Experimental Infection of the Opossum Didelphis aurita by Rickettsia felis, Rickettsia bellii, and Rickettsia parkeri and Evaluation of the Transmission of the Infection to Ticks Amblyomma cajennense and Amblyomma dubitatum

This work evaluated the infection of opossums (Didelphis aurita) by Rickettsia felis, Rickettsia bellii, and Rickettsia parkeri and their role as amplifier hosts for horizontal transmission to Amblyomma cajennense and/or Amblyomma dubitatum ticks. Infection in D. aurita was induced by intraperitoneal inoculation with R. felis (n = 4 opossums), R. bellii (n = 4), and R. parkeri (n = 2). Another group of six opossums were inoculated intraperitoneally with Leibovitz-15 sterile culture medium, representing the uninfected groups (n = 2 opossums simultaneously to each infected group). Opossum blood samples collected during the study were used for DNA extraction, followed by real-time polymerase chain reaction targeting the rickettsial gene gltA, hematology, and detection of Rickettsia spp.-reactive antibodies by indirect immunofluorescence assay. Opossums were infested with uninfected A. cajennense and/or A. dubitatum for 30 days postinoculation (DPI). Flat ticks molted from ticks fed on opossums were allowed to feed on uninfected rabbits, which were tested for seroconversion by immunofluorescence assay. Samples of flat ticks were also tested by real-time polymerase chain reaction. Inoculated opossums showed no clinical abnormalities. Antibodies to Rickettsia spp. were first detected at the second to fourth DPI, with detectable titers until the 150th DPI. Rickettsemia was detected only in one opossum inoculated with R. parkeri, at the eighth DPI. Only one A. cajennense tick (2.0%) previously fed on a R. parkeri-inoculated opossum became infected. None of the rabbits infested with opossum-derived ticks seroconverted. The study demonstrated that R. felis, R. bellii, and R. parkeri were capable to produce antibody response in opossums, however, with undetectable rickettsemia for R. felis and R. bellii, and very low rickettsemia for R. parkeri. Further studies must be done with different strains of these rickettsiae, most importantly the strains that have never gone through in vitro passages.

Experimental Infection of Opossums Didelphis aurita by Rickettsia rickettsii and Evaluation of the Transmission of the Infection to Ticks Amblyomma cajennense

The present study evaluated the infection of opossums (Didelphis aurita) by Rickettsia rickettsii and their role as amplifier hosts for horizontal transmission of R. rickettsii to Amblyomma cajennense ticks. Three groups of opossums were evaluated: on day 0, group 1 (G1) was inoculated intraperitoneally with R. rickettsii; group 2 (G2) was infested by R. rickettsii-infected ticks; and group 3 (G3) was the uninfected control group. Opossum rectal temperature was measured daily. Blood samples were collected every 2 to 4 days during 30 days, and used to (1) inoculate guinea pigs intraperitoneally; (2) extract DNA followed by real-time polymerase chain reaction (PCR) targeting the rickettsial gene gltA; (3) study hematology; (4) detect R. rickettsii-reactive antibodies by indirect direct immunofluorescence assay (IFA). Blood was also collected every 10 days from days 30 to 180, to be tested by serology. Opossums were infested by uninfected A. cajennense larvae and nymphs from days 3 to 15. Engorged ticks were collected and allowed to molt in an incubator. Thereafter, the subsequent flat ticks were allowed to feed on uninfected rabbits, which were tested for seroconversion by IFA. Samples of flat ticks were also tested by real-time PCR. All G1 and G2 opossums became infected by R. rickettsii, as demonstrated by real-time PCR or/and guinea pig inoculation, but they showed no clinical abnormality. Rickettsemia was first detected at days 2 to 8, lasting intermittently till days 1 to 30. Approximately 18% and 5% of the flat ticks previously fed on G1 and G2 opossums, respectively, became infected by R. rickettsii, but only the rabbits infested with G1-derived ticks seroconverted. The study demonstrated that R. rickettsii was capable of infecting opossums without causing illness and developing rickettsemia capable of causing infection in guinea pigs and ticks, although the infection rate in ticks was low.

Hepatitis B Infection Is Associated with Asymptomatic Malaria in the Brazilian Amazon

Background: Areas that are endemic for malaria are also highly endemic for hepatitis B virus (HBV) infection. Nevertheless, it is unknown whether HBV infection modifies the clinical presentation of malaria. This study aimed to address this question. Methodology and Findings: An observational study of 636 individuals was performed in Rondonia, western Amazon, Brazil between 2006 and 2007. Active and passive case detections identified Plasmodium infection by field microscopy and nested Polymerase Chain Reaction (PCR). HBV infections were identified by serology and confirmed by real-time PCR. Epidemiological information and plasma cytokine profiles were studied. The data were analyzed using adjusted multinomial logistic regression. Plasmodium-infected individuals with active HBV infection were more likely to be asymptomatic (OR: 120.13, P < 0.0001), present with lower levels of parasitemia and demonstrate a decreased inflammatory cytokine profile. Nevertheless, co-infected individuals presented higher HBV viremia. Plasmodium parasitemia inversely correlated with plasma HBV DNA levels (r=-0.6; P=0.0003). Conclusion: HBV infection diminishes the intensity of malaria infection in individuals from this endemic area. This effect seems related to cytokine balance and control of inflammatory responses. These findings add important insights to the understanding of the factors affecting the clinical outcomes of malaria in endemic regions.

Copper sprays and windbreaks for control of citrus canker on young orange trees in southern Brazil

The benefit of windbreaks and copper sprays for control of citrus canker caused by Xanthomonas axonopodis pv. citri was investigated in a commercial citrus orchard located in a citrus canker endemic area in southern Brazil. Control of canker was evaluated as incidence and severity of lesions on foliage and by the effect on premature leaf and fruit drop for three production seasons. Effect of the treatments on fruit production was evaluated as incidence of citrus canker on prematurely abscised fruits and harvested fruits. Copper application significantly reduced damage to foliage and fruit, while windbreaks made little contribution to disease control. Copper sprays increased fruit yield for 3 years. This could be attributed to lower incidence of fruit with lesions and fewer fruits abscised due to canker infection. Incidence and severity on the leaves were inversely related to the number of fruits harvested per tree and directly related to the number of fruits abscised per tree. Published by Elsevier Ltd.

Splenectomy Increases Mortality in Murine Trypanosoma cruzi Infection

The spleen is a secondary lymphoid organ that harbours a variety of cells such as T and B lymphocytes and antigen-presenting cells important to immune response development. In this study, we evaluated the impact of spleen removal in the immune response to experimental Trypanosoma cruzi infection. C57BL/6 mice were infected with Y strain of the parasite and infection was followed daily. Mice that underwent splenectomy had fewer parasites in peripheral blood at the peak of infection; however, mortality was increased. Histological analysis of heart and liver tissues revealed an increased number of parasites and inflammatory infiltrates at these sites. Spleen removal was associated with reduction in IFN-gamma and TNF-alpha production during infection as well as with a decrease in specific antibody secretion. Haematological disorders were also detected. Splenectomized mice exhibited severe anaemia and decreased bone marrow cell numbers. Our results indicate that spleen integrity is critical in T. cruzi infection for the immune response against the parasite, as well as for the control of bone marrow haematological function.

Antileishmanial activity screening of 5-nitro-2-heterocyclic benzylidene hydrazides

A series of 53 nitro derivatives rationally designed were obtained by parallel synthesis and screened against Leishmania donovani. Six compounds exhibited IC(50) values lower than standard drugs. Brief SAR analysis revealed that substitution is important to the activity. Nitrothiophene analogues were more potent than the nitrofuran ones. This was attributed to the ability of sulfur atoms in accommodating electrons from nitro group, which facilitate its reduction and therefore the formation of free radicals lethal to parasites. (c) 2008 Elsevier Ltd. All rights reserved.

Lipid microspheres loaded with antigenic membrane proteins of the Leishmania amazonensis as a potential biotechnology application

Lipid microspheres (LM) are excellent drug delivery or vaccines adjuvant systems and are relatively stable. The aim of this work is to develop and characterize a system that is able to encapsulate and present antigenic membrane proteins from Leishmania amazonensis. Membrane proteins are important for vaccine`s formulation because these proteins come in contact with the host cell first, triggering the cell mediated immune response. This is a useful tool to avoid or inactivate the parasite invasion. The LM are constituted by soybean oil (SO), dipalmitoylphosphatidilcholine (DPPC), cholesterol and solubilized protein extract (SPE). The particles formed presented an average diameter of 200 run, low polydispersion and good stability for a period of 30 days, according to dynamic light scattering assays. Isopycnic density gradient centrifugation of LM-protein showed that proteins and lipids floated in the sucrose gradient (5-50%w/v) suggesting that the LM-protein preparation was homogeneous and that the proteins are interacting with the system. The results show that 85% of SPE proteins were encapsulated in the LM. Studies of cellular viability of murine peritoneal macrophages show that our system does not present cytotoxic effect for the macrophages and still stimulates their NO production (which makes its application as a vaccine adjuvant possible). LM-protein loaded with antigenic membrane proteins from L. amazonensis seems to be a promising vaccine system for immunization against leishmaniasis. (C) 2009 Elsevier Inc. All rights reserved.

Dynamics of Hepatitis D (delta) virus genotype 3 in the Amazon region of South America

Hepatitis delta virus (HDV) is widely distributed and associated with fulminant hepatitis epidemics in areas with high prevalence of HBV. Several studies performed in the 1980s showed data on HDV infection in South America, but there are no studies on the viral dynamics of this virus. The aim of this study was to conduct an evolutionary analysis of hepatitis delta genotype 3 (HDV/3) prevalent in South America: estimate its nucleotide substitution rate, determine the time of most recent ancestor (TMRCA) and characterize the epidemic history and evolutionary dynamics. Furthermore, we characterized the presence of HBV/HDV infection in seven samples collected from patients who died due to fulminant hepatitis from Amazon region in Colombia and included them in the evolutionary analysis. This is the first study reporting HBV and HDV sequences from the Amazon region of Colombia. Of the seven Colombian patients, five were positive for HBV-DNA and HDV-RNA. Of them, two samples were successfully sequenced for HBV (subgenotypes F3 and Fib) and the five samples HDV positive were classified as HDV/3. By using all HDV/3 available reference sequences with sampling dates (n = 36), we estimated the HDV/3 substitution rate in 1.07 x 10(-3) substitutions per site per year (s/s/y), which resulted in a time to the most recent common ancestor (TMRCA) of 85 years. Also, it was determined that HDV/3 spread exponentially from early 1950s to the 1970s in South America. This work discusses for the first time the viral dynamics for the HDV/3 circulating in South America. We suggest that the measures implemented to control HBV transmission resulted in the control of HDV/3 spreading in South America, especially after the important raise in this infection associated with a huge mortality during the 1950s up to the 1970s. The differences found among HDV/3 and the other HDV genotypes concerning its diversity raises the hypothesis of a different origin and/or a different transmission route. (C) 2011 Elsevier B.V. All rights reserved.

Hepatitis D and B virus genotypes in chronically infected patients from the Eastern Amazon Basin

Hepatitis D virus (HDV) is a defective hepatotropic virus whose infectivity is dependent on hepatitis B virus (HBV). HDV super- or co-infiection leads to an increased risk of fulminant hepatitis or progression to severe chronic liver disease in HBV infected patients. The Brazilian Amazon Basin has been reported to be endemic for HBV and HDV, especially in the Western Amazon Basin. In this region, HDV infection is frequently associated with acute fulminant hepatitis with characteristic histologic features. HDV is classified into seven major clades (HDV-1 to HDV-7) and HBV is subdivided into eight genotypes (A-H). HDV and HBV genotypes have been shown to have a distinct geographic distribution. The aim of this study was to determine the HBV and HDV genotypes harbored by chronically infected patients from the Eastern Amazon Basin, Brazil. We studied 17 serum samples from HBV and HDV chronically infected patients admitted to a large public hospital (Santa Casa de Misericordia) at Belem, state of Para, Brazil, between 1994 and 2002. HDV-3 and HBV genotype A (subtype adw2) have been identified in all cases, in contrast to previous studies from other regions of the Amazon, where HBV genotype F has been found co-infecting patients that harbored HDV-3. The HDV-3/HBV-A co-infection suggests that there is not a specific interaction between HBV and HDV genotypes, and co-infection might merely reflect the most frequent genotypes found in a particular geographic area. The analysis of the carboxy-terminal region of the large hepatitis D antigen (L-HDAg), which interacts with the hepatitis B surface antigen (HBsAg) and is essential for HDV assembly, showed some diversity between the different isolates from the Eastern Amazon. This diversity is not observed among HDV-3 sequences from other South American regions. (C) 2008 Elsevier B.V. All rights reserved.

In vitro effect of Aloe vera, Coriandrum sativum and Ricinus communis fractions on Leishmania infantum and on murine monocytic cells

In South America, visceral leishmaniasis is a zoonosis caused by the protozoan species Leishmania infantum (syn. L. chagasi) and is primarily transmitted through the bite of the female Lutzomyia longipalpis. Its main reservoir in urban areas is the dog. The application of control measures recommended by health agencies have not achieved significant results in reducing the incidence of human cases, and the lack of effective drugs to treat dogs resulted in the prohibition of this course of action in Brazil. Therefore, it is necessary to search new alternatives for the treatment of canine and human visceral leishmaniasis. The objectives of this study were to evaluate the in vitro effect of fractions from Aloe vera (aloe), Coriandrum sativum (coriander), and Ricinus communis (castor) on promastigotes and amastigotes of L. infantum and to analyze the toxicity against the murine monocytic cells RAW 264.7. To determine the viability of these substances on 50% parasites (IC50), we used a tetrazolium dye (MU) colorimetric assay (bromide 3-4.5-dimethylthiazol-2-yl-2,5-dephenyltetrazolium), and on amastigotes we performed an in situ ELISA. All fractions were effective against L. infantum promastigotes and did not differ from the positive control pentamidine (p > 0.05). However, the R. communis ethyl acetate and chloroform fractions, as well as the C. sativum methanol fraction, were the most effective against amastigotes and did not differ from the positive control amphotericin B (p > 0.05). The R. communis ethyl acetate fraction was the least toxic, presenting 83.5% viability of RAW 264.7 cells, which was similar to the results obtained with amphotericin B (p > 0.05). Based on these results, we intend to undertake in vivo studies with R. communis ethyl acetate fractions due the high effectiveness against amastigotes and promastigotes of L. infantum and the low cytotoxicity towards murine monocytic cells. (C) 2011 Elsevier B.V. All rights reserved.

Esophageal cancer associated with chronic mucocutaneous candidiasis. Could chronic candidiasis lead to esophageal cancer?

Chronic mucocutaneous candidiasis (CMC) is a rare disease associated with immunodeficiency and characterized by persistent and refractory infections of the skin, appendages and mucous membranes caused by members of the genus Candida. Several different disorders are classified under this common denominator, including chronic and recurrent mucocutaneous infections due to Candida spp., which are sometimes linked to autoimmune endocrinopathies. These fungal infections are usually confined to the mucocutaneous surface, with little propensity for systemic disease or septicemia. We describe a patient with CMC who had an esophageal candidiasis refractory to treatment for decades and who developed an epidermoid esophageal cancer. No risk factors such as familiar susceptibility, smoking, alcohol drinking, or living in an endemic area were verified. This case report suggests the participation of nitrosamine compounds produced by chronic Candida infections as a risk factor for esophageal cancer in a patient with autosomal-dominant chronic mucocutaneous candidiasis.

Antileishmanial and antitrypanosomal activity of bufadienolides isolated from the toad Rhinella jimi parotoid macrogland secretion

Amphibian skin secretions are considered a rich source of biologically active compounds and are known to be rich in peptides, bufadienolides and alkaloids. Bufadienolides are cardioactive steroids from animals and plants that have also been reported to possess antimicrobial activities. Leishmaniasis and American Trypanosomiasis are parasitic diseases found in tropical and subtropical regions. The efforts toward the discovery of new treatments for these diseases have been largely neglected, despite the fact that the only available treatments are highly toxic drugs. In this work, we have isolated, through bioguided assays, the major antileishmanial compounds of the toad Rhinella jimi parotoid macrogland secretion. Mass spectrometry and (1)H and (13)C NMR spectroscopic analyses were able to demonstrate that the active molecules are telocinobufagin and hellebrigenin. Both steroids demonstrated activity against Leishmania (L.) chagasi promastigotes, but only hellebrigenin was active against Trypanosoma cruzi trypomastigotes. These steroids were active against the intracellular amastigotes of Leishmania, with no activation of nitric oxide production by macrophages. Neither cytotoxicity against mouse macrophages nor hemolytic activities were observed. The ultrastructural studies with promastigotes revealed the induction of mitochondrial damage and plasma membrane disturbances by telocinobufagin, resulting in cellular death. This novel biological effect of R. jimi steroids could be used as a template for the design of new therapeutics against Leishmaniasis and American Trypanosomiasis. (C) 2008 Elsevier Ltd. All rights reserved.

Loxosceles venom-induced cytokine activation, hemolysis, and acute kidney injury

Herein, we describe a confirmed case of Loxosceles spider bite that illustrates the critical complications seen in loxoscelism, including skin necrosis, rhabdomyolysis, hemolysis, coagulopathy, acute kidney failure, and electrolyte disorders. Upon initial assessment, laboratory studies revealed the following: the white blood cell count was 29 400 WBCs/mm(3), hemoglobin was 9.2g/dL, and the platelet count was 218000cells/mm(3). Coagulation studies revealed the following: international normalized ratio, 1.83; activated partial-thromboplastin time, 62s; D-dimer, 600 ng/mL (normal range < 500 ng/mL); free protein S, 37% (normal range = 64-114%); protein C, negative; and antithrombin III, negative. Various serum levels were abnormal: urea, 110mg/dL; creatinine, 3.1 mg/dL; indirect bilirubin, 3.8 mg/dL; creatine kinase, 1631 U/L, lactate dehydrogenase, 6591 U/L; potassium 6.2mmol/L. Urine tests were positive for hemoglobin and bilirubin. In addition, concentrations of interleukin-6 and tumor necrosis factor-alpha were notably elevated in the serum. In conclusion, physicians must be alert to the possibility of loxoscelism when a patient presents with the clinical and laboratory findings described above, especially if the patient resides in an endemic area. Advances in our understanding of multiple pathways and mediators that orchestrate the response to Loxosceles venom might reveal new possibilities for the management of loxoscelism. (C) 2007 Elsevier Ltd. All rights reserved.

Exacerbation of Leishmania (Viannia) shawi infection in BALB/c mice after immunization with soluble antigen from amastigote forms

The present study aimed to evaluate the effects of immunization with soluble amastigote (AmaAg) and promastigote (ProAg) antigens from Leishmania (Viannia) shawi on the course of infection in BALB/c mice. After immunization with AmaAg, the challenged group showed greater lesion size and parasite load in the skin and lymph nodes, associated with diminished interleukin (IL)-2, IL-4, IL-10, interferon (IFN)-gamma and nitrate levels in the supernatant of lymph node cell cultures, together with increases in transforming growth factor (TGF)-beta concentrations and humoral immune response. In contrast, immunization with ProAg led to smaller lesion size with reduced numbers of viable parasites in the skin. Protection was associated with increases in IL-12, IFN-gamma, TGF-beta and nitrates and decreases in IL-4 and IL-10 levels. Concerning humoral immune response, a significant reduction in anti-leishmania immunoglobulin G was verified in the ProAg-challenged group. Analysis of these results suggests that AmaAg induced a suppressive cellular immune response in mice, favouring the spread of infection, whereas ProAg induced partial protection associated with increased cellular immune response.

Infectious diseases in paediatric pathology: experience from a developing country

Infectious and parasitic diseases have always challenged man. Although many of them are typically seen in some areas of the world and can be adequately managed by just improving socioeconomic status and sanitary conditions, they are still quite prevalent and may sometimes be seen outside their original geographical areas. Human migration due to different reasons, tourism, blood transfusion and solid organ transplantation has created new concerns for health professionals all over the world. If not for diagnostic purposes, at least these tropical and infectious diseases should be largely known because their epidemiology, pathogenesis, host/parasite interaction, inflammatory and reparative responses are quite interesting and teach us about human biology. Curiosity is inherent to pathology practice and so we are compelled to look for things other than tumours or degenerative diseases. This review focuses on infectious and parasitic diseases found in a developing country and brings up-to-date information on diseases caused by viruses (dengue, yellow fever), bacteria (typhoid fever, leprosy), parasites (Chagas` disease, cutaneous and visceral leishmaniasis, amoebiasis, Capillaria hepatica, schistosomiasis, cysticercosis) and caused by fungi (paracoccidioidomycosis, cryptococcosis, histoplasmosis) that may be useful for pathologists when facing somewhat strange cases from developing countries.