Experimental Infection of Calomys callosus (Rodentia, Cricetidae) by Toxoplasma gondii

Calomys callosus, Rengger 1830 (Rodentia, Cricetidae), a wild rodent found in Central Brazil, was studied to investigate its susceptibility to Toxoplasma gondii experimental infection and its humoral immune response against this protozoa. The electrophoretic profile of the serum proteins of C. callosus showed that IgG, which shows no affinity to Protein A, has higher cross reactivity with rat IgG than with IgG from other rodents. The susceptibility assay was performed by inoculation groups of animals with various suspensions of T. gondii tachyzoites from 102 to 106 parasites. All animals died between 3 and 9 days after infection and the kinetics of antibody synthesis was determined. Basically, they recognized predominantly the immunodominant antigen SAG-1 (P30). The immunohistochemistry assays revealed that the liver was the most heavily infected organ, followed by the spleen, lungs, intestine, brain and kidneys. It can be concluded that C. callosus is an excellent experimental model for acute phase of Toxoplasma infection

Ultrastructural study of the TG180 murine sarcoma cell invasion by Toxoplasma gondii: comparison between in vivo and in vitro cell cultures

Infection of non-adherent TG180 murine sarcoma cells with Toxoplasma gondii was compared, at the ultrastructural level, in both in vivo and in vitro conditions. Suspensions of 3.0 x 10(6) TG180 cells infected in vitro with 1.0 x 10(6) parasites of the RH strain were harvested between the first and 6th day post-infection and processed for transmission electron microscopy. In vivo infection was made by intraperitoneal inoculation in mice of 1.0 x 10(6) TG180 cells, that were co-inoculated with a parasite suspension at the same cell concentration. Cells were harvested 10, 20, 30 min and 24, 48 h post-inoculation and processed for transmission electron microscopy at the same conditions of the in vitro culture. It was observed TG180 murine sarcoma cells with intense and equivalent intracellular parasitism in both conditions. Host cells with parasitophorous vacuoles containing up to 16 parasites, as well as parasites undergoing mitoses or presenting a bradyzoite-like morphology, were frequently seen in both culture methods.

One-tube nested Polymerase Chain Reaction for detection of Chlamydia trachomatis

Here we present a one-tube nested PCR test, which allows the detection of minimal quantities of Chlamydia trachomatis in human fluids. This assay includes the use of an internal control to avoid false negative results due to the presence of inhibitors. The results obtained show that this assay is robust enough to be used for clinical diagnosis.

Preparation of sand fly (Diptera: Psychodidae: Phlebotominae) specimens for histological studies

Phlebotominae sand fly specimens were prepared for histological and physiological studies. Different fixatives were tested on sectioned and whole bodied adult females in order to obtain good fixation and provide satisfactory penetration of the embedding media. All fixed specimens were infiltrated (up to seven days under 5ºC) and embedded in hydroxyethyl metacrylate. Two-three µm sections were stained, mounted in Canada balsam and observed by light microscopy. Best results were achieved when whole bodied insects were double fixed in Bouin's and Carnoy's fluids (4 h/2 h) and stained in Hematoxilin/Eosin or fixed in calcium formaldehyde and stained in mercury bromophenol blue.

Differential expression of notch signaling-related transcripts accompanies Pro-thymocyte proliferation and phenotype transition induced by epidermal growth factor plus insulin in fetal thymus organ cultures

Thymus regression upon stressing stimuli, such as infectious diseases, is followed by organ reconstitution, paralleling its development in ontogeny. A narrow window of thymus development was here studied, encompassing the pro-T lymphoid precursor expansion during specification stages, by the use of epidermal growth factor plus insulin (INS) in murine fetal thymus organ cultures. Aiming to disclose signaling pathways related to these stages, cultured thymus lobes had their RNA extracted, for the search of transcripts differentially expressed using RNAse protection assays and reverse transcriptase-polymerase chain reactions. We found no difference that could explain INS-driven thymocyte growth, in the pattern of transcripts for death/proliferation mediators, or for a series of growth factor receptors and transcriptional regulators known as essential for thymus development. Thymocyte suspensions from cultured lobes, stained for phenotype analysis by fluorescence activated cell sorting, showed a decreased staining for Notch1 protein at cell surfaces upon INS addition. We analyzed the expression of Notch-related elements, and observed the recruitment of a specific set of transcripts simultaneous and compatible with INS-driven thymocyte growth, namely, transcripts for Notch3, for its ligand Jagged2, and for Deltex1, a mediator of a poorly characterized alternative pathway downstream of the Notch receptor.

Experimental transmission of the parasitic flagellates Trypanosoma cruzi and Trypanosoma rangeli between triatomine bugs or mice and captive neotropical bats

Trypanosoma cruzi and Trypanosoma rangeli-like trypanosomes have been found in a variety of neotropical bat species. In this study, bats (Artibeus lituratus, Carollia perspicillata, Desmodus rotundus, Glossophaga soricina, Molossus molossus, Phyllostomus hastatus) were maintained under controlled conditions, and experiments were conducted to determine how they might become infected naturally with trypanosomes. All bats were first screened for existing infections by hemoculture and the examination of blood smears, and only apparently uninfected animals were then used in the experiments. Proof was obtained that the triatomine bug Rhodnius prolixus would readily feed upon some of the bats, and two species became infected after being bitten by bugs infected with T. rangeli. Some bats also became infected by ingesting R. prolixus carrying T. cruzi, or following subcutaneous or intragastic inoculation with fecal suspensions of R. prolixus containing T. cruzi. P. hastatus became infected after ingesting mice carrying T. cruzi. All of the bats studied inhabit roosts that may be occupied by triatomine bugs and, with the exception of D. rotundus, all also feed to at least some extent upon insects. These findings provide further evidence of how bats may play significant roles in the epidemiology of T. cruzi and T. rangeli in the New World tropics.

Immunodetection of Helicobacter sp. and the associated expression of ABO blood group antigens in the gastric mucosa of captive and free-living New World primates in the Amazon region

The histo-blood group ABH antigens were first described in humans. These antigens are only present on erythrocytes from great apes and humans, while in more primitive animals they are found in tissues and body fluids. The ABH antigens are mainly distributed in tissues exposed to the external environment and potentially serve as ligands for pathogens or inhibitors of tissue connections. The objective of this paper was two-fold: (i) to determine the presence of Helicobacter sp. in the gastric mucosa of 16 captive and 24 free-living New World monkeys and (ii) to evaluate the presence of histopathological alterations related to bacterial infection and the associated expression of ABH antigens in the tissue. Stomach tissues from 13 species of monkey were assessed using haematoxylin-eosin and modified Gram staining (Hucker) methods. An immunohistochemical analysis of the tissue revealed the presence of infectious bacteria that were characteristic of the genus Helicobacter sp. The results demonstrate that various species of monkey might be naturally infected with the Helicobacter sp. and that there is an increased susceptibility to infection. This study serves as a comparative analysis of infection between human and non-human primates and indicates the presence of a new species of Helicobacter.

Comparison of two PCR methods for detection of Leptospira interrogans in formalin-fixed and paraffin-embedded tissues

In this study we compared two polymerase chain reaction (PCR) methods using either 16S ribosomal RNA (rRNA) or 23S rRNA gene primers for the detection of different Leptospira interrogans serovars. The performance of these two methods was assessed using DNA extracted from bovine tissues previously inoculated with several bacterial suspensions. PCR was performed on the same tissues before and after the formalin-fixed, paraffin-embedding procedure (FFPE tissues). The 23S rDNA PCR detected all fresh and FFPE positive tissues while the 16S rDNA-based protocol detected primarily the positive fresh tissues. Both methods are specific for pathogenic L. interrogans. The 23S-based PCR method successfully detected Leptospira in four dubious cases of human leptospirosis from archival tissue specimens and one leptospirosis-positive canine specimen. A sensitive method for leptospirosis identification in FFPE tissues would be a useful tool to screen histological specimen archives and gain a better assessment of human leptospirosis prevalence, especially in tropical countries, where large outbreaks can occur following the rainy season.

Hepatitis C virus quantification in serum and saliva of HCV-infected patients

The hepatitis C virus (HCV) can be detected in blood and other bodily fluids, such as saliva, semen and gastric juices. The aim of this study was to compare the HCV viral loads in the serum and saliva of infected patients. Twenty-nine patients with detectable HCV RNA in their serum and saliva were included in this study. The HCV viral loads were determined through quantitative real-time polymerase chain reactions. The median viral RNA levels were 5.78 log10 copies in the serum and 3.32 log10 copies in the saliva. We observed that the salivary HCV viral load was significantly lower than the viral load in the serum. Further studies are required to understand the role of saliva in the diagnosis, management and potential transmission of HCV.

The infection of microvascular endothelial cells with ExoU-producing Pseudomonas aeruginosa triggers the release of von Willebrand factor and platelet adhesion

An increased plasma concentration of von Willebrand factor (vWF) is detected in individuals with many infectious diseases and is accepted as a marker of endothelium activation and prothrombotic condition. To determine whether ExoU, a Pseudomonas aeruginosa cytotoxin with proinflammatory activity, enhances the release of vWF, microvascular endothelial cells were infected with the ExoU-producing PA103 P. aeruginosa strain or an exoU-deficient mutant. Significantly increased vWF concentrations were detected in conditioned medium and subendothelial extracellular matrix from cultures infected with the wild-type bacteria, as determined by enzyme-linked immunoassays. PA103-infected cells also released higher concentrations of procoagulant microparticles containing increased amounts of membrane-associated vWF, as determined by flow cytometric analyses of cell culture supernatants. Both flow cytometry and confocal microscopy showed that increased amounts of vWF were associated with cytoplasmic membranes from cells infected with the ExoU-producing bacteria. PA103-infected cultures exposed to platelet suspensions exhibited increased percentages of cells with platelet adhesion. Because no modulation of the vWF mRNA levels was detected by reverse transcription-polymerase chain reaction assays in PA103-infected cells, ExoU is likely to have induced the release of vWF from cytoplasmic stores rather than vWF gene transcription. Such release is likely to modify the thromboresistance of microvascular endothelial cells.

Metabolomics in the fight against malaria

Metabolomics uses high-resolution mass spectrometry to provide a chemical fingerprint of thousands of metabolites present in cells, tissues or body fluids. Such metabolic phenotyping has been successfully used to study various biologic processes and disease states. High-resolution metabolomics can shed new light on the intricacies of host-parasite interactions in each stage of the Plasmodium life cycle and the downstream ramifications on the host’s metabolism, pathogenesis and disease. Such data can become integrated with other large datasets generated using top-down systems biology approaches and be utilised by computational biologists to develop and enhance models of malaria pathogenesis relevant for identifying new drug targets or intervention strategies. Here, we focus on the promise of metabolomics to complement systems biology approaches in the quest for novel interventions in the fight against malaria. We introduce the Malaria Host-Pathogen Interaction Center (MaHPIC), a new systems biology research coalition. A primary goal of the MaHPIC is to generate systems biology datasets relating to human and non-human primate (NHP) malaria parasites and their hosts making these openly available from an online relational database. Metabolomic data from NHP infections and clinical malaria infections from around the world will comprise a unique global resource.

Using a top predator as a sentinel for environmental contamination with pathogenic bacteria: the Iberian wolf and leptospires

The Iberian wolf (Canis lupus) is the top predator in the Iberian environments in which it lives, feeding on a wide range of species, thus encountering a wide range of disease agents. Therefore, the wolf can serve as sentinel of environmental contamination with pathogens. We investigated the exposure of free-living wolves to 14 serovars of Leptospira interrogans sensu lato. Kidney samples from 49 wolves collected from 2010-2013 in northwestern Spain were analysed by culture, direct immunofluorescence and polymerase chain reaction. Tissue fluids were analysed for antibodies by a microscopic agglutination test. Ten wolves (observed prevalence: 20%, 95% confidence interval = 11-33%) showed evidence of contact with leptospires, eight through direct detection and nine through serology (7 wolves were positive according to both techniques). Titres below the cut-off level were also detected in seven cases. Serovars confirmed were Canicola (n = 4), Icterohaemorrhagiae (n = 3) and Sejroë, Ballum and Grippotyphosa (n = 1 each), indicating that wolves were infected with serovars for which dogs, rodents and ungulates, are the natural hosts and supporting the utility of the wolf and other large predators as environmental sentinels for pathogens.