Effect of salivary CYP4EM1 and CYP4EM2 gene silencing on the life span of Chagas disease vector Rhodnius prolixus (Hemiptera, Reduviidae) exposed to sublethal dose of deltamethrin.

Control of Chagas disease in endemic countries is primarily accomplished through insecticide spraying for triatomine vectors. In this context, pyrethroids are the first-choice insecticide, and the evolution of insect resistance to these insecticides may represent an important barrier to triatomine control. In insects, cytochrome P450s are enzymes involved in the metabolism of xenobiotics and endogenous chemicals that are encoded by genes divided into different families. In this work, we evaluated the role of three Rhodnius prolixus CYP4EM subfamily genes during blood meal and after deltamethrin exposure. CYP4 gene members were expressed in different insect organs (integument, salivary glands (SGs), midgut, fat body and malpighian tubules) at distinct transcriptional levels. CYP4EM1 gene was highly expressed in the SG and was clearly modulated after insect blood meal. Injection of CYP4EM1dsRNA promoted significant reduction in mRNA levels of both CYP4EM1 and CYP4EM2 genes and induced deleterious effects in R. prolixus nymphs subsequently exposed to sublethal doses of deltamethrin (3.4 or 3.8 ng/nymph treated). The higher dose reduced the survival over time and increased susceptibility of R. prolixus nymphs to deltamethrin. A better understanding of this mechanism can help in developing of more efficient strategies to reduce Trypanosoma cruzi vector transmission in Americas.

Maternal and fetal immune response patterns in heifers experimentally infected with Neospora caninum in the second trimester of pregnancy--a descriptive study.

Fetal and maternal immune responses 3, 6 and 9 weeks post infection (wpi) were investigated in cows experimentally infected with Neospora caninum on day 110 of gestation. Descriptive analysis showed that the fetuses had lower percentages of spleen T cell subpopulations (CD3+, CD4+ and CD8+) at 6 wpi compared to 3 wpi and/or 9 wpi, with the lowest percentages observed in a dead fetus found upon euthanasia at that time. Increased expression of most cytokines over levels recorded at 3 and 9 wpi were found in fetuses that were alive at 6 wpi. Up-regulated Th1, Th2 and Treg expression was also observed at 6 wpi in the spleen and in the lymph nodes draining the placenta of the cows. At the placental level, while most cytokines were down-regulated from 6 wpi, up-regulation of IL-4 expression was observed at 6 wpi in the caruncle. Our results suggest that the immune response at 6 wpi was crucial for fetal survival in this model of bovine neosporosis.

Cytokine gene expression at the materno-foetal interface after experimental Neospora caninum infection of heifers at 110 days of gestation.

Neospora caninum is a major cause of abortion in cattle, but the reasons why only some animals abort remain unclear. The immunological control of the parasite in the placenta or by the foetus could be the key to determining the mechanism of abortion and/or transplacental transmission to the foetus. In this study, cytokine gene expression, analysed by real-time RT-PCR, at the maternal (caruncle) and foetal placenta (cotyledon) of heifers infected at 110 days of gestation by intravenous inoculation of N. caninum tachyzoites was compared with the responses in uninfected heifers. Animals were euthanized 3 weeks after infection. Upregulated Th1, Th2 and T-regulatory (Treg) cytokine gene expression was observed in both the maternal and the foetal placenta in the infected group. In the caruncle of infected animals, the main changes included upregulation of IFN-γ, IL-12p40, IL-6 and IL-10. In the cotyledon, the main changes included upregulation of IFN-γ and downregulation of TGF-ß, being the later the only cytokine downregulated in the infected group. The observed cytokine expression pattern was associated with alive but transplacentally infected foetuses, suggesting that such cytokine pattern is beneficial to foetal survival, but could have a role in the transplacental transmission of the parasite.

Cytokines profile in cystic fibrosis patients with nasal polyps.

UNLABELLED: Nasal polyposis often complicates the progress of patients with cystic fibrosis and there has been little study about the importance of cytokines in the polyps of such individuals. OBJECTIVE: To assess RNAm expression for interleukins 4, 5, 6, 8, GM-CSF and IFN-gamma by RT-PCR in eosinophilic and non-eosinophilic polyps of patients with cystic fibrosis. MATERIAL AND METHOD: A total of 124 persons were evaluated, of which thirteen patients with cystic fibrosis and nasosinusal polyps were selected--three were eosinophilic and ten were non-eosinophilic. The control group was composed of eleven individuals with normal otorhinolaryngological exam and the mean age was 18 years (3-57). The middle turbinate mucosa and nasal polyps were biopsied from the control group and the cystic fibrosis group respectively, and these were analyzed with RT-PCR. The middle turbinate mucosa was biopsied in the control group and in the cystic fibrosis group polyps that was analyzed to RT-PCR. The polyps of cystic fibrosis patients were also further anaylsed for subjected to a second biopsy in order to determine the percentage of eosinophils. IL-4, IL-5, IL-6, IL-8, IFN-gamma and GM-CSF transcriptions were analyzed. RESULTS: There was no difference in IL-5, IL-8 and GM-CSF when compared to the eosinophilic, non-eosinophilic and control groups (p>0.05). When compared to the eosinophilic and non-eosinophilic groups, higher IL-4 and IL-6 values (p=0.01 and p=0.01 respectively) were observed. When analyzed separately with the control group, IL-4 (p=0.01) expression was higher in the eosinophilic group, while IFN-gamma (p=0.03) was lower in the non-eosinophilic group. IL-5, IL-8, GM-CSF are non-specific cytokines present in the nasosinusal sinonasal polyps of cystic fibrosis patients. IL-4 and IL-6 are important mediators in the eosinophilic sinonasal polyps, while low IFN-gamma may be related to lower eosinophils in non-eosinophilic polyps.

Brasiliensin: A novel intestinal thrombin inhibitor from Triatoma brasiliensis (Hemiptera: Reduviidae) with an important role in blood intake.

Every hematophagous invertebrate studied to date produces at least one inhibitor of coagulation. Among these, thrombin inhibitors have most frequently been isolated. In order to study the thrombin inhibitor from Triatoma brasiliensis and its biological significance for the bug, we sequenced the corresponding gene and evaluated its biological function. The T. brasiliensis intestinal thrombin inhibitor, termed brasiliensin, was sequenced and primers were designed to synthesize double strand RNA (dsRNA). Gene knockdown (RNAi) was induced by two injections of 15mug of dsRNA into fourth instar nymphs. Forty-eight hours after the second injection, bugs from each group were allowed to feed on hamsters. PCR results showed that injections of dsRNA reduced brasiliensin expression in the anterior midgut by approximately 71% in knockdown nymphs when compared with controls. The reduction in gene expression was confirmed by the thrombin inhibitory activity assay and the citrated plasma coagulation time assay which showed activity reductions of approximately 18- and approximately 3.5-fold, respectively. Knockdown nymphs ingested approximately 39% less blood than controls. In order to confirm the importance of brasiliensin in blood ingestion, fourth instar nymphs were allowed to ingest feeding solution alone or feeding solution containing 15U of thrombin prior to blood feeding. Fifty-five percent less blood was ingested by nymphs which were fed thrombin prior to blood feeding. The results suggest that anticoagulant activity in the midgut is an important determinant of the amount of blood taken from the host. The role of anticoagulants during blood ingestion is discussed in the light of this novel insight.

RNA interference of the salivary gland nitrophorin 2 in the triatomine bug Rhodnius prolixus (Hemiptera: Reduviidae) by dsRNA ingestion or injection.

Mass sequencing of cDNA libraries from salivary glands of triatomines has resulted in the identification of many novel genes of unknown function. The aim of the present work was to develop a functional RNA interference (RNAi) technique for Rhodnius prolixus, which could be widely used for functional genomics studies in triatomine bugs. To this end, we investigated whether double-stranded RNA (dsRNA) can inhibit gene expression of R. prolixus salivary nitrophorin 2 (NP2) and what impact this might have on anticoagulant and apyrase activity in the saliva. dsRNA was introduced by two injections or by ingestion. RT-PCR of the salivary glands showed that injections of 15 microg of NP2 dsRNA in fourth-instar nymphs reduced gene expression by 75+/-14% and that feeding 1 microg/microL of NP2 dsRNA into second-instar nymphs (approx. 13 microg in total) reduced gene expression by 42+/-10%. Phenotype analysis showed that saliva of normal bugs prolonged plasma coagulation by about four-fold when compared to saliva of knockdown bugs. These results and the light color of the salivary gland content from some insects are consistent with the knockdown findings. The findings suggest that RNAi will prove a highly valuable functional genomics technique in triatomine bugs. The finding that feeding dsRNA can induce knockdown is novel for insects.

Infecções helmínticas em um rebanho leiteiro na região Campo das Vertentes de Minas Gerais / Helminth infectíon pattern of a dairy herd in Campo das Vertentes region in Minas Gerais state

Bezerros traçadores foram utilizados para avaliar a contaminação sazonal das pastagens por helmintos gastrintestinais e pulmonares em uma propriedade de exploração leiteira na região Campo das Vertentes,Minas Gerais. Os animais se infectaram durante todos os meses do ano. As maiores cargas parasitárias.foram recuperadas no período chuvoso (setembro - abril), e o pico foi observado em abril-maio, final do período chuvoso. As espécies recuperadas foram Cooperia punctata, C. spatulata, Haemonchus contortus, H. similis, Oesophagostomum radiatum, Trichostrongylus axei, T. colubriformis, Trichuris discolor, T. globulosa, Dictyocaulus viviparus e Agriostomum vryburgi. Para avaliar a dinâmica das infecções, foi acompanhado um grupo de vacas com seus bezerros lactantes no período de janeiro de 1999 a fevereiro de 2000. Os valores da contagem de ovos por grama de fezes (OPG) dos bezerros apresentaram dois picos, em maio de 1999 e fevereiro de 2000, enquanto o pico da contagem de OPG das vacas ocorreu em julho e agosto de 2000. Nas coproculturas, os gêneros de maior ocorrência foram Cooperia e Haemonchus nos bezerros, e Haemonchus e Trichostrongylus nas vacas.