranging from 1, 2, 3, 7, 15, 21 to 30 days after injection. Histochemical, immunohistochemical, immunofluorescence and transmission electron microscopical techniques were used to analyze the development of the lesions. Within the sciatic nerves, Schwann cells initially showed signs of intoxication and rejected their sheaths; after seven days, some thin newly formed myelin sheaths with uneven compactness and redundant loops (tomacula) were conspicuous. We concluded that the regeneration of lost myelin sheaths within the PNS followed the pattern already reported for this model in other laboratory species. Therefore, these results suggest that absence of Cx 32 did not interfere with the normal pattern of remyelination in this model in young mice.

Entomological characterization and natural infection of anophelines in an area of the Atlantic Forest with autochthonous malaria cases in mountainous region of Espírito Santo State, Brazil

No Espírito Santo, os casos de malária autóctone estão distribuídos na região serrana próximo aos fragmentos de Mata Atlântica. Uma vez que alguns aspectos da doença são obscuros, a detecção das possíveis espécies de vetores pode auxiliar na elucidação de incertezas epidemiológicas. Estudos entomológicos e de infecção natural foram realizados com anofelinos (Diptera: Culicidae) capturados no município de Santa Tereza, ES. Capturas mensais foram realizadas de março de 2004 a fevereiro de 2006. Armadilhas CDC-CO2 foram utilizadas do crepúsculo (18:00h) ao amanhecer (6:00h), para capturar anofelinos nos seguintes habitats: próximo ao domicílio e área aberta (solo), margem e interior da mata (solo e copa). Armadilhas Shannon também foram utilizadas nos mesmos locais que as de CDC-CO2. Capturou-se o total de 2.290 anofelinos distribuídos em 10 espécies. A maior frequência relativa foi de Anopheles (Kerteszia) cruzii Dyar & Knab / A.(K.) homunculus Komp, sendo a maioria capturada em CDC-CO2 instalada na copa da mata. A principal espécie capturada em armadilha Shannon foi A.(Nyssorhynchus) strodei Root. O maior número de anofelinos foi capturado entre julho e setembro das 18:00h às 22:00h. Provavelmente A.(K.) cruzii é responsável pela transmissão da malária dentro ou próximo aos fragmentos de Mata Atlântica. Entretanto, a participação de outras espécies não pode ser ignorada, visto que 53 por cento da amostragem foi constituída pelo subgênero Nyssorhynchus. A detecção de Plasmodium vivax no tórax de A. cruzii, A. parvus (Chagas) e A. galvaoi Causey, Deane & Deane por meio de PCR reforça esse argumento