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Find video protocols related to scientific articles indexed in Pubmed.
Abortion and foetal lesions induced by Neospora caninum in experimentally infected water buffalos (Bubalus bubalis).
Parasitol. Res.
PUBLISHED: 09-19-2014
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The water buffalo (Bubalus bubalis) is an important species in several countries for its milk and meat production, as well as for transport and other agricultural activities. It is, in general, considered more resistant than cattle to different parasitic diseases, also less demanding for forage quality. It has been postulated that buffalo may be resistant to abortion caused by neosporosis, because of high serological prevalences found in buffalo herds from different localities, with no description of Neospora caninum-related abortion. Recent studies have demonstrated the potential impact of neosporosis in pregnant water buffalo cows. In this work, three pregnant buffalo cows were experimentally infected with Nc-1 strain of N. caninum, and abortion was detected 35 days post-infection. Molecular and histopathological results found in post-mortem tissues are described and discussed, confirming the susceptibility of water buffalos to abortion caused by N. caninum.
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Genetic Multipartitions Based on D-Loop Sequences and Chromosomal Patterns in Brown Chromis, Chromis multilineata (Pomacentridae), in the Western Atlantic.
Biomed Res Int
PUBLISHED: 05-20-2014
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Connectivity levels among Brazilian reef fish fauna populations have attracted growing interest, mainly between mainland shores and oceanic islands. The Pomacentridae, whose phylogeographic patterns are largely unknown in the Atlantic, are a family of dominant fish in reef regions. We present data on the variability and population structure of damselfish Chromis multilineata in different areas along the northeast coast of Brazil and in the waters around the oceanic islands of Fernando de Noronha (FNA) and Saint Peter and Saint Paul Archipelago (SPSPA) through analysis of the HVR1 mtDNA sequence of the control region. The remote SPSPA exhibits the highest level of genetic divergence among populations. Conventional and molecular cytogenetic analysis showed similar karyotype patterns (2n = 48 acrocentrics) between these insular areas. Our estimates reveal three genetically different population groups of C. multilineata on the Brazilian coast. The level of genetic structure is higher than previous data suggested, indicating complex panel of interactions between the oceanic island and coastal populations of Brazil.
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Structural comparison of lipophosphoglycan from Leishmania turanica and L. major, two species transmitted by Phlebotomus papatasi.
Parasitol. Int.
PUBLISHED: 05-13-2014
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The lipophosphoglycan (LPG) of Leishmania major has a major role in the attachment to Phlebotomus papatasi midgut. Here, we investigated the comparative structural features of LPG of L. turanica, another species transmitted by P. papatasi. The mAb WIC 79.3, specific for terminal Gal(?1,3) side-chains, strongly reacted with L. turanica LPG. In contrast, L. turanica LPG was not recognized by arabinose-specific mAb 3F12. In conclusion, LPGs from L. major and L. turanica are similar, with the latter being less arabinosylated than L. major's. The high galactose content in L. turanica LPG is consistent with its predicted recognition by P. papatasi lectin PpGalec.
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Understanding the mechanisms controlling Leishmania amazonensis infection in vitro: the role of LTB4 derived from human neutrophils.
J. Infect. Dis.
PUBLISHED: 03-14-2014
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Neutrophils are rapidly recruited to the site of Leishmania infection and play an active role in capturing and killing parasites. They are the main source of leukotriene B4 (LTB4), a potent proinflammatory lipid mediator. However, the role of LTB4 in neutrophil infection by Leishmania amazonensis is not clear. In this study, we show that L. amazonensis or its lipophosphoglycan can induce neutrophil activation, degranulation, and LTB4 production. Using pharmacological inhibitors of leukotriene synthesis, our findings reveal an LTB4-driven autocrine/paracrine regulatory effect. In particular, neutrophil-derived LTB4 controls L. amazonensis killing, degranulation, and reactive oxygen species production. In addition, L. amazonensis infection induces an early increase in Toll-like receptor 2 expression, which facilitates parasite internalization. Nuclear factor kappa B (NFkB) pathway activation represents a required upstream event for L. amazonensis-induced LTB4 synthesis. These leishmanicidal mechanisms mediated by neutrophil-derived LTB4 act through activation of its receptor, B leukotriene receptor 1 (BLT1).
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Anti-tuberculosis evaluation and conformational study of N-acylhydrazones containing the thiophene nucleus.
Arch. Pharm. (Weinheim)
PUBLISHED: 03-10-2014
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A series of N-acylhydrazonyl-thienyl derivatives (compounds 2 and 3), mainly of the type 2-(aryl-CH=N-NHCOCH2 )-thiene (2: aryl?=?substituted-phenyl; 3: aryl?=?heteroaryl) were evaluated against Mycobacterium tuberculosis. Particularly active compound was 3 (heteroaryl?=?5-nitrothien-2-yl or 5-nitrofuran-2-yl) with MIC values of 8.5 and 9.0??M, respectively. Moderately active compounds were compound 3 (heteroaryl?=?pyridin-2-yl) and compound 2 containing aryl?=?2- or 4-hydroxyphenyl groups, with MIC values between 170 and 408??M. Compound 2 containing OMe, H, F, Cl, Br, CN, and NO2 substituents and compound 3 (heteroaryl?=?furan-2-yl, thien-2-yl, pyrrol-2-yl, imidazol-2-yl, pyridin-3-yl, and pyridin-4-yl) were all inactive. Clearly, there is no correlation of activity with the electronic effects of the substituents. The activities suggest different modes of biological action of the compounds having nitro-heteroaryl groups, on the one hand, and the 2-hydroxyphenyl or pyridin-2-yl substituents, on the other hand. Compounds having 2- or 4-hydroxyphenyl, 2-hydroxy-5-nitrophenyl, or 4-hydroxy-3-chlorophenyl were less cytotoxic than ethambutol. It is important to notice that compound 3 (aryl?=?5-NO2 -furan-2-yl) exhibited a promising therapeutic index (TI?=?1093.90), with a value 4.4 less than that of ethambutol. Compounds 2 and 3 exist in DMSO or MeOD solutions as mixtures of EC(O)N /EC=N and ZC(O)N /EC=N conformers.
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Epidermal growth factor receptor gene polymorphisms are associated with prognostic features of breast cancer.
BMC Cancer
PUBLISHED: 03-03-2014
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The epidermal growth factor receptor (EGFR) is differently expressed in breast cancer, and its presence may favor cancer progression. We hypothesized that two EGFR functional polymorphisms, a (CA)n repeat in intron 1, and a single nucleotide polymorphism, R497K, may affect EGFR expression and breast cancer clinical profile.
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Polymorphisms of DNA repair genes are related to the pathogenesis of myelodysplastic syndrome.
Hematol Oncol
PUBLISHED: 01-29-2014
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Some studies show that alterations in DNA repair genes polymorphisms are associated with the pathogenesis and susceptibility of Myelodysplastic Syndrome (MDS). We genotyped 60 MDS patients for six DNA repair gene polymorphisms: BRCA1 rs4793191, BRCA2 rs9567623, RAD51 rs1801320, XRCC5 rs3835, XRCC6 rs2267437 and LIG4 rs1805388. The G/C heterozygote genotype of rs1801320 polymorphism was associated with a decreased chance of developing MDS (p?=?0.05). Additionally, the G/G homozygous genotype was associated with the presence of one cytopenia in whole blood. The genotype C/G and CG?+?GG of the rs2267437 polymorphism was associated with normal karyotype (p?=?0.010) and bone marrow cellularity normocellular?+?hypercellular (p?=?0.023). We found that the A/G heterozygous genotype of the rs3835 polymorphism is associated with decreased chance of developing MDS (p?
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Shotgun proteomics to unravel the complexity of the Leishmania infantum exoproteome and the relative abundance of its constituents.
Mol. Biochem. Parasitol.
PUBLISHED: 01-27-2014
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The exoproteome of some Leishmania species has revealed important insights into host-parasite interaction, paving the way for the proposal of novel disease-oriented interventions. The focus of the present investigation constituted the molecular profile of the L. infantum exoproteome revealed by a shotgun proteomic approach. Promastigotes under logarithmic phase of growth were obtained and harvested by centrifugation at different time points. Cell integrity was evaluated through the counting of viable parasites using propidium iodide labeling, followed by flow cytometry analysis. The 6h culture supernatant, operationally defined here as exoproteome, was then conditioned to in solution digestion and the resulting peptides submitted to mass spectrometry. A total of 102 proteins were identified and categorized according to their cellular function. Their relative abundance index (emPAI) allowed inference that the L. infantum exoproteome is a complex mixture dominated by molecules particularly involved in nucleotide metabolism and antioxidant activity. Bioinformatic analyses support that approximately 60% of the identified proteins are secreted, of which, 85% possibly reach the extracellular milieu by means of non-classic pathways. At last, sera from naturally infected animals, carriers of differing clinical forms of Canine Visceral Leishmaniasis (CVL), were used to test the immunogenicity associated to the L. infantum exoproteome. Western blotting experiments revealed that this sub-proteome was useful at discriminating symptomatic animals from those exhibiting other clinical forms of the disease. Collectively, the molecular characterization of the L. infantum exoproteome and the preliminary immunoproteomic assays opened up new research avenues related to treatment, prognosis and diagnosis of CVL.
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Immunological profile of resistance and susceptibility in naturally infected dogs by Leishmania infantum.
Vet. Parasitol.
PUBLISHED: 01-23-2014
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Visceral leishmaniasis has a great impact on public health, and dogs are considered the main domestic reservoir of Leishmania infantum, the causal parasite. In this study, 159 animals naturally infected by L. infantum from an endemic area of Brazil were evaluated through an analysis of cellular responses, using flow cytometry, and of the hematological parameters. The results confirmed that disease progression is associated with anemia and reductions in eosinophils, monocytes and lymphocytes. The investigation of the immune response, based on the immunophenotypic profile of peripheral blood, showed declines in the absolute numbers of T lymphocytes CD5(+) and their subsets (CD4(+) and CD8(+)) and a drop of B lymphocytes in asymptomatic seropositive (AD-II) and symptomatic seropositive (SD) dogs. Neutrophils, when stimulated with soluble antigen of L. infantum, showed higher synthesis of interferon (IFN)-?(+) in AD-II and SD groups, with decreased production of interleukin (IL)-4(+) in asymptomatic seronegative dogs positive for L. infantum infection based on polymerase chain reaction testing (AD-I group). In the AD-II and SD groups, subpopulations of stimulated lymphocytes (CD4(+) and CD8(+)) also exhibited greater synthesis of IFN-?(+) and IL-4(+) in culture. These results suggest that the animals of the AD-II and SD groups exhibited a mixed immune response (Type 1 and 2) and the AD-I group presenting an immune profile very similar to normal control animals.
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LBSapSal-vaccinated dogs exhibit increased circulating T-lymphocyte subsets (CD4? and CD8?) as well as a reduction of parasitism after challenge with Leishmania infantum plus salivary gland of Lutzomyia longipalpis.
Parasit Vectors
PUBLISHED: 01-18-2014
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The development of a protective vaccine against canine visceral leishmaniasis (CVL) is an alternative approach for interrupting the domestic cycle of Leishmania infantum. Given the importance of sand fly salivary proteins as potent immunogens obligatorily co-deposited during transmission of Leishmania parasites, their inclusion in an anti-Leishmania vaccine has been investigated in the last few decades. In this context, we previously immunized dogs with a vaccine composed of L. braziliensis antigens plus saponin as the adjuvant and sand fly salivary gland extract (LBSapSal vaccine). This vaccine elicited an increase in both anti-saliva and anti-Leishmania IgG isotypes, higher counts of specific circulating CD8? T cells, and high NO production.
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Host Modulation by a Parasite: How Leishmania infantum Modifies the Intestinal Environment of Lutzomyia longipalpis to Favor Its Development.
PLoS ONE
PUBLISHED: 01-01-2014
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Some reports have described the interference of Leishmania on sand flies physiology, and such behavior most likely evolved to favor the development and transmission of the parasite. Most of these studies showed that Leishmania could modulate the level of proteases in the midgut after an infective blood meal, and decreased proteolytic activity is indeed beneficial for the development of promastigotes in the gut of sand flies. In the present study, we performed a detailed investigation of the intestinal pH in Lutzomyia longipalpis females naturally infected with Leishmania infantum and investigated the production of trypsin by these insects using different approaches. Our results allowed us to propose a mechanism by which these parasites interfere with the physiology of L. longipalpis to decrease the production of proteolytic enzymes. According to our hypothesis L. infantum promastigotes indirectly interfere with the production of trypsin by modulating the mechanism that controls the intestinal pH via the action of a yet non-identified substance released by promastigote forms inside the midgut. This substance is not an acid, whose action would be restrict on to release H+ to the medium, but is a substance that is able to interfere with midgut physiology through a mechanism involving pH control. According to our hypothesis, as the pH decreases, the proteolytic enzymes efficiency is also reduced, leading to a decline in the supply of amino acids to the enterocytes: this decline reduces the stimulus for protease production because it is regulated by the supply of amino acids, thus leading to a delay in digestion.
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Immunotherapy and Immunochemotherapy in Visceral Leishmaniasis: Promising Treatments for this Neglected Disease.
Front Immunol
PUBLISHED: 01-01-2014
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self-healing or chronic cutaneous leishmaniasis or post-kala-azar dermal leishmaniasis; mucosal leishmaniasis; visceral leishmaniasis (VL), which is fatal if left untreated. The epidemiology and clinical features of VL vary greatly due to the interaction of multiple factors including parasite strains, vectors, host genetics, and the environment. Human immunodeficiency virus infection augments the severity of VL increasing the risk of developing active disease by 100-2320 times. An effective vaccine for humans is not yet available. Resistance to chemotherapy is a growing problem in many regions, and the costs associated with drug identification and development, make commercial production for leishmaniasis, unattractive. The toxicity of currently drugs, their long treatment course, and limited efficacy are significant concerns. For cutaneous disease, many studies have shown promising results with immunotherapy/immunochemotherapy, aimed to modulate and activate the immune response to obtain a therapeutic cure. Nowadays, the focus of many groups centers on treating canine VL by using vaccines and immunomodulators with or without chemotherapy. In human disease, the use of cytokines like interferon-? associated with pentavalent antimonials demonstrated promising results in patients that did not respond to conventional treatment. In mice, immunomodulation based on monoclonal antibodies to remove endogenous immunosuppressive cytokines (interleukin-10) or block their receptors, antigen-pulsed syngeneic dendritic cells, or biological products like Pam3Cys (TLR ligand) has already been shown as a prospective treatment of the disease. This review addresses VL treatment, particularly immunotherapy and/or immunochemotherapy as an alternative to conventional drug treatment in experimental models, canine VL, and human disease.
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Evaluation of change in canine diagnosis protocol adopted by the visceral leishmaniasis control program in Brazil and a new proposal for diagnosis.
PLoS ONE
PUBLISHED: 01-01-2014
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The techniques used for diagnosis of canine visceral leishmaniasis (CVL) in Brazil ELISA and IFAT have been extensively questioned because of the accuracy of these tests. A recent change in the diagnosis protocol excluded IFAT and included the Dual-Path Platform (DPP). We evaluated the prevalence and incidence rates of Leishmania spp. before and after the change in the protocol. In addition, based on our results, we propose a new alternative that is less expensive for the screening and confirmation of CVL. Plasma samples were obtained from a serobank from dogs evaluated in a cross-sectional study (1,226 dogs) and in a cohort study of susceptible animals (n = 447), followed for 26 months. Serology testing was performed using ELISA, IFAT, and DPP. The incidence and prevalence of CVL were determined by using the protocol of the Visceral Leishmaniasis Control and Surveillance Program until 2012 (ELISA and IFAT using filter paper) and the protocol used after 2012 (DPP and ELISA using plasma). The prevalence was 6.2% and the incidence was 2.8 per 1,000 dog-months for the protocol used until 2012. For the new diagnosis protocol for CVL resulted in an incidence of 5.4 per 1,000 dog-months and a prevalence of 8.1%. Our results showed that the prevalence and incidence of infection were far greater than suggested by the previously used protocol and that the magnitude of infection in endemic areas has been underestimated. As tests are performed sequentially and euthanasia of dogs is carried out when the serological results are positive in both tests, the sequence does not affect the number of animals to be eliminated by the Control Program. Then we suggest to municipalities with a large demand of exams to use ELISA for screening and DPP for confirmation, since this allows easier performance and reduced cost.
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Nandrolone decanoate inhibits gluconeogenesis and decreases fasting glucose in Wistar male rats.
J. Endocrinol.
PUBLISHED: 01-01-2014
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The use of anabolic-androgenic steroids to improve physical performance or appearance has increased notably. The doses used are 10- to 100- fold higher than the therapeutic dose (TD), and this abuse can cause several side effects. Glucose metabolism is significantly affected by anabolic-androgenic steroid abuse, but studies about glycemic regulation during fasting are scarce. There are some evidences showing that testosterone can antagonize glucocorticoids action, which are crucial to glucose production during fasting. Thus, the aim of this study was to determine the impact of supraphysiological doses (SDs) of nandrolone decanoate (DECA) on rat glucose metabolism during fasting. Male Wistar rats were treated with i.m. injections of vehicle, a low TD (0.016?mg/100?g b.w.-TD group) or a high SD (1?mg/100?g b.w.-SD group) of DECA, once a week for 8 weeks. After 12?h fasting, we evaluated glucose and pyruvate tolerance tests, liver glycogen content, serum levels of gluconeogenic substrates, insulin and corticosterone, glucose uptake and hexokinase (HK) activity in skeletal muscle, and the adrenal catecholamine content. SD group had increased serum insulin levels and a blunted response to insulin regarding glucose uptake in skeletal muscle. Fasting serum glucose decreased significantly in SD group, as well as the pyruvate tolerance test and liver glycogen content. Moreover, serum levels of glycerol were increased in SD group. Our data indicate that SDs of DECA exert effects on different regulatory points of glucose metabolism, resulting in defective gluconeogenesis and decreased skeletal muscle glucose uptake in response to insulin.
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Brazilians have different views on when abortion should be legal, but most do not agree with imprisoning women for abortion.
Reprod Health Matters
PUBLISHED: 12-10-2013
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Unsafe abortions remain a major public health problem in countries with very restrictive abortion laws. In Brazil, parliamentarians - who have the power to change the law - are influenced by "public opinion", often obtained through surveys and opinion polls. This paper presents the findings from two studies. One was carried out in February-December 2010 among 1,660 public servants and the other in February-July 2011 with 874 medical students from three medical schools, both in São Paulo State, Brazil. Both groups of respondents were asked two sets of questions to obtain their opinion about abortion: 1) under which circumstances abortion should be permitted by law, and 2) whether or not women in general and women they knew who had had an abortion should be punished with prison, as Brazilian law mandates. The differences in their answers were enormous: the majority of respondents were against putting women who have had abortions in prison. Almost 60% of civil servants and 25% of medical students knew at least one woman who had had an illegal abortion; 85% of medical students and 83% of civil servants thought this person(s) should not be jailed. Brazilian parliamentarians who are currently reviewing a reform in the Penal Code need to have this information urgently.
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Evaluation of a prototype flow cytometry test for serodiagnosis of canine visceral leishmaniasis.
Clin. Vaccine Immunol.
PUBLISHED: 10-09-2013
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Diagnosing canine visceral leishmaniasis (CVL) is a critical challenge since conventional immunoserological tests still present some deficiencies. The current study evaluated a prototype flow cytometry serology test, using antigens and fluorescent antibodies that had been stored for 1 year at 4°C, on a broad range of serum samples. Noninfected control dogs and Leishmania infantum-infected dogs were tested, and the prototype test showed excellent performance in differentiating these groups with high sensitivity, specificity, positive and negative predictive values, and accuracy (100% in all analyses). When the CVL group was evaluated according to the dogs clinical status, the prototype test showed outstanding accuracy in all groups with positive serology (asymptomatic II, oligosymptomatic, and symptomatic). However, in dogs which had positive results by PCR-restriction fragment length polymorphism (RFLP) but negative results by conventional serology (asymptomatic I), serological reactivity was not observed. Additionally, sera from 40 dogs immunized with different vaccines (Leishmune, Leish-Tec, or LBSap) did not present serological reactivity in the prototype test. Eighty-eight dogs infected with other pathogens (Trypanosoma cruzi, Leishmania braziliensis, Ehrlichia canis, and Babesia canis) were used to determine cross-reactivity and specificity, and the prototype test performed well, particularly in dogs infected with B. canis and E. canis (100% and 93.3% specificities, respectively). In conclusion, our data reinforce the potential of the prototype test for use as a commercial kit and highlight its outstanding performance even after storage for 1 year at 4°C. Moreover, the prototype test efficiently provided accurate CVL serodiagnosis with an absence of false-positive results in vaccinated dogs and minor cross-reactivity against other canine pathogens.
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Essential roles of endogenous glucocorticoids and TNF/TNFR1 in promoting bone-marrow eosinopoiesis in ovalbumin-sensitized, airway-challenged mice.
Life Sci.
PUBLISHED: 07-19-2013
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Stress mechanisms paradoxically contribute to allergic episodes in humans and mice. Glucocorticoids (GC) and interleukin (IL)-5 synergically upregulate murine bone-marrow eosinophil production. Here we explored the role of endogenous GC in allergen-stimulated bone-marrow eosinophil production in ovalbumin-sensitized/challenged mice.
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Analysis using canine peripheral blood for establishing in vitro conditions for monocyte differentiation into macrophages for Leishmania chagasi infection and T-cell subset purification.
Vet. Parasitol.
PUBLISHED: 07-11-2013
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Canine visceral leishmaniasis (CVL) is a parasitic disease endemic in many countries, and dogs present as the major natural reservoir of the parasite, Leishmania chagasi (syn. L. infantum). Biomarkers in the canine immune system is an important technique in the course of developing vaccines and treatment strategies against CVL. New methodologies for studying the immune response of dogs during Leishmania infection and after receiving vaccines and treatments against CVL would be useful. In this context, we used peripheral blood mononuclear cells (PBMCs) from healthy dogs to evaluate procedures related to (i) establishment of in vitro conditions of monocytes differentiated into macrophages infected with L. chagasi and (ii) purification procedures of T-cell subsets (CD4(+) and CD8(+)) using microbeads. Our data demonstrated that after 5 days of differentiation, macrophages were able to induce significant phagocytic and microbicidal activity after L. chagasi infection and also showed increased frequency of parasitism and a higher parasite load. Although N-acetyl-?-d-glucosaminidase (NAG) levels presented similar levels of macrophage culture and L. chagasi infection, a progressive decrease in myeloperoxidase (MPO) levels was a hallmark over 5 days of culture. High purity levels (>90%) of CD4 and CD8 T cells were obtained on a magnetic separation column. We concluded that monocytes differentiated into macrophages at 5 days and displayed an intermediate frequency of parasitism and parasite load 72 h after L. chagasi infection. Furthermore, the purification system using canine T-lymphocyte subsets obtained after 5 days of monocyte differentiation proved efficient for CD4 or CD8 T-cell purification (?90%). The in vitro analysis using L. chagasi-infected macrophages and purified T cells presented a prospective methodology that could be incorporated in CVL vaccine and treatment studies that aim to analyze the microbicidal potential induced by specific CD4(+) and/or CD8(+) T cells.
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Sexual dimorphism of thyroid reactive oxygen species production due to higher NADPH oxidase 4 expression in female thyroid glands.
Thyroid
PUBLISHED: 06-26-2013
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Dual oxidases (DUOX1 and DUOX2) are NADPH oxidases (NOX) involved in hydrogen peroxide production necessary for thyroid hormonogenesis, but recently, the NOX4 has also been described in the thyroid gland. The prevalence of thyroid disease is higher in women, and the basis for this difference might involve a higher oxidative stress level in the female thyroid gland. Hence, we aimed at evaluating whether the function and the expression of enzymes involved in the thyroid redox balance differ between females and males.
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A new set of primers directed to 18S rRNA gene for molecular identification of Cryptosporidium spp. and their performance in the detection and differentiation of oocysts shed by synanthropic rodents.
Exp. Parasitol.
PUBLISHED: 06-18-2013
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Cryptosporidium spp. are cosmopolitan protozoa that infect fishes, reptiles, amphibians, birds and mammals. More than 20 species are recognized within this genus. Rodents are a group of abundant and ubiquitous organisms that have been considered reservoirs of Cryptosporidium for humans and livestock. The aim of this study was to design specific primers for the gene encoding 18S rRNA, potentially capable of amplifying any species or genotype of Cryptosporidium spp. and evaluate the diagnostic attributes of the nested-PCR based on such probes. The primers were designed to amplify the shortest segment as possible to maximize the sensitivity of the test, but preserving the discriminatory potential of the amplified sequences for phylogenetic inferences. The nested-PCR standardized in this study (nPCR-SH) was compared in terms of sensitivity with another similar assay (nPCR-XIAO) that has been largely used for the detection and identification of Cryptosporidium spp. worldwide. We also aimed to molecularly characterize samples of Cryptosporidum spp. isolated from synanthropic rodents using these probes. Forty-five rodents were captured in urban areas of the municipality of Umuarama, Paraná State, Brazil. Fecal samples were submitted to three molecular tests (nested-PCRs), two of them targeted to the 18S rDNA gene (nPCR-SH and nPCR-XIAO) and the third targeted to the gene encoding actin (nPCR-actin). The nPCR-SH was tested positive on samples of Cryptosporidum parvum, Cryptosporidum andersoni, Cryptosporidum meleagridis, Cryptosporidum hominis, Cryptosporidum canis, and Cryptosporidum serpentis. Sixteen samples of rodents were positive by nPCR-SH, six by nPCR-XIAO and five by nPCR-actin. Sequencing of amplified fragments allowed the identification of Cryptosporidum muris in three samples of Rattus rattus, and two genotypes of Cryptosporidium, the genotypes mouse II and III. Cryptosporidium genotype mouse II was found in one sample of Mus musculus and genotype mouse III, in twelve samples, being five from R. rattus and seven from M. musculus. The results of this study demonstrated that the primers designed for detection of Cryptosporidium spp. were more efficient than those used in the nPCR-XIAO. Genotypes or species of Cryptosporidium that can be usually transmitted for human beings and livestock were not found in synanthropic rodents, suggesting that the importance of these animals in zoonotic transmission of cryptosporidiosis should be revisited.
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[Cross-cultural adaptation of the World Health Organization Disability Assessment Schedule (WHODAS 2.0) into Portuguese].
Rev Assoc Med Bras
PUBLISHED: 05-17-2013
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The World Health Organization Disability Assessment Schedule (WHODAS 2.0) was designed to assess the functioning level in six life domains (cognition, mobility, self-care, getting along, life activities, and participation in community activities). There are different versions, from the simplest to the most complete, various presentations (either interviews or self-administered), comprehending the domains of the International Classification of Functioning, Disability and Health (ICF). This study aimed to make a cross-cultural adaptation of the complete version into Portuguese.
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Performance of Glutamate Dehydrogenase and Triose Phosphate Isomerase Genes in the Analysis of Genotypic Variability of Isolates of Giardia duodenalis from Livestocks.
Biomed Res Int
PUBLISHED: 04-16-2013
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Giardia duodenalis is a small intestinal protozoan parasite of several terrestrial vertebrates. This work aims to assess the genotypic variability of Giardia duodenalis isolates from cattle, sheep and pigs in the Southeast of Brazil, by comparing the standard characterization between glutamate dehydrogenase (gdh) and triose phosphate isomerase (tpi) primers. Fecal samples from the three groups of animals were analyzed using the zinc sulphate centrifugal flotation technique. Out of 59 positive samples, 30 were from cattle, 26 from sheep and 3 from pigs. Cyst pellets were stored and submitted to PCR and nested-PCR reactions with gdh and tpi primers. Fragment amplification of gdh and tpi genes was observed in 25 (42.4%) and 36 (61.0%) samples, respectively. Regarding the sequencing, 24 sequences were obtained with gdh and 20 with tpi. For both genes, there was a prevalence of E specific species assemblage, although some isolates have been identified as A and B, by the tpi sequencing. This has also shown a larger number of heterogeneous sequences, which have been attribute to mixed infections between assemblages B and E. The largest variability of inter-assemblage associated to the frequency of heterogeneity provided by tpi sequencing reinforces the polymorphic nature of this gene and makes it an excellent target for studies on molecular epidemiology.
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Dogs immunized with LBSap vaccine displayed high levels of IL-12 and IL-10 cytokines and CCL4, CCL5 and CXCL8 chemokines in the dermis.
Mol. Immunol.
PUBLISHED: 03-27-2013
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The complex interplay between cytokines and chemokines regulates innate and adaptive immune responses against pathogens; specifically, cytokine and chemokine expression drives activation of immune effector cells and their recruitment to tissue infection sites. Herein, we inoculated dogs with Leishmania braziliensis antigens plus saponin (the LBSap vaccine), as well as with the vaccine components, and then used real-time PCR to evaluate the kinetics of dermal expression of mRNAs of cytokines (IL-12, IFN-?, TNF-?, IL-4, IL-13, TGF-? and IL-10) and chemokines (CCL2, CCL4, CCL5, CCL21 and CXCL8) 1, 12, 24 and 48 h after inoculation. We also evaluated the correlation between cytokine and chemokine expression and dermal cellularity. The LBSap vaccine induced high levels of IL-12 and IL-10 expression at 12 and 24 h, respectively. Furthermore, we observed positive correlations between IL-12 and IL-13 expression, IFN-? and IL-13 expression, and IL-13 and TGF-? expression, suggesting that a mixed cytokine microenvironment developed after immunization with the vaccine. Inoculation with the saponin adjuvant alone induced a chemokine and cytokine expression profile similar to that observed in the LBSap group. CCL4 and CXCL8 chemokine expression was up regulated by the LBSap vaccine. CCL5 expression was initially highest in the LBSap group, but at 48 h, expression was highest in the LB group. Information about the kinetics of the immune response to this vaccine gained using this dog model will help to elucidate the mechanisms of and factors involved in a protective response against Leishmania infection and will aid in establishing rational approaches for the development of vaccines against canine visceral leishmaniasis.
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Effect of adding single-joint exercises to a multi-joint exercise resistance-training program on strength and hypertrophy in untrained subjects.
Appl Physiol Nutr Metab
PUBLISHED: 03-18-2013
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The aim of this study was to examine the effect of adding single-joint (SJ) exercises to a multi-joint (MJ) exercise resistance-training program on upper body muscle size and strength. Twenty-nine untrained young men participated in a 10-week training session. They were randomly divided in 2 groups: the MJ group performed only MJ exercises (lat pulldown and bench press); the MJ+SJ group performed the same MJ exercises plus SJ exercises (lat pulldown, bench press, elbow flexion, and elbow extension). Before and after the training period, the muscle thickness (MT) of the elbow flexors was measured with ultrasound, and peak torque (PT) was measured with an isokinetic dynamometer. There was a significant (p < 0.05) increase in MT (6.5% for MJ and 7.04% for MJ+SJ) and PT (10.40% for MJ and 12.85% for MJ+SJ) in both groups, but there were no between-group differences. Therefore, this study showed that the inclusion of SJ exercises in a MJ exercise training program resulted in no additional benefits in terms of muscle size or strength gains in untrained young men.
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Canine visceral leishmaniasis: incidence and risk factors for infection in a cohort study in Brazil.
Vet. Parasitol.
PUBLISHED: 03-05-2013
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Zoonotic visceral leishmaniasis in Brazil is caused by Leishmania infantum parasites and is transmitted by sand flies of the Phlebotominae family. Dogs are the main urban reservoirs and represent the major source of contagion for the vectors. Studies have shown that most infected dogs are polymerase chain reaction-positive months before seroconversion. Herein, we describe a cohort study designed to identify the incidence of and risk factors for L. infantum infection as detected by polymerase chain reaction-restriction fragment length polymorphism. To determine the risk factors for infection, we conducted a baseline canine survey (n=1443) from which dogs were selected for the cohort study (n=282) involving three evaluations over the course of a 26-month follow-up period. Serology, molecular tests, and a structured questionnaire were used. The risk factors for infection were identified by means of the Cox regression model. The overall infection incidence was 5.8 per 100 dog-months (95% confidence interval 5.1-6.5). Increased risk of infection was associated with the presence of previous cases of canine visceral leishmaniasis in the domiciles (hazard ratio [HR] 1.4; 95% confidence interval [CI] 1.1-1.8) and unplastered house walls (HR 3.6; 95% CI 1.6-8.1). These risk factors suggest that insecticide spraying in cracks and crevices in unplastered walls can reduce biting rates within and around homes. Furthermore, our results demonstrate that the Visceral Leishmaniasis Control and Surveillance Program should adopt environmental management measures in homes with previous cases of canine visceral leishmaniasis, because these homes are more likely to maintain the transmission cycle.
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Cytokine and nitric oxide patterns in dogs immunized with LBSap vaccine, before and after experimental challenge with Leishmania chagasi plus saliva of Lutzomyia longipalpis.
Vet. Parasitol.
PUBLISHED: 02-20-2013
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In the studies presented here, dogs were vaccinated against Leishmania (Leishmania) chagasi challenge infection using a preparation of Leishmania braziliensis promastigote proteins and saponin as adjuvant (LBSap). Vaccination with LBSap induced a prominent type 1 immune response that was characterized by increased levels of interleukin (IL-) 12 and interferon gamma (IFN-?) production by peripheral blood mononuclear cells (PBMC) upon stimulation with soluble vaccine antigen. Importantly, results showed that this type of responsiveness was sustained after challenge infection; at day 90 and 885 after L. chagasi challenge infection, PBMCs from LBSap vaccinated dogs produced more IL-12, IFN-? and concomitant nitric oxide (NO) when stimulated with Leishmania antigens as compared to PBMCs from respective control groups (saponin, LB- treated, or non-treated control dogs). Moreover, transforming growth factor (TGF)-? decreased in the supernatant of SLcA-stimulated PBMCs in the LBSap group at 90 days. Bone marrow parasitological analysis revealed decreased frequency of parasitism in the presence of vaccine antigen. It is concluded that vaccination of dogs with LBSap vaccine induced a long-lasting type 1 immune response against L. chagasi challenge infection.
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Two biochemically distinct lipophosphoglycans from Leishmania braziliensis and Leishmania infantum trigger different innate immune responses in murine macrophages.
Parasit Vectors
PUBLISHED: 02-19-2013
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The dominant, cell surface lipophosphoglycan (LPG) of Leishmania is a multifunctional molecule involved in the interaction with vertebrate and invertebrate hosts. Although the role of LPG on infection has been extensively studied, it is not known if LPG interspecies variations contribute to the different immunopathologies of leishmaniases. To investigate the issue of interspecies polymorphisms, two Leishmania species from the New World that express structural variations of side chains of LPG repeat units were examined. In this context, the procyclic form of L. braziliensis LPG (strain M2903), is devoid of side chains, while the L. infantum LPG (strain BH46) has up to three glucoses residues in the repeat units.
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Cytotoxicity and In Vitro Antileishmanial Activity of Antimony (V), Bismuth (V), and Tin (IV) Complexes of Lapachol.
Bioinorg Chem Appl
PUBLISHED: 01-30-2013
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Leishmania amazonensis is the etiologic agent of the cutaneous and diffuse leishmaniasis often associated with drug resistance. Lapachol [2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone] displays a wide range of antimicrobial properties against many pathogens. In this study, using the classic microscopic in vitro model, we have analyzed the effects of a series of lapachol and chlorides complexes with antimony (V), bismuth (V), and tin (IV) against L. amazonensis. All seven compounds exhibited antileishmanial activity, but most of the antimony (V) and bismuth (V) complexes were toxic against human HepG2 cells and murine macrophages. The best IC50 values (0.17 ± 0.03 and 0.10 ± 0.11??g/mL) were observed for Tin (IV) complexes (3) [(Lp)(Ph3Sn)] and (6) (Ph3SnCl2), respectively. Their selective indexes (SIs) were 70.65 and 120.35 for HepG2 cells, respectively. However, while analyzing murine macrophages, the SI decreased. Those compounds were moderately toxic for HepG2 cells and toxic for murine macrophages, still underlying the need of chemical modification in this class of compounds.
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Effect of the preservative and temperature conditions on the stability of Leishmania infantum promastigotes antigens applied in a flow cytometry diagnostic method for canine visceral leishmaniasis.
Diagn. Microbiol. Infect. Dis.
PUBLISHED: 01-16-2013
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The control of canine visceral leishmaniasis (CVL) is imperative, but euthanasia of seropositive dogs has been highly criticized. Commonly used, immunodiagnostic tests, including Dual-Path Platform®, enzyme-linked immunosorbent assay, and immunofluorescent antibody test, have failed at detecting asymptomatic dogs in endemic areas. In this context, new serological methods are needed. Flow cytometry serology has demonstrated potential as a test with excellent performance for CVL. In this study, we proposed to establish the best conditions for preserving Leishmania infantum promastigote antigens employed in this serology test. During 12 months of follow-up, promastigotes were maintained in different preservatives (phosphate-buffered saline with 3% fetal bovine serum, phenol 0.35%, thimerosal 0.01%, and formaldehyde 0.5%) and stored at 3 distinct temperatures (25 °C, 4 °C, and -20 °C). During the study period, the morphological characteristics of the promastigotes were assessed by flow cytometry according to the forward and side scatter parameters and also under optical microscopic analysis. Reactivity performance was evaluated as the percentage of positive fluorescent parasites in the sera of naturally infected and noninfected dogs. Microbiological analysis was performed at 2 time points, the first and sixth months, to rule out contamination of stored promastigotes. Taken together, our results indicated that the best conditions to preserve fixed L. infantum antigens were storage in formaldehyde at 4 °C. Promastigotes presented the best morphological profile, with appropriate antigenic stability even at 4 °C, in an inexpensive preservative for a long period of conservation.
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An alternative in vitro drug screening test using Leishmania amazonensis transfected with red fluorescent protein.
Diagn. Microbiol. Infect. Dis.
PUBLISHED: 01-10-2013
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Fluorescent and colorimetric reporter genes are valuable tools for drug screening models, since microscopy is labor intensive and subject to observer variation. In this work, we propose a fluorimetric method for drug screening using red fluorescent parasites. Fluorescent Leishmania amazonensis were developed after transfection with integration plasmids containing either red (RFP) or green fluorescent protein (GFP) genes. After transfection, wild-type (LaWT) and transfected (LaGFP and LaRFP) parasites were subjected to flow cytometry, macrophage infection, and tests of susceptibility to current antileishmanial agents and propranolol derivatives previously shown to be active against Trypanosoma cruzi. Flow cytometry analysis discriminated LaWT from LaRFP and LaGFP parasites, without affecting cell size or granulosity. With microscopy, transfection with antibiotic resistant genes was not shown to affect macrophage infectivity and susceptibility to amphotericin B and propranolol derivatives. Retention of fluorescence remained in the intracellular amastigotes in both LaGFP and LaRFP transfectants. However, detection of intracellular RFP parasites was only achieved in the fluorimeter. Murine BALB/c macrophages were infected with LaRFP parasites, exposed to standard (meglumine antimoniate, amphotericin B, Miltefosine, and allopurinol) and tested molecules. Although it was possible to determine IC(50) values for 4 propranolol derivatives (1, 2b, 3, and 4b), all compounds were considered inactive. This study is the first to develop a fluorimetric drug screening test for L. amazonensis RFP. The fluorimetric test was comparable to microscopy with the advantage of being faster and not requiring manual counting.
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Clinical Forms of Canine Visceral Leishmaniasis in Naturally Leishmania infantum-Infected Dogs and Related Myelogram and Hemogram Changes.
PLoS ONE
PUBLISHED: 01-01-2013
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Hematological analysis has limited applications for disease diagnosis in Leishmania infantum-infected dogs, but it can be very important in evaluating the clinical forms of the disease and in understanding the evolution of canine visceral leishmaniasis (CVL) pathogenesis. Recently, we demonstrated that alterations in leucopoiesis and erythropoiesis are related to clinical status and bone marrow parasite density in dogs naturally infected by L. infantum. To further characterize these alterations, we evaluated the association between the hematological parameters in bone marrow and peripheral blood alterations in groups of L. infantum-infected dogs: asymptomatic I (AD-I: serum negative/PCR+), asymptomatic II (AD-II: serum positive), oligosymptomatic (OD), and symptomatic (SD). Results were compared with those from noninfected dogs (NID). The SD group was found to present a decrease in erythropoietic lineage with concomitant reductions in erythrocytes, hemoglobin, and hematocrit parameters, resulting in anemia. The SD group also had increased neutrophils and precursors and decreased band eosinophils and eosinophils, leading to peripheral blood leucopenia. In the AD-II group, lymphocytosis occurred in both the peripheral blood and the bone marrow compartments. The SD group exhibited lymphocytosis in the bone marrow, with lymphopenia in the peripheral blood. In contrast, the AD-I group, showed no significant changes suggestive of CVL, presenting normal counts in bone marrow and peripheral blood. Our results showed for the first time that important changes in hematopoiesis and hematological parameters occur during ongoing CVL in naturally infected dogs, mainly in symptomatic disease. Taken together, our results based on myelogram and hemogram parameters enable better understanding of the pathogenesis of the anemia, lymphocytosis, and lymphopenia, as well as the leucopenia (eosinopenia and monocytopenia), that contribute to CVL prognosis.
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Inhibitory activity of Syzygium aromaticum and Cymbopogon citratus (DC.) Stapf. essential oils against Listeria monocytogenes inoculated in bovine ground meat.
Braz. J. Microbiol.
PUBLISHED: 01-01-2013
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This research evaluated the antimicrobial effect of the clove (Syzygium aromaticum) and lemongrass (Cymbopogon citratus (DC.) Stapf.) essential oils (EOs) against Listeria monocytogenes ATCC 19117 growth added to bovine ground meat stored under refrigeration (5 ± 2 °C) for three days. The EOs, extracted by hydrodistillation and analyzed by gas chromatography-mass spectrometry (GC-MS), were tested in vitro using an agar well diffusion methodology for determination of Minimum Inhibitory Concentration (MIC). The MIC concentrations for both essential oils on culture tested of L. monocytogenes were 1.56%. The EOs concentrations applied in contaminated ground beef were 1.56, 3.125 and 6.25% (w/v) based on MIC levels and possible activity reductions by food constituents. The bacteria populations were significantly reduced (p ? 0.05) after one day of storage in ground meat samples treated with clove and lemongrass EOs at concentrations of 1.56%. There were no significant counts of L. monocytogenes in samples at the other concentrations of the two oils applied after the second day of storage. The sensory acceptability evaluation of the bovine ground meat samples treated with EOs showed that the addition at concentrations higher than 1.56% promote undesirable alterations of taste, odor and characteristic color. The application of EOs at low concentrations in food products can be used in combination with other preservation methods, such as refrigeration, to control pathogens and spoilage bacteria during shelf-life; which goes according to current market trends, where consumers are requesting natural products.
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Flavonoid rutin increases thyroid iodide uptake in rats.
PLoS ONE
PUBLISHED: 01-01-2013
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Thyroid iodide uptake through the sodium-iodide symporter (NIS) is not only an essential step for thyroid hormones biosynthesis, but also fundamental for the diagnosis and treatment of different thyroid diseases. However, part of patients with thyroid cancer is refractory to radioiodine therapy, due to reduced ability to uptake iodide, which greatly reduces the chances of survival. Therefore, compounds able to increase thyroid iodide uptake are of great interest. It has been shown that some flavonoids are able to increase iodide uptake and NIS expression in vitro, however, data in vivo are lacking. Flavonoids are polyhydroxyphenolic compounds, found in vegetables present in human diet, and have been shown not only to modulate NIS, but also thyroperoxidase (TPO), the key enzyme in thyroid hormones biosynthesis, besides having antiproliferative effect in thyroid cancer cell lines. Therefore, we aimed to evaluate the effect of some flavonoids on thyroid iodide uptake in Wistar rats in vivo. Among the flavonoids tested, rutin was the only one able to increase thyroid iodide uptake, so we decided to evaluate the effect of this flavonoid on some aspects of thyroid hormones synthesis and metabolism. Rutin led to a slight reduction of serum T4 and T3 without changes in serum thyrotropin (TSH), and significantly increased hypothalamic, pituitary and brown adipose tissue type 2 deiodinase and decreased liver type 1 deiodinase activities. Moreover, rutin treatment increased thyroid iodide uptake probably due to the increment of NIS expression, which might be secondary to increased response to TSH, since TSH receptor expression was increased. Thus, rutin might be useful as an adjuvant in radioiodine therapy, since this flavonoid increased thyroid iodide uptake without greatly affecting thyroid function.
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American tegumentary leishmaniasis: effectiveness of an immunohistochemical protocol for the detection of Leishmania in skin.
PLoS ONE
PUBLISHED: 01-01-2013
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American tegumentary leishmaniasis (ATL) is endemic in Latin America, where Brazil has over 27 thousand cases per year. The aim of the present study was to develop an immunohistochemical method (IHC) for ATL diagnosis. For this purpose, we used serum from a dog naturally infected with Leishmania (L) infantum (canine hyperimmune serum) as the primary antibody, followed by a detection system with a secondary biotinylated antibody.
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Genetic polymorphism of MUC7 in individuals with aggressive or chronic periodontitis.
J Oral Sci
PUBLISHED: 12-15-2011
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Individuals with periodontitis exhibit differential expression of mucin-glycoprotein-2 (MG2), a protein encoded by the MUC7 gene. It is well known that MG2 exerts bactericidal activity as well as exhibiting genetic polymorphism involving a variable number of tandem repeats (VNTR). In the present study, we assessed the distribution of allelic variants of the MUC7 gene in 22 individuals with aggressive periodonitis, 68 with chronic periodonitis, and 87 without periodonitis. Oral mucosal cells were collected, the DNA was extracted, and specific primers were used to amplify the region encoding the MUC7 tandem repeats (TRs). Polymerase chain reaction products were subjected to electrophoresis and analyzed on polyacrylamide gels stained with silver nitrate. Although the percentage distribution of homozygosity (6-6TR) and heterozygosity (5-6TR) showed variation among the groups, the observed differences were not statistically significant (P > 0.05; Fishers Exact Test). The present results indicate that the expression of different numbers of TRs in this salivary mucin in the oral environment does not interfere with the etiopathogenesis of aggressive or chronic periodontitis.
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Genotypic characterization of Cryptosporidium hominis from water samples in Sao Paulo, Brazil.
Am. J. Trop. Med. Hyg.
PUBLISHED: 11-04-2011
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The protozoan parasite Cryptosporidium has emerged as one of the most important water contaminants, causing waterborne outbreaks of diarrheal diseases worldwide. The small size of oocysts under the microscope and the possibility of changes in characteristics of oocysts, mainly in environmental samples, make the taxonomy of the genus difficult if morphologic characteristics are considered. This limitation encouraged the application of molecular methods to identify this microorganism. The aim of this study was to detect and identify by nested-polymerase chain reaction oocysts of Cryptosporidium present in water samples in the state of São Paulo, Brazil. Water samples were concentrated through a membrane filter, DNA was extracted by using a standard technique, and both amplification reactions used forward and reverse oligonucleotides that were complementary to Cryptosporidium 18S ribosomal RNA gene sequences. Thirty water samples from different sites of collection in the state of São Paulo were evaluated. Cryptosporidium oocysts were detected in 30% of the samples. By genoptyping, C. hominis and Cryptosporidium sp. were identified in recreational water and C. meleagridis was identified in surface water samples. This is the first report of C. hominis in environmental samples in Brazil. Although identification of Cryptosporidium is still a difficult task, molecular methods are essential for specific identification and are a helpful tool to aid to understand the epidemiology of this parasite in Brazil.
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Detection of assemblages A and B of Giardia duodenalis in water and sewage from São Paulo state, Brazil.
J Water Health
PUBLISHED: 09-28-2011
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Giardia duodenalis is a protozoan that parasitizes humans and other mammals and causes giardiasis. Although its isolates have been divided into seven assemblages, named A to G, only A and B have been detected in human faeces. Assemblage A isolates are commonly divided into two genotypes, Al and All. Even though information about the presence of this protozoan in water and sewage is available in Brazil, it is important to verify the distribution of different assemblages that might be present, which can only be done by genotyping techniques. A total of 24 raw and treated sewage, surface and spring water samples were collected, concentrated and purified. DNA was extracted, and a nested PCR was used to amplify an 890 bp fragment of the gdh gene of G. duodenalis, which codes for glutamate dehydrogenase. Positive samples were cloned and sequenced. Ten out of 24 (41.6%) samples were confirmed to be positive for G. duodenalis by sequencing. Phylogenetic analysis grouped most sequences with G. duodenalis genotype All from GenBank. Only two raw sewage samples presented sequences assigned to assemblage B. In one of these samples genotype All was also detected. As these assemblages/genotypes are commonly associated to human giardiasis, the contact with these matrices represents risk for public health.
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Glycoconjugates in New World species of Leishmania: polymorphisms in lipophosphoglycan and glycoinositolphospholipids and interaction with hosts.
Biochim. Biophys. Acta
PUBLISHED: 09-22-2011
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Protozoan parasites of the genus Leishmania cause a number of important diseases in humans and undergo a complex life cycle, alternating between a sand fly vector and vertebrate hosts. The parasites have a remarkable capacity to avoid destruction in which surface molecules are determinant for survival. Amongst the many surface molecules of Leishmania, the glycoconjugates are known to play a central role in host-parasite interactions and are the focus of this review.
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Human cutaneous leishmaniasis: interferon-dependent expression of double-stranded RNA-dependent protein kinase (PKR) via TLR2.
FASEB J.
PUBLISHED: 08-16-2011
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We investigated the type I interferon (IFN-1)/PKR axis in the outcome of the Leishmania (Leishmania) amazonensis infection, along with the underlying mechanisms that trigger and sustain this signaling pathway. Reporter assays of cell extracts from RAW-264.7 macrophages infected with L. (L.) amazonensis or HEK-293T cells cotransfected with TLR2 and PKR promoter constructions were employed. Primary macrophages of TLR2-knockout (KO) or IFNR-KO mice were infected, and the levels of PKR, IFN-1, and superoxide dismutase 1 (SOD1) transcript levels were investigated and compared. Immunohistochemical analysis of human biopsy lesions was evaluated for IFN-1 and PKR-positive cells. Leishmania infection increased the expression of PKR and IFN-? on induction of PKR-promoter activity. The observed effects required the engagement of TLR2. TLR2-KO macrophages expressed low IFN-? and PKR levels postinfection with a reduced parasite load. We also revealed the requirement of PKR signaling for Leishmania-induced IFN-1 expression, responsible for sustaining PKR expression and enhancing infection. Moreover, during infection, SOD1 transcripts increased and were also enhanced when IFN-1 was added to the cultures. Remarkably, SOD1 expression was abrogated in infected, dominant-negative PKR-expressing cells. Finally, lesions of patients with anergic diffuse cutaneous leishmaniasis exhibited higher levels of PKR/IFN-1-expressing cells compared to those with single cutaneous leishmaniasis. In summary, we demonstrated the mechanisms and relevance of the IFN-1/PKR axis in the Leishmania infection.
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A survey of Neospora caninum and Toxoplasma gondii infection in urban rodents from Brazil.
J. Parasitol.
PUBLISHED: 07-26-2011
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Toxoplasma gondii is a protozoan parasite that infects humans and other warm-blooded animals; it uses feral and domestic cats as the definitive hosts. Neospora caninum is a protozoan parasite of animals whose life cycle is very similar to T. gondii but uses canids as definitive hosts. Small rodents play an important role in the life cycle of T. gondii , and a few findings indicated that they may be natural intermediate hosts for N. caninum . The present study was aimed at identifying infections by T. gondii and N. caninum in urban rodents. Infections by T. gondii were quantified using isolation of the parasite by bioassay in mice; molecular methods were also used for both parasites. Overall, 217 rodents were captured. Brain and heart tissues of all rodents were bioassayed in mice for the detection of T. gondii infection. Brain and heart tissues of 121 rodents had the DNA extracted for molecular analysis. Toxoplasma gondii was isolated by bioassay from a single rodent. From the 121 rodents tested for the presence of T. gondii DNA, 2 animals were positive. In contrast, DNA of N. caninum was not detected in any of the samples. In conclusion, the surveys of N. caninum and T. gondii infection in Rattus rattus , Rattus norvegicus , and Mus musculus captured in urban areas of São Paulo reveal a striking low frequency of occurrence of these infections.
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Effect of vegetable oil (Brazil nut oil) and mineral oil (liquid petrolatum) on dental biofilm control.
Braz Oral Res
PUBLISHED: 06-08-2011
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Dental biofilm control represents a basic procedure to prevent caries and the occurrence of periodontal diseases. Currently, toothbrushes and dentifrices are used almost universally, and the employment of good oral hygiene allows for appropriate biofilm removal by both mechanical and chemical control. The aim of this study was to evaluate the effectiveness of adding vegetable or mineral oil to a commercially available dentifrice in dental biofilm control. A comparison using the Oral Hygiene Index Simplified (OHI-S) was performed in 30 individuals who were randomly divided into three groups. Group 1 (G1) received a commercially available dentifrice; the composition of this dentifrice was modified by addition of mineral oil (Nujol®) for group 2 (G2) or a vegetable oil (Alpha Care®) for group 3 (G3) at 10% of the total volume, respectively. The two-way repeated-measures analysis of variance (two-way ANOVA) was used to test the effect of group (G1, G2 and G3) or time (baseline, 45 days and 90 days) on the OHI-S index scores. Statistical analysis revealed a significant reduction in the OHI-S at day 90 in G2 (p < 0.05) and G3 (p < 0.0001) in comparison to G1. Therefore, the addition of a vegetable or a mineral oil to a commercially available dentifrice improved dental biofilm control, suggesting that these oils may aid in the prevention and/or control of caries and periodontal disease.
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Occurrence and molecular diagnosis of Cryptosporidium serpentis in captive snakes in São Paulo, Brazil.
J. Zoo Wildl. Med.
PUBLISHED: 06-01-2011
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The present study aimed to determine whether Cryptosporidium oocysts were present in stools from captive snakes at Fundação Parque Zoológico (Zoological Park Foundation) in São Paulo, Brazil. Two collections were performed; the first in July 2008 and the second in February 2009. Fecal samples were collected from 74 enclosures that housed 101 individuals of 23 snake species. The stool specimens collected from 16 out of the 74 enclosures (21.6%) contained Cryptosporidium spp. oocysts; all of them were confirmed as Cryptosporidium serpentis, using molecular techniques. Only in three (18.7%) out of the 16 enclosures with positive samples were there animals with clinical signs compatible with infection by C. serpentis, such as regurgitation and significant progressive weight loss. From the results, it was concluded that diagnostic examinations need to be performed periodically, even on clinically healthy animals, as a preventive measure.
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Population genetic analyses of the AmpFlSTR® NGM™ in Brazil.
Int. J. Legal Med.
PUBLISHED: 05-19-2011
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Population data of 15 short tandem repeat loci of the AmpFlSTR® next generation multiplex (NGM)™ were obtained from a sample of 835 individuals. The loci are the ten short tandem repeats (STRs) in the SGM Plus® Kit plus the EDNAP- and ENSFI-recommended STRs D10S1248, D22S1045, D2S441, D1S1656, and D12S391. Allele frequency and other forensically relevant statistics data were generated for the NGM loci into five current country macroregions of Brazil (North, Northeast, Central West, Southeast, and South). All the analyzed loci meet Hardy-Weinberg equilibrium expectations and no linkage disequilibrium in all pairs of loci. The observed and expected heterozygosity, power of discrimination, polymorphic information content, and the other population-genetic indices were calculated. The overall power of discrimination was greater than 0.99999999999999999996 and the combined power of exclusion was greater than 0.9999998 in all Brazilian populations. Comparative analysis between populations from different Brazilian macroregions as well as between Brazil and Caucasian, African Americans, and Hispanic US populations are presented.
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Toll receptors type-2 and CR3 expression of canine monocytes and its correlation with immunohistochemistry and xenodiagnosis in visceral leishmaniasis.
PLoS ONE
PUBLISHED: 05-16-2011
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The aim of the present study was to investigate TLR2 expression in peripheral blood monocytes from dogs naturally infected with Leishmania (Leishmania) infantum to determine whether it correlates with CD11b/CD18 (CR3) expression, and to evaluate the potential of dogs as sources of infection using phlebotomine xenodiagnosis. Forty eight dogs were serologically diagnosed with L. infantum infection by indirect immunofluorescence antibody test (IFAT) and enzyme linked immunosorbent assay (ELISA). Parasitological exams from bone-marrow aspirates were positive by PCR analysis. All dogs were clinical defined as symptomatic. Ear skin tissue samples were obtained for immunohistochemistry (IHQ) analysis. The potential of these dogs as a source of infection using phlebotomine xenodiagnosis (XENO) was evaluated. Flow cytometry was carried out on peripheral blood mononuclear cells using superficial receptors including CD14, CD11b, TLR2 and MHCII. IHQ ear skin tissue parasite load and XENO where done where we found a strict correlation (r?=?0.5373). Dogs with higher expression of MFI of CD11b inside CD14 monocytes were represented by dogs without parasite ear tissue load that were unable to infect phlebotomines (IHQ?/XENO?). Dogs with lower expression of MFI of CD11b inside CD14 monocytes were represented by dogs with parasite ear tissue load and able to infect phlebotomines (IHQ?/XENO?) (p?=?0,0032). Comparable results were obtained for MFI of MHCII (p?=?0.0054). In addition, considering the population frequency of CD11b?TLR2? and CD11b?MHCII?, higher values were obtained from dogs with IHQ?/XENO? than dogs with IHQ?/XENO? (p?=?0.01; p?=?0.0048, respectively). These data, together with the TLR2 and NO assays results (CD11b?TLR2? and NO with higher values for dogs with IHQ?/XENO? than dogs with IHQ?/XENO?, led to the conclusion that IHQ?/XENO? dogs are more resistant or could modulate the cellular immune response essential for Leishmania tissue clearance.
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Synthesis, cytotoxicity, and in vitro antileishmanial activity of mono-t-butyloxycarbonyl-protected diamines.
Diagn. Microbiol. Infect. Dis.
PUBLISHED: 04-24-2011
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Leishmania amazonensis is the etiologic agent of the cutaneous and diffuse leishmaniasis. This species is often associated with drug resistance, and the conventional treatments exhibit high toxicity for patients. Therefore, the search for new antileishmanial compounds is urgently needed since there is no vaccine available. In this study, using the in vitro traditional drug screening test, we have analyzed the effects of a series of diaminoalkanes monoprotected with t-butyloxycarbonyl (BOC) against L. amazonensis. Among the 18 tested compounds, 6 exhibited antileishmanial activity (2, 7-9, 17, and 18). Best IC(50) values (10.39 ± 0.27 and 3.8 ± 0.42 ?g/mL) were observed for compounds 17 and 18 (H(2)N(CH(2))nNHBoc, n = 10 and 12), respectively. Although those compounds had higher lipophilicity as indicated by their cLog P values, compound 17 was very toxic. Determination of the selective indexes indicated that 50% of the active compounds were very toxic for HepG2 cells. However, compounds 2, 8, and 18 had good lipophilicity and were less toxic among all polyamine derivatives tested. The chemical properties of antileishmanial diamine derivatives, such as lipophilicity and cytotoxicity, are relevant factors for the design of new drugs. A higher lipophilicity is likely to improve the chances of reaching this intracellular parasite.
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An unusual glycoform of human salivary mucin MG2.
Clin Oral Investig
PUBLISHED: 04-14-2011
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Since in a previous study we encountered a subject with an unusual split MG2 banding pattern, the aim of this study was to investigate the molecular basis of this observation. Submandibular/sublingual secretion was collected under resting and stimulated conditions and examined on Western blots probed with anti-MG2 antibodies or on gels stained with periodic acid-Schiff reagent. Genomic DNA was isolated and the N-, tandem repeat (TR), and C-terminal regions of MUC7 were amplified by PCR since MG2 is known to display a genetic polymorphism. Although the typical appearance of MG2 on blots and gels is a single 180 kDa band, salivary secretions from the subject exhibited doublet immunoreactive bands of approximately 180 and 125 kDa. Additionally, under resting conditions the 180 kDa band was predominant whereas upon stimulation the 125 kDa band became predominant. Genomic DNA analysis showed that MUC7 in the individual with split MG2 bands was not truncated and that the MUC7 genotype in this individual was (6/6) where both alleles encoded six TRs. The MG2 split banding pattern observed in this subject was not derived from proteolytic degradation of this salivary mucin in whole saliva or from genetic polymorphism. The expression of two isoforms of MG2 could in principle improve or reduce the activity of this key component of the oral host defense system.
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Genotyping of potentially zoonotic Giardia duodenalis from exotic and wild animals kept in captivity in Brazil.
Vet. Parasitol.
PUBLISHED: 02-08-2011
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We have studied the variability of glutamate dehydrogenase (gdh) and small subunit ribosomal (SSU) rRNA coding genes of Giardia species in fecal samples isolated from wild and exotic animals in Brazil, and compared with homologous sequences of isolates from human and domestic animals characterized in previous studies. Cysts of Giardia duodenalis were obtained from feces of naturally infected monkeys (Alouatta fusca) (n=20), chinchillas (Chinchilla lanigera) (n=3), ostriches (Struthio camelus) (n=2) and jaguar (Panthera onca) (n=1). Assemblage AI was assigned to the unique isolate of jaguar. All the samples from monkeys, chinchillas, and ostriches were assigned to Assemblage B. There was little evolutionary divergence between the referred isolates and isolates described elsewhere. The Assemblage B isolates identified in this study were closely related to Assemblage BIV isolated from humans. The molecular identification of Assemblages A and B of G. duodenalis isolates from exotic and wild animals demonstrates that such hosts may be a potential reservoir for zoonotic transmission of G. duodenalis.
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Prevalence and factors associated with Leishmania infantum infection of dogs from an urban area of Brazil as identified by molecular methods.
PLoS Negl Trop Dis
PUBLISHED: 01-21-2011
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Various factors contribute to the urbanization of the visceral leishmaniasis (VL), including the difficulties of implementing control measures relating to the domestic reservoir. The aim of this study was to determine the prevalence of canine visceral leishmaniasis in an urban endemic area in Brazil and the factors associated with Leishmania infantum infection among seronegative and PCR-positive dogs.
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Genotyping of Toxoplasma gondii isolates from free range chickens in the Pantanal area of Brazil.
Vet. Parasitol.
PUBLISHED: 01-11-2011
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The aim of this paper was to genetically characterize Toxoplasma gondii isolates from free range chickens in regions of Brazilian territory in the state of Mato Grosso do Sul (MS) where T. gondii strains have never been studied. In total, T. gondii isolates from 22 free range chickens were included in this study. Fifty chickens from Eldorado, thirty from Rio Verde and ten from Aquidauana were sampled between January and April 2007. In relation to the genetic diversity of T. gondii isolates from chickens in MS, the magnitude of the diversity in the isolates sampled in this study was comparable to the overall diversity in a composite data set. These 22 isolates in MS revealed 11 genotypes, whereas the 321 isolates ever genotyped in Brazil have revealed 95 genotypes. The values of Simpsons Diversity Index for the whole population of T. gondii isolates in Brazil, the whole population of T. gondii isolates from chickens in Brazil and the population surveyed in this study were 0.97, 0.95 and 0.90, respectively. Seven of the 11 genotypes revealed from chicken isolates from MS are newly described genotypes and six of them each have a single isolate. In conclusion, the results obtained from isolates in MS corroborate previous studies on T. gondii isolates in Brazil, thus confirming their diversity and atypicality. Nonetheless, the applicability of PCR-RFLP markers for epidemiological inferences remains controversial.
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Evaluation of Vickers hardness of different types of acrylic denture base resins with and without glass fibre reinforcement.
Gerodontology
PUBLISHED: 11-11-2010
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To evaluate the Vickers hardness of different acrylic resins for denture bases with and without the addition of glass fibres.
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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.
Rev Bras Parasitol Vet
PUBLISHED: 09-24-2010
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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.
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Evidence of congenital transmission of Neospora caninum in naturally infected water buffalo (Bubalus bubalis) fetus from Brazil.
Parasitol. Res.
PUBLISHED: 09-16-2010
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The aim of this study was to determine the congenital infection by Neospora caninum in the water buffalo (Bubalus bubalis), a natural intermediate host. Nine pregnant water buffalos, raised under free-grazing condition, were slaughtered, and their fetuses were collected. Samples of brain and thoracic fluid were obtained from those fetuses, with gestational ages ranging from 2 to 5 months. The DNA of N. caninum was detected and identified in the brain of one of those fetuses, using two PCR assays, one directed to the Nc5 gene and the other, to the common toxoplasmatiid ITS1 sequence. The DNA fragments produced on PCR were sequenced, and N. caninum was confirmed in the samples. No antibodies to N. caninum were detected on any sample of thoracic fluid by immunofluorescent antibody test (IFAT < 25). This is the first confirmation of congenital transmission of N. caninum in water buffalos.
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Identification of Hammondia heydorni oocysts by a heminested-PCR (hnPCR-AP10) based on the H. heydorni RAPD fragment AP10.
Vet. Parasitol.
PUBLISHED: 09-15-2010
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Toxoplasma gondii, Hammondia hammondi, Neospora caninum, Neospora hughesi and Hammondia heydorni are members of the Toxoplasmatinae sub-family. They are closely related coccidians with similarly sized oocysts. Molecular diagnostic techniques, especially those based on polymerase chain reaction (PCR), can be successfully applied for the differentiation of Hammondia-like oocysts. In this paper, we describe a rapid and simple method for the identification of H. heydorni oocysts among other members of the Toxoplasmatinae sub-family, using a heminested-PCR (hnPCR-AP10) based on a H. heydorni RAPD fragment available in molecular database. DNA of oocysts of H. heydorni yielded a specific fragment of 289-290 bp in the heminested-PCR assay. No product was yielded when the primers were used for the amplification of DNA extracted from T. gondii, N. caninum, N. hughesi and H. hammondi, thus allowing the differentiation of H. heydorni among other members of the Toxoplasmatinae sub-family. The hnPCR-AP10 was capable of detecting H. heydorni genetic sequences from suspensions with at least 10 oocysts. In conclusion, the hnPCR-AP10 proved to be a reliable method to be used in the identification of H. heydorni oocysts from feces of dogs.
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Functional consequences of dual oxidase-thyroperoxidase interaction at the plasma membrane.
J. Clin. Endocrinol. Metab.
PUBLISHED: 09-08-2010
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Thyroperoxidase (TPO) and dual oxidase (DUOX) are present at the apical membrane of thyrocytes, where TPO catalyzes thyroid hormone biosynthesis in the presence of H2O2 produced by DUOX. Both enzymes are colocalized and associated, but the consequences of this interaction remain obscure.
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Viability of sporulated oocysts of Neospora caninum after exposure to different physical and chemical treatments.
J. Parasitol.
PUBLISHED: 09-03-2010
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The aim of the present study was to evaluate the viability of Neospora caninum sporulated oocysts after various chemical and physical treatments. Bioassays in gerbils and molecular techniques (PCR-RFLP) were used for identification of the oocysts shed by experimentally infected dogs. Sporulated oocysts were purified and divided into 11 treatment groups as follows: absolute ethanol for 1 hr; 20 C for 6 hr; 4 C for 6 hr; 60 C for 1 min; 100 C for 1 min; 10% formaldehyde for 1 hr; 10% ammonia for 1 hr; 2% iodine for 1 hr; 10% sodium hypochlorite for 1 hr; 70% ethanol for 1 hr; and one group was left untreated and kept as a positive control. All chemical treatments were performed at room temperature (37 C). A total of 33 gerbils, or 3 gerbils per treatment, were used for bioassays. After treatment, the oocysts were divided into aliquots of 1,000 oocysts and orally administered to gerbils. After 63 days, the gerbils were anesthetized and killed with 0.2 ml of T61; blood and tissue samples were collected for serological (IFAT and western blotting), molecular (real-time PCR), histopathology, and immunohistochemical tests. Treatments were considered effective only if all 5 detection techniques tested negative. High temperatures at 100 C for 1 min and 10% sodium hypochlorite for 1 hr were the only treatments that met this condition, effectively inactivating all oocysts.
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Evaluating the impact of community-based health interventions: evidence from Brazils Family Health Program.
Health Econ
PUBLISHED: 08-31-2010
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This paper analyzes the direct and indirect impacts of Brazils Family Health Program, using municipality level mortality data from the Brazilian Ministry of Health, and individual level data from the Brazilian household survey. We estimate the effects of the program on mortality and on household behavior related to child labor and schooling, employment of adults, and fertility. We find consistent effects of the program on reductions in mortality throughout the age distribution, but mainly at earlier ages. Municipalities in the poorest regions of the country benefit particularly from the program. For these regions, implementation of the program is also robustly associated with increased labor supply of adults, reduced fertility, and increased school enrollment. Evidence suggests that the Family Health Program is a highly cost-effective tool for improving health in poor areas.
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Evaluation of the flexural strength and elastic modulus of resins used for temporary restorations reinforced with particulate glass fibre.
Gerodontology
PUBLISHED: 08-23-2010
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The flexural strength and the elastic modulus of acrylic resins, Dencor, Duralay and Trim Plus II, were evaluated with and without the addition of silanised glass fibre.
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Antimicrobial activity of Satureja montana L. essential oil against Clostridium perfringens type A inoculated in mortadella-type sausages formulated with different levels of sodium nitrite.
Int. J. Food Microbiol.
PUBLISHED: 08-06-2010
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This research evaluated the antimicrobial effect of the winter savory (Satureja montana L.) essential oil (EO) against Clostridium perfringens type A (ATCC 3624) inoculated in mortadella-type sausages formulated with different levels of sodium nitrite (NaNO?: 0 ppm, 100 ppm and 200 ppm) in addition to EO at concentrations of 0.0%, 0.78%, 1.56% and 3.125% stored at 25°C for 30 days. The EO extracted by hydrodistillation and analyzed by gas chromatography-mass spectrometry (CG-MS) was tested in vitro using an agar well diffusion method for determination of minimum inhibitory concentration (MIC) on C. perfringens. According to compositional analysis of the winter savory EO, 26 chemical compounds were identified, and the major constituents were thymol (28.99%), p-cymene (12.00%), linalool (11.00%) and carvacrol (10.71%). The results obtained showed that EO applied at a concentration of 1.56%, which was defined as the MIC, exhibited antimicrobial activity against C. perfringens in the in vitro assays, and the transmission electron microscopy (TEM) revealed structural damage and cell lysis of C. perfringens caused by EO treatment. A synergistic effect between NaNO? and EO was observed. In mortadella-type sausages formulated with 100 ppm of NaNO? and EO at all concentrations tested, the population of target microorganisms was reduced (p?0.05) compared to control samples during all storage period. This data suggests the potential combined use of savory EO and minimal amounts of the synthetic additive, NaNO? to control C. perfringens in mortadella, which goes according to current market trends, where consumers are requesting natural products.
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A novel bioactive glass-ceramic for treating dentin hypersensitivity.
Braz Oral Res
PUBLISHED: 06-16-2010
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Dentin hypersensitivity (DH) is a painful response to stimulus applied to the open dentinal tubules of a vital tooth. Its a common oral condition, however, without an ideal treatment available yet. This work evaluated in vitro the effect of micron-sized particles from a novel bioactive glass-ceramic (Biosilicate) in occluding open dentinal tubules. A dentin disc model was employed to observe comparatively, using scanning electron microscopy (SEM), dentinal tubule occlusion by different products and deposition of hydroxyl carbonate apatite (HCA) on dentin surface by Biosilicate, after a single application: G1 - Dentifrice with potassium nitrate and fluoride; G2 - Two-step calcium phosphate precipitation treatment; G3 - Water-free gel containing Biosilicate particles (1%); G4 - Biosilicate particles mixed with distilled water in a 1:10 ratio; all of them after 1, 12 and 24 hours of immersion in artificial saliva. Fourier transform infrared spectroscopy (FTIR) was performed to detect HCA formation on dentin discs filled with Biosilicate after 2 minutes, 30 minutes and 12 hours of immersion in artificial saliva. SEM showed a layer of HCA formed on dentin surface after 24 hours by G4. G1, G2 and G3 promoted not total occlusion of open dentinal tubules after 24 hours. FTIR showed HCA precipitation on the dentin surface induced by Biosilicate after 30 minutes. The micron-sized particles from the bioactive glass-ceramic thus were able to induce HCA deposition in open dentinal tubules in vitro. This finding suggests that Biosilicate may provide a new option for treating DH.
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Genotyping of Cryptosporidium spp. from free-living wild birds from Brazil.
Vet. Parasitol.
PUBLISHED: 04-20-2010
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In wild and domestic birds, cryptosporidiosis is often associated with infections by Cryptosporidium galli, Cryptosporidium baileyi and Cryptosporidium meleagridis. In addition to these species, a number of avian Cryptosporidium species yet to be fully characterized are commonly found among exotic and wild avian isolates. The present study aimed to detect and identify samples of Cryptosporidium spp. from free-living wild birds, in order to contribute to the knowledge of the variability of this parasite in the free-living population of Brazil. Stool samples were collected from 242 birds, with the following proportions of individuals: 50 Emberizidae (20.7%), 112 Psittacidae (46.3%), 44 Cardinalidae (18.2%), 12 Turdidae (5.0%), eight Ramphastidae (3.3%), seven Icteridae (2.9%), three Estrilididae (1.2%), two Contigidae (0.8%), two Thraupidae (0.8%) and two Fringilidae (0.8%). Among the 242 fecal samples from wild birds, 16 (6.6%) were positive for the presence of oocysts of Cryptosporidium. Molecular characterization of the 16 samples of Cryptosporidium, were performed with phylogenetic reconstructions employing 292 positions of 18S rDNA. None of the samples of birds was characterized as C. meleagridis. C. galli was identified in one rufous-bellied thrush (Turdus rufiventris), five green-winged saltators (Saltator similis), one slate-coloured seedeater (Sporophila schistacea), one goldfinch (Carduelis carduelis) and three saffron finches (Sicalis flaveola). One goldfinch isolate, one buffy-fronted seedeater (Sporophila frontalis), one red-cowled cardinal (Paroaria dominicana) and one other saffron finch (S. flaveola) were identified as C. baileyi. Avian genotype II was found in an isolate from a white-eyed parakeet (Aratinga leucophthalma). Clinical symptoms of cryptosporidiosis in birds have already been described and the number of wild birds which were shedding parasites was high. Therefore, further epidemiological research and disease surveillance of birds in the wild is warranted.
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Evaluation of 4 polymerase chain reaction protocols for cultured Leishmania spp. typing.
Diagn. Microbiol. Infect. Dis.
PUBLISHED: 04-02-2010
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Leishmaniasis is a disease caused by the protozoan Leishmania resulting in a variety of clinical manifestations, from self-healing skin lesions to fatal visceral disease. The development of polymerase chain reaction (PCR)-based techniques has made species identification easier, faster, and less labor intensive. The main targets for PCR amplification include kinetoplastid DNA (kDNA), miniexon, and conserved regions such as the internal transcribed spacer. The objective of this work was to evaluate 4 different PCR techniques designed to type Leishmania using laboratory strains. Parasites were subjected to 4 PCR procedures using specific Leishmania primers for miniexon (designated A1 and A2) and kDNA (designated B1 and B2, C1 and C2, and D1, D2 and D3). Discrimination between some species and the 2 main subgenera Leishmania and Viannia was achieved. Unweighted pair group method analysis resulted in the expected clustering of the 2 species from the subgenus Leishmania. However, some species in the subgenus Viannia could not be distinguished, representing a continued challenge for PCR-based protocols. Results are discussed in terms of advantages, limitations, and reproducibility of these 4 PCR-based techniques in the taxonomy of Leishmania.
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[Endometrial osseous metaplasia: clinical presentation and follow-up].
Rev Bras Ginecol Obstet
PUBLISHED: 03-09-2010
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to describe the clinical signs and symptoms of patients with bone metaplasia and to assess the risk factors for changes in these symptoms after removal of the bone fragment.
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Laparoscopic ureteral reimplant for ureteral stricture.
Int Braz J Urol
PUBLISHED: 03-06-2010
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Evaluate the initial experience of laparoscopic ureteral reimplant for ureteral stenosis.
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Occurrence of antibodies anti-Neospora caninum, anti-Toxoplasma gondii, and anti-Leishmania chagasi in serum of dogs from Pará State, Amazon, Brazil.
Parasitol. Res.
PUBLISHED: 03-05-2010
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A cross-sectional study was conducted to determine the occurrence of anti-Toxoplasma gondii, anti-Neospora caninum, and anti- Leishmania chagasi antibodies in dogs of the state of Pará, Brazil. For this purpose, 129 blood samples were collected from dogs of different ages and gender. Samples of 72 dogs were collected from 39 rural properties from 19 municipalities, and 57 samples were from stray dogs, collected after captivity by the Center of Zoonosis Control from the municipality of Santarém. The sera were analyzed for anti-T. gondii and anti-N. caninum antibodies by indirect fluorescent antibody tests with cutoff values of 1:16 and 1:50, respectively. For the presence of L. chagasi antibodies, enzyme-linked immunosorbent assay was used and positive results were confirmed by immunochromatographic method using the recombinant antigen K39. Of the total of 129 dogs, 90 (69.8%) were positive for T. gondii, 16 (12.4%) for N. caninum, and 30 (23.3%) for L. chagasi. Antibodies for all three parasites were found simultaneously in seven dogs (5.4%), mostly in urban dogs (six of seven). No association was observed related to gender and location (urban or rural) of dogs and occurrence of N. caninum and T. gondii antibodies although, regarding L. chagasi, higher prevalence was found in females (P < 0.02) and in dogs from urban location (P < 0.001). From the 39 farms, in 30 (76.9%) at least one dog was positive for T. gondii or N. caninum or both. Higher occurrence of Leishmania antibodies was observed in N. caninum-negative dogs (P < 0.05).
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Serological evidence of Paracoccidioides brasiliensis infection in chickens from Paraná and Mato Grosso do Sul States, Brazil.
Mycopathologia
PUBLISHED: 02-11-2010
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The objective of this study was to detect antibodies against Paracoccidioides brasiliensis in free-range and caged chickens Gallus domesticus. Initially, the humoral immune response of two chickens immunized with P. brasiliensis was evaluated. Both animals showed the production of antibodies to gp43, the major P. brasiliensis antigen. The seroepidemiological survey was conducted in chickens from the Pantanal region in Mato Grosso do Sul State (free-range n = 40) and from northern region of Paraná State (free-range n = 100, caged n = 43). The serum samples were analyzed by indirect ELISA using gp43 as antigen. The positivity observed in free-range chickens from Mato Grosso do Sul (55%) was significantly higher (P = 0.0001) than in free-range chickens from Paraná State (16%). In contrast to the free-range chickens, no positivity was observed in the caged chickens (P = 0.003). This is the first report showing serological evidence of P. brasiliensis infection in chickens. The results suggest that free-range chickens are more frequently infected by P. brasiliensis, probably due to the constant contact with soil than caged chickens and could be useful as epidemiological markers of paracoccidioidomycosis.
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Prevalence of Toxoplasma gondii and Neospora caninum infections in sheep from Federal District, central region of Brazil.
Trop Anim Health Prod
PUBLISHED: 06-19-2009
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Serum samples from 1028 sheep were collected from 32 herds within Federal District, in the central region of Brazil. The samples were examined by indirect fluorescent antibody test (IFAT) using sera diluted 1:64 and 1:50 as cut-off values for the detection of antibodies against Toxoplasma gondii and Neospora caninum, respectively. The observed prevalence for T. gondii infection was 38.22% (26.81%
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Genetic analysis of the cause of endometrial osseous metaplasia.
Obstet Gynecol
PUBLISHED: 06-11-2009
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To analyze solitary bone fragments from the uterine cavity through DNA genotyping, thus elucidating whether they originate from metaplasia, from previous abortion, or both.
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Comparison of agar gel immunodiffusion test, rapid slide agglutination test, microbiological culture and PCR for the diagnosis of canine brucellosis.
Res. Vet. Sci.
PUBLISHED: 04-22-2009
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The performance of the rapid slide agglutination test, with and without 2-mercaptoethanol (RSAT and 2ME-RSAT) and agar gel immunodiffusion test (AGID) was evaluated for the diagnosis of brucellosis in naturally infected dogs. The microbiological culture, PCR and clinical parameters were used as reference. A total of 167 dogs were clinically examined and tested by blood culture, culture of semen/vaginal swab and PCR in blood and semen/vaginal swab. According to the results observed the 167 dogs were divided into three groups: Brucella canis infected dogs (Group 1), B. canis non-infected dogs (Group 2) and dogs with suspected brucellosis (Group 3). The dogs were then tested by RSAT, 2ME-RSAT and AGID. Groups 1 and 2 were used to calculate the diagnostic sensitivity and specificity of the serological tests and the results observed in Group 3 were also discussed. The diagnostic sensitivity of RSAT, 2ME-RSAT and AGID was respectively 70.58%, 31.76%, and 52.94%. The diagnostic specificity of RSAT, 2ME-RSAT and AGID was respectively 83.34%, 100%, and 100%. In dogs with suspected brucellosis 15% were RSAT positive, none was 2ME-RSAT positive and 5% were AGID positive. Although the serological tests are the most commonly used methods for brucellosis diagnosis, a significant proportion of false-negative results were observed highlighting the importance of the direct methods of diagnosis, like blood culture and PCR to improve the diagnosis of canine brucellosis.
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