Molecular detection of Paracoccidioides brasiliensis in road-killed wild animals

Paracoccidioides brasiliensis infections have been little studied in wild and/or domestic animals, which may represent an important indicator of the presence of the pathogen in nature. Road-killed wild animals have been used for surveillance of vectors of zoonotic pathogens and may offer new opportunities for eco-epidemiological studies of paracoccidiodomycosis (PCM). The presence of P. brasiliensis infection was evaluated by Nested-PCR in tissue samples collected from 19 road-killed animals; 3 Cavia aperea (guinea pig), 5 Cerdocyon thous (crab-eating-fox), 1 Dasypus novemcinctus (nine-banded armadillo), 1 Dasypus septemcinctus (seven-banded armadillo), 2 Didelphis albiventris (white-eared opossum), 1 Eira barbara (tayra), 2 Gallictis vittata (grison), 2 Procyon cancrivorus (raccoon) and 2 Sphiggurus spinosus (porcupine). Specific P. brasiliensis amplicons were detected in (a) several organs of the two armadillos and one guinea pig, (b) the lung and liver of the porcupine, and (c) the lungs of raccoons and grisons. P. brasiliensis infection in wild animals from endemic areas might be more common than initially postulated. Molecular techniques can be used for detecting new hosts and mapping `hot spot` areas of PCM.

Paracoccidioides brasiliensis infection in dogs from Western Brazilian Amazon

The objective of the study was to evaluate Paracoccidioides brasiliensis infection in urban dogs from the municipality of Monte Negro, Rondonia, Western Brazilian Amazon. The serum samples (n=126) were analyzed by indirect ELISA and the immunodiffusion test using P. brasiliensis gp43 and exoantigen as antigens, respectively. A positivity of 54.8% was observed only in the ELISA test and no statistical difference was observed in the seroprevalence in relation to age or sex. This is the first paracoccidioidomycosis survey carried out with dogs from the Western Brazilian Amazon. The higher positivity rates of P. brasiliensis infection observed in this study suggest that veterinarians must be alert to detect new cases of natural disease in dogs living in paracoccidioidomycosis endemic areas.

Hepatic Involvement In Paediatric Patients With Paracoccidioidomycosis: A Histological Study.

Paracoccidioidomycosis is a systemic mycosis that is endemic to certain countries in Latin America. This study aimed to describe the histological features of liver involvement in patients with paracoccidioidomycosis aged <16 years of age who were treated between 1980 and 2010, with a diagnosis that was confirmed by detection of the fungus by pathological examination. Liver tissue was obtained from one necropsy and 12 biopsies. Throughout 2007, biopsies were taken from patients with persistent jaundice or portal hypertension, after which biopsies became indicated due to elevated aminotransferase and low albumin levels. Using haematoxylin and eosin (H&E), Masson's trichrome and immunohistochemical (CK7 and CK19) staining, we noted degenerative alterations in bile duct cells and inflammatory injury to the bile ducts in 10 biopsies. Using immunohistochemistry for CK7 and CK19, we observed ductal proliferation in all 12 samples. Bile duct injuries by inflammatory cells might explain the predominant increase in canalicular enzymes; immunohistochemistry is more sensitive in demonstrating ductular reactions and might show changes that are not apparent on H&E staining.

Synchronous Oral Paracoccidioidomycosis And Pulmonary Tuberculosis In An Immunocompetent Patient.

Paracoccidioidomycosis (PCM) and tuberculosis (TB) are chronic granulomatous infectious diseases, in which the main form of contraction is through inhalation of the microorganism-Paracoccidioides brasiliensis and Mycobacterium tuberculosis. Oral involvement of PCM is observed in up to 70 % of the cases and usually presents clinically as ulcerations with granular surface showing tiny hemorrhagic areas. Oral presentation of TB is rare with prevalence smaller than 0.5 % of all cases. Clinical presentation of oral TB mainly consists of single ulcers with irregular limits and necrotic base. A 70-year-old immunocompetent man presented simultaneously oral PCM and pulmonary TB. Medical history revealed a previous diagnosis of pulmonary TB; however, even under treatment for TB, the patient remained with oral lesions and intense pulmonary fibrosis. The physician requested P. brasiliensis serological analysis, which resulted positive. Although the combination of PCM and TB has been reported in the literature, it is still considered an uncommon condition and their diagnosis may represent a challenge to healthcare professionals because of the similarity between their clinical and radiological presentations.

Masticatory afficiency in denture wearers with bilateral balanced occlusion and canine guidance

The maintenance of masticatory function is especially important for patients wearing complete dentures due to their limitations. Thus, the bilateral balanced occlusal concept is used to achieve greater masticatory efficiency. However, a critical review of the literature reveals that there is not sufficient scientific evidence to support bilateral balanced occlusion as the most appropriate occlusal concept in complete dentures. Therefore, the aim of this study was to evaluate the masticatory efficiency in complete dentures wearers with bilateral balanced occlusion and canine guidance. A double-blinded controlled crossover clinical trial was conducted. The sample was composed by 24 edentulous patients who wore sets of complete dentures with both occlusal concepts during equal periods of 3 months. Objective data were collected through the masticatory efficiency test performed by the colorimetric method with the beads, in which capsules of a synthetic material enclosing fuchsine-containing granules were used. Subjective data were recorded by patient's ratings of their chewing function. No significant statistical difference was found for masticatory efficiency (p=0.095) between the two occlusal concepts studied. The results suggest that bilateral balanced occlusion does not improve the masticatory efficiency in complete denture wearers.

Bottle feeding, increased overjet and Class 2 primary canine relationship: is there any association?

The aim of this study was to investigate the association between bottle feeding and prevalence rates of increased overjet and Class 2 primary canine relationship. The sample consisted of 911 children (461 boys, 450 girls) aged 3 (13.9%), 4 (40.8%), 5 (34%) and 6 (11.3%) years, with complete primary dentition. Information about nutritive and nonnutritive (pacifier and/or digit) sucking habits was collected through questionnaires. Three calibrated dentists (κ: 0.9-1.0 and Rs > 0.90) performed the clinical assessments. The children were divided into four groups: G1 - not bottle-fed; G2 - exclusively bottle-fed; G3 - breast- and bottle-fed, bottle feeding ceased before 3 years of age; and G4 - breast- and bottle-fed, bottle feeding ceased between 3 and 4 years of age. Associations between nutritive and nonnutritive sucking behaviors and the malocclusions studied were analyzed by multiple binary logistic regression (α= 0.05). The frequencies of increased overjet were: 25.3% (G1), 38.8% (G2), 39.2% (G3) and 47.8% (G4). The percentages of Class 2 canine relationship were: 27.9% (G1), 48.8% (G2), 43.4% (G3) and 43% (G4). No significant effect of bottle feeding was found. The chances of diagnosing increased overjet (O.R. = 4.42, p < 0.001) and Class 2 canine relationship (O.R. = 4.02, p < 0.001) were greater for children with pacifier and/or digit-sucking habits, compared to those without a history of nonnutritive sucking behavior. It may be suggested that bottle feeding alone is not directly associated with higher prevalence rates of increased overjet and Class 2 canine relationship in the primary dentition.

Sensitivity evaluation of a single-step PCR assay using Ehrlichia canis p28 gene as a target and its application in diagnosis of canine ehrlichiosis

The aim of this study was to optimize a PCR assay that amplifies an 843 pb fragment from the p28 gene of Ehrlichia canis and compare it with two other PCR methods used to amplify portions of the 16S rRNA and dsb genes of Ehrlichia. Blood samples were collected from dogs suspected of having a positive diagnosis for canine ehrlichiosis. Amplification of the p28 gene by PCR produced an 843-bp fragment and this assay could detect DNA from one gene copy among 1 billion cells. All positive samples detected by the p28-based PCR were also positive by the 16S rRNA nested-PCR and also by the dsb-based PCR. Among the p28-based PCR negative samples, 55.3% were co-negatives, but 27.6% were positive in 16S rRNA and dsb based PCR assays. The p28-based PCR seems to be a useful test for the molecular detection of E. canis, however improvements in this PCR sensitivity are desired, so that it can become an important alternative in the diagnosis of canine ehrlichiosis.

Microbiological and histopathological aspects of canine pyometra

As pyometra is recognized as one of the main causes of disease and death in the bitch the purposes of this study were to evaluate microbiological and histopathological aspects of canine pyometra and to research the virulence factors of the E. coli isolates identifying possible risks to human health. The microbiological isolation from the intrauterine contents of 100 dogs with pyometra was carried out and the virulence factors in the E. coli strains were identified using PCR method. This study also consisted of the counting of microorganisms colonies forming units in samples of intrauterine content, tests of antimicrobial susceptibility of the E. coli isolates and the histological examination of the uterus. E. coli was the most prevalent microorganism isolated (76.6%) and 120 strains (79.5%) were positive for sfa, 86 (56.9%) were positive for cnf, 87 (57.6%) were positive for pap, 52 (34.4%) were positive for hly, 51 (33.8%) were positive for iuc and 5 (3.3%) were positive for afa genes. One observed more sensitivity of E. coli to norfloxacin, polimixin B, sulphazotrin, chloranfenicol and enrofloxacin. In 42% of the samples of uterine walls where microorganisms were isolated, the sizes of the areas of the inflammatory responses corresponded to 39-56%. Virulence factors were identified in 98.0% of the strains evaluated, demonstrating a high frequency of potentially pathogenic E. coli. It must be considered that dogs are animals that are living in close proximity to man for thousands of years and have an important role in the transmission of E. coli to other animals and to man.

The finding of Lutzomyia almerioi and Lutzomyia longipalpis naturally infected by Leishmania spp. in a cutaneous and canine visceral leishmaniases focus in Serra da Bodoquena, Brazil

To identify natural infections by Leishmania spp. in insect vectors of cutaneous and visceral leishmaniasis, we performed field studies in natural and anthropic environments in the Guaicurus Settlement (Bodoquena Range) of the Bonito municipality, Mato Grosso do Sul state, Brazil. From October 2002 to October 2003, a total of 1395 sandfly females were captured with Shannon and light traps and dissected in search of flagellates. The sample is composed of a total of 13 species, with Lutzomyia almerioi (59.9%) and Lutzomyia longipalpis (31.4%) predominant. Infections by flagellates were directly observed in three of the dissected of Lu. almerioi females (0.36%). To increase the sensitivity of detection, DNA extracted from pools of the 1220 dissected females (Lu. almerioi 808, Lu. longipalpis 399 and Nyssomyia whitmani 13) was subjected to small subunit rRNA-based polymerase chain reactions (SSU-PCR). DNA from Leishmania (L.) infantum chagasi was detected in at least 0.37% of Lu. almerioi females and in 0.25% of Lu. longipalpis females. The DNA of the Leishmania (Viannia) sp. was detected in 0.12% of Lu. almerioi and in 0.70% of Lu. longipalpis. Leishmania (L.) amazonensis was found in 1.25% of Lu. longipalpis. Mixed infections of L. (Leishmania) sp. and L. (Viannia) sp. were found in 0.50% of Lu. longipalpis. When considering that each positive pool contained at least a single infected specimen, we found a 1.23% rate of Leishmania spp. infection among the total population of dissected female sand flies as determined by PCR. This is the first report of natural infection by L. (L.) infantum chagasi and L. (Viannia) sp. in Lu. almerioi. It is also the first report of infection by L. (Viannia) sp. in Lu. longipalpis. The observation that Lu. longipalpis and Lu. almerioi are naturally infected by agents of both cutaneous and visceral leishmaniases suggests that these two species play a role in the transmission (continua) (continuação) of these diseases within the study area. Furthermore, the finding that Lu. longipalpis has been naturally infected by L. (L.) amazonensis and L. (Viannia) sp., and Lu. almerioi by L. (L.) infantum chagasi and L. (Viannia), suggests their participation as permissive vectors

Dendritic Cells Transfected with scFv from Mab 7.B12 Mimicking Original Antigen gp43 Induces Protection against Experimental Paracoccidioidomycosis

Paracoccidioidomycosis (PCM), endemic in Latin America, is a progressive systemic mycosis caused by Paracoccidioides brasiliensis (P. brasiliensis), which primarily attacks lung tissue. Dendritic cells (DCs) are able to initiate a response in naive T cells, and they also participate in Th-cell education. Furthermore, these cells have been used for therapy in several disease models. Here we transfected DCs with a plasmid (pMAC/PS-scFv) encoding a single chain variable fragment (scFv) of an anti-Id antibody that is capable of mimicking gp43, the main antigenic component of P. brasiliensis. First, Balb/c mice were immunized subcutaneously with pMAC/PS-scFv and, after seven days, scFv protein was presented to the regional lymph nodes cells. Moreover, we showed that the DCs transfected with scFv were capable of efficiently activating proliferation of total lymph node cells and inducing a decrease in lung infection. Therefore, our results suggested that the use of scFv-transfected DCs may be a promising therapy in the paracoccidioidomycosis (PCM) model.

T Helper 1-Inducing Adjuvant Protects against Experimental Paracoccidioidomycosis

Immunostimulatory therapy is a promising approach to improving the treatment of systemic fungal infections such as paracoccidioidomycosis (PCM), whose drug therapy is usually prolonged and associated with toxic side effects and relapses. The current study was undertaken to determine if the injection of a T helper (Th) 1-stimulating adjuvant in P. brasiliensis infected mice could have a beneficial effect on the course of experimental PCM. For this purpose, mice were infected and treated with complete Freund's adjuvant (CFA), a well-established Th1 experimental inductor, or incomplete Freund's adjuvant (IFA - control group) on day 20 postinfection. Four weeks after treatment, the CFA-treated mice presented a mild infection in the lungs characterized by absence of epithelioid cell granulomas and yeast cells, whereas the control mice presented multiple sites of focal epithelioid granulomas with lymphomonocytic halos circumscribing a high number of viable and nonviable yeast cells. In addition, CFA administration induced a 2.4 log reduction (>99%) in the fungal burden when compared to the control group, and led to an improvement of immune response, reversing the immunosuppression observed in the control group. The immunotherapy with Th1-inducing adjuvant, approved to be used in humans, might be a valuable tool in the treatment of PCM and potentially useful to improve the clinical cure rate in humans.

Cell-Free Antigens from Paracoccidioides brasiliensis Drive IL-4 Production and Increase the Severity of Paracoccidioidomycosis

The thermally dimorphic fungus Paracoccidioides brasiliensis (Pb) is the causative agent of paracoccidioidomycosis (PCM), one of the most frequent systemic mycosis that affects the rural population in Latin America. PCM is characterized by a chronic inflammatory granulomatous reaction, which is consequence of a Th1-mediated adaptive immune response. In the present study we investigated the mechanisms involved in the immunoregulation triggered after a prior contact with cell-free antigens (CFA) during a murine model of PCM. The results showed that the inoculation of CFA prior to the infection resulted in disorganized granulomatous lesions and increased fungal replication in the lungs, liver and spleen, that paralleled with the higher levels of IL-4 when compared with the control group. The role of IL-4 in facilitating the fungal growth was demonstrated in IL-4-deficient- and neutralizing anti-IL-4 mAb-treated mice. The injection of CFA did not affect the fungal growth in these mice, which, in fact, exhibited a significant diminished amount of fungus in the tissues and smaller granulomas. Considering that in vivo anti-IL-4-application started one week after the CFA-inoculum, it implicates that IL-4-CFA-induced is responsible by the mediation of the observed unresponsiveness. Further, the characterization of CFA indicated that a proteic fraction is required for triggering the immunosuppressive mechanisms, while glycosylation or glycosphingolipids moieties are not. Taken together, our data suggest that the prior contact with soluble Pb antigens leads to severe PCM in an IL-4 dependent manner.

First Description of a Cluster of Acute/Subacute Paracoccidioidomycosis Cases and Its Association with a Climatic Anomaly

Background: Identifying clusters of acute paracoccidioidomycosis cases could potentially help in identifying the environmental factors that influence the incidence of this mycosis. However, unlike other endemic mycoses, there are no published reports of clusters of paracoccidioidomycosis. Methodology/Principal Findings: A retrospective cluster detection test was applied to verify if an excess of acute form (AF) paracoccidioidomycosis cases in time and/or space occurred in Botucatu, an endemic area in Sao Paulo State. The scan-test SaTScan v7.0.3 was set to find clusters for the maximum temporal period of 1 year. The temporal test indicated a significant cluster in 1985 (P<0.005). This cluster comprised 10 cases, although 2.19 were expected for this year in this area. Age and clinical presentation of these cases were typical of AF paracccidioidomycosis. The space-time test confirmed the temporal cluster in 1985 and showed the localities where the risk was higher in that year. The cluster suggests that some particularities took place in the antecedent years in those localities. Analysis of climate variables showed that soil water storage was atypically high in 1982/83 (similar to 2.11/2.5 SD above mean), and the absolute air humidity in 1984, the year preceding the cluster, was much higher than normal (similar to 1.6 SD above mean), conditions that may have favored, respectively, antecedent fungal growth in the soil and conidia liberation in 1984, the probable year of exposure. These climatic anomalies in this area was due to the 1982/83 El Nino event, the strongest in the last 50 years. Conclusions/Significance: We describe the first cluster of AF paracoccidioidomycosis, which was potentially linked to a climatic anomaly caused by the 1982/83 El Nino Southern Oscillation. This finding is important because it may help to clarify the conditions that favor Paracoccidioides brasiliensis survival and growth in the environment and that enhance human exposure, thus allowing the development of preventive measures.

Effect of the effluent released from the canine internal mammary artery after intraluminal and extraluminal perfusion of acetylcholine and adenosine diphosphate

Segments of the canine internal mammary artery (35 mm in length) were suspended in vitro in an organ chamber containing physiological salt solution (95% O(2)/5% CO(2), pH = 7.4, 37 degrees C). Segments were individually cannulated and perfused at 5 ml/minute using a roller pump. Vasorelaxant activity of the effluent from the perfused internal mammary arteries was bioassayed by measuring the decrease in tension induced by the effluent of the coronary artery endothelium-free ring which had been contracted with prostaglandin F(2 alpha) (2 x 10(-6) M). Intraluminal perfusion of adenosine diphosphate (10(-5) M) induced significant increase in relaxant activity in the effluent from the perfused blood vessel. However, when adenosine diphosphate (10(-5) M) was added extraluminally to the internal mammary artery, no change in relaxant activity in the effluent was noted. In contrast, acetylcholine produced significant increase in the relaxant activity on the effluent of the perfused internal mammary artery with both intraluminal and extraluminal perfusion. The intraluminal and extraluminal release of endothelium-derived relaxing factor (EDRF) by acetylcholine (10(-5) M) can be inhibited by site-specific administration of atropine (10(-5) M). These experiments indicate that certain agonists can induce the release of EDRF only by binding to intravascular receptors while other agonists can induce endothelium-dependent vasodilatation by acting on neural side receptors.

Vascular relaxation of canine visceral arteries after ischemia by means of supraceliac aortic cross-clamping followed by reperfusion

Background: The supraceliac aortic cross-clamping can be an option to save patients with hipovolemic shock due to abdominal trauma. However, this maneuver is associated with ischemia/reperfusion (I/R) injury strongly related to oxidative stress and reduction of nitric oxide bioavailability. Moreover, several studies demonstrated impairment in relaxation after I/R, but the time course of I/R necessary to induce vascular dysfunction is still controversial. We investigated whether 60 minutes of ischemia followed by 30 minutes of reperfusion do not change the relaxation of visceral arteries nor the plasma and renal levels of malondialdehyde (MDA) and nitrite plus nitrate (NOx). Methods: Male mongrel dogs (n = 27) were randomly allocated in one of the three groups: sham (no clamping, n = 9), ischemia (supraceliac aortic cross-clamping for 60 minutes, n = 9), and I/R (60 minutes of ischemia followed by reperfusion for 30 minutes, n = 9). Relaxation of visceral arteries (celiac trunk, renal and superior mesenteric arteries) was studied in organ chambers. MDA and NOx concentrations were determined using a commercially available kit and an ozone-based chemiluminescence assay, respectively. Results: Both acetylcholine and calcium ionophore caused relaxation in endothelium-intact rings and no statistical differences were observed among the three groups. Sodium nitroprusside promoted relaxation in endothelium-denuded rings, and there were no inter-group statistical differences. Both plasma and renal concentrations of MDA and NOx showed no significant difference among the groups. Conclusion: Supraceliac aortic cross-clamping for 60 minutes alone and followed by 30 minutes of reperfusion did not impair relaxation of canine visceral arteries nor evoke biochemical alterations in plasma or renal tissue.