Short-chain fatty acids and FFAR2 as suppressors of bone resorption.

Short-chain fatty acids (SCFAs) exert a variety of immune and metabolic functions by binding to G-protein-coupled receptors, mainly free fatty acid receptor 2 (FFAR2). However, the effects of SCFAs and FFARs on bone remodeling, especially in alveolar bone, have been less explored. In this study, we investigated the influence of the SCFA/FFAR2 axis on alveolar bone. Bone samples from wild-type (WT) and FFAR2-deficient mice (FFAR2-/-) were analyzed using micro-CT, histology and qPCR. WT and FFAR2-/- animals received a high-fiber diet (HFD) reported to increase circulating levels of SCFAs. Additionally, we analyzed the effects of SCFAs and a synthetic FFAR2 agonist, phenylacetamide-1 (CTMB), on bone cell differentiation. The participation of histone deacetylase inhibitors (iHDACs) in the effects of SCFAs was further assessed in vitro. CTMB treatment was also evaluated in vivo during orthodontic tooth movement (OTM). FFAR2-/- mice exhibited deterioration of maxillary bone parameters. Consistent with this, FFAR2-/- mice exhibited a significant increase of OTM and changes in bone cell numbers and in the expression of remodeling markers. The HFD partially reversed bone loss in the maxillae of FFAR2-/- mice. In WT mice, the HFD induced changes in the bone markers apparently favoring a bone formation scenario. In vitro, bone marrow cells from FFAR2-/- mice exhibited increased differentiation into osteoclasts, while no changes in osteoblasts were observed. In line with this, differentiation of osteoclasts was diminished by SCFAs and CTMB. Moreover, CTMB treatment significantly reduced OTM. Pretreatment of osteoclasts with iHDACs did not modify the effects of SCFAs on these cells. In conclusion, SCFAs function as regulators of bone resorption. The effects of SCFAs on osteoclasts are dependent on FFAR2 activation and are independent of the inhibition of HDACs. FFAR2 agonists may be useful to control bone osteolysis.

Unbalanced inflammatory reaction could increase tissue destruction and worsen skin infectious diseases - a comparative study of leishmaniasis and sporotrichosis.

The clinical presentations of skin diseases produced by different pathogens, as American tegumentary leishmaniasis (ATL) and sporotrichosis can be similar and possibly influenced by the skin immune system (SIS). The aim of the study was to understand the underlying mechanisms of skin inflammation produced by different pathogens. We used immunohistochemistry to analyze 96 patients: a- localized cutaneous leishmaniasis (LCL-ATL); b- sporotrichoid cutaneous leishmaniasis (SCL-ATL); c-lymphocutaneous (LC-SP); d- fixed (F-SP) sporotrichosis. LCL-ATL and SCL-ATL had a significantly higher percentage of CD8, FasL and NOS2 than sporotrichosis. In contrast, LC-SP had a substantially higher percentage of CD4, BCl2 and neutrophils than ATL lesions. These results indicated some differences in the profile of the in situ immune response suggesting that SIS is a complex, adaptable system capable of different responses to intracellular or extracellular pathogens. However, regardless of the etiological agents, the inflammatory reaction and clinical manifestations can be similar. SCL-ATL and LC-SP presented similarities in both clinical presentation and in situ inflammatory profile (CD3, CD22, neutrophils, macrophages). The clinical presentation of ATL and sporotrichosis could be explained by a combination of factors both of the host SIS and the etiological agent. The unbalanced host parasite relationship could result in atypical manifestations of skin disease.

Accuracy of quantitative polymerase chain reaction in samples of frozen and paraffin-embedded healthy skin for the diagnosis of canine visceral leishmaniasis / Acurácia da reação em cadeia da polimerase quantitativa em amostras de pele íntegra congelada e parafinada no diagnóstico da leishmaniose visceral canina

The purpose of the present work was to evaluate the accuracy of quantitative polymerase chain reaction (qPCR) performed on samples of fresh frozen tissue (FT) and formalin-fixed, paraffin-embedded (FFPE) healthy skin. This is a validation study conducted with samples from 46 dogs from an endemic area in Brazil. After sample collection, DNA extractions were conducted using commercial kits and qPCR was oriented to kinetoplast DNA (kDNA) targets of the Leishmania infantum species. The results obtained for the FFPE samples showed 63.6% sensitivity and 77.1% specificity, whereas those obtained for the FT samples showed 100% and 48.6%, respectively. Poor agreement was observed for the results of the qPCR technique with FT and FFPE samples. Our results suggest freezing as the most suitable conservation method for the formation of sample databases considering DNA recovery.(AU)

O objetivo deste trabalho foi avaliar a acurácia da PCR quantitativa (qPCR) realizada em amostras de pele íntegra congelada (FT) e parafinada (FFPE). Trata-se de um estudo de validação, com amostras provenientes de 46 cães de uma área endêmica no Brasil. Após as coletas de amostras, as extrações de DNA foram realizadas utilizando-se kits comerciais, e a qPCR foi orientada para alvos do kDNA da espécie Leishmania infantum. Os resultados obtidos para as amostras FFPE foram 63,6% de sensibilidade e 77,1% de especificidade; para as amostras FT, 100% e 48,6%, respectivamente. A concordância dos resultados da técnica de qPCR com amostras FT e FFPE foi pobre. Os resultados sugerem que o congelamento é o método mais adequado de conservação para banco de amostras para recuperação de DNA.(AU)

Avaliação da confiabilidade entre dois observadores em exames citopatológico e imunocitoquímico de aspirado de medula óssea no diagnóstico da leishmaniose visceral canina / Reliability evaluation between two evaluators in the cytopathologic and immunocytochemical exams from bone marrow aspirate in the canine visceral leishmaniasis diagnosis

Visceral leishmaniasis is a zoonosis in which the dog appears as the main source of infection in urban areas. Its diagnosis is complex and the cytopathological exam is a fast and cheap alternative to parasite direct visualization and its sensitivity can be increased by immunocytochemistry, though with a higher cost. The accuracy of such methods is dependent on the microscopist's experience and therefore, this study evaluated the reliability of such techniques between two observers, from bone marrow aspirates of 50 dogs from an endemic area for the disease. The parasitological culture in Novy-MacNeal-Nicolle medium was used as the reference standard. Among the main findings, the sensitivities obtained by observers I and II were respectively 62.5% and 37.5%, while specificities were 81.1% and 100%. On immunocytochemistry evaluation, the sensitivity was 0% for both evaluators and the specificity 97.3% and 100%. The agreement between evaluators was weak (κ = 0.167) for the cytopathological test and it could not be evaluated for immunocytochemistry, for which there was no detection by the evaluator II. The agreements among the diagnostic methods and the standard reference for the observer I were reasonable (κ = 0.364) for cytopathological examination and bad (κ = -0.041) for immunocytochemistry. For observer II, such agreement could be assessed only for the cytopathological test, being moderate (κ = 0.497). The results point to the possible expertise difference between evaluators, with the evaluator II demonstrating greater experience when interpreting the citopathological test. Although there was the expected sensitivity increase with immunocytochemistry, the technique used in this study was not effective for the diagnosis of infection, regardless of the evaluator.(AU)

Cell-block Immunohistochemistry of Bone Marrow Aspirates: a Novel Tool to Improve the Diagnosis of Leishmania Infection in Dogs.

Parasitological methods are the most specific procedures used for the diagnosis of Leishmania spp. infection, but their limited sensitivity poses a disadvantage and prompts the need for alternatives. The choice of site for sample collection influences diagnostic sensitivity. The combination of an accurate diagnostic method and a technique that allows large-scale field studies is highly desirable to enhance the investigation of Leishmania spp. infection in dogs, especially in endemic regions. The bone marrow is a good target for the detection of Leishmania spp. in dogs. In this context, bone marrow aspiration is rapid and less invasive compared with biopsy procedures, and also enables cell block processing, paraffin wax embedding and the sectioning of samples for further histological and immunohistochemical analyses. The aim of this study was to describe for the first time parasitological methods (immunohistochemistry [IHC] and histopathology) using the cell block technique with bone marrow aspirates for the diagnosis of Leishmania spp. infection in dogs. Bone marrow aspiration was performed in 45 dogs from an area endemic for visceral leishmaniosis for parasitological culture and the cell block technique (histopathology and IHC). Fourteen (31.1%) dogs tested positive for Leishmania spp. by IHC, six (13.3%) by parasitological culture and four (8.9%) by histopathology. Cell block IHC was a useful tool for the diagnosis of canine visceral leishmaniosis. Further studies should be conducted to validate this method for routine epidemiological screening.

Protective effects of the angiotensin type 1 receptor antagonist losartan in infection-induced and arthritis-associated alveolar bone loss.

BACKGROUND AND OBJECTIVE: The angiotensin type 1 (AT1) receptor has been implicated in the pathogenesis of inflammatory bone disorders. This study aimed to investigate the effect of an AT1 receptor antagonist in infection-induced and arthritis-associated alveolar bone loss in mice. MATERIAL AND METHODS: Mice were subjected to Aggregatibacter actinomycetemcomitans oral infection or antigen-induced arthritis and treated daily with 10 mg/kg of the prototype AT1 antagonist, losartan. Treatment was conducted for 30 d in the infectious condition and for 17 d and 11 d in the preventive or therapeutic regimens in the arthritic model, respectively. The mice were then killed, and the maxillae, serum and knee joints were collected for histomorphometric and immunoenzymatic assays. In vitro osteoclast assays were performed using RAW 264.7 cells stimulated with A. actinomycetemcomitans lipopolysacharide (LPS). RESULTS: Arthritis and A. actinomycetemcomitans infection triggered significant alveolar bone loss in mice and increased the levels of myeloperoxidase and of TRAP(+) osteoclasts in periodontal tissues. Losartan abolished such a phenotype, as well as the arthritis joint inflammation. Both arthritis and A. actinomycetemcomitans conditions were associated with the release of tumor necrosis factor alpha (TNF-α), interferon-gamma, interleukin-17 and chemokine (C-X-C motif) ligand 1 and an increased RANKL/osteoprotegerin ratio in periodontal tissues, but such expression decreased after losartan treatment, except for TNF-α. The therapeutic approach was as beneficial as the preventive one. In vitro, losartan prevented LPS-induced osteoclast differentiation and activity. CONCLUSION: The blockade of AT1 receptor exerts anti-inflammatory and anti-osteoclastic effects, thus protecting periodontal tissues in distinct pathophysiological conditions of alveolar bone loss.

The relevance of leukotrienes for bone resorption induced by mechanical loading.

5-Lipoxygenase (5-LO) metabolites are important pro-inflammatory lipid mediators. However, much still remains to be understood about the role of such mediators in bone remodeling. This study aimed to investigate the effect of 5-LO metabolites, LTB4 and CysLTs, in a model of mechanical loading-induced bone remodeling. Strain-induced tooth movement and consequently alveolar bone resorption/apposition was achieved by using a coil spring placed on molar and attached to incisors of C57BL6 (wild-type-WT), 5-LO deficient mice (5-LO(-/-)) and mice treated with 5-LO inhibitor (zileuton-ZN) or with antagonist of CysLTs receptor (montelukast-MT). The amount of bone resorption and the number of osteoclasts were determined morphometrically. The expression of inflammatory and bone remodeling markers in periodontium was analyzed by qPCR. Osteoclast differentiation and TNF-α production were evaluated in vitro using RAW 264.7 cells treated with LTB4 or LTD4. Bone resorption, TRAP(+) cells and expression of Tnfa, Il10 and Runx2 were significantly diminished in 5-LO(-/-), ZN- and MT-treated mice. The expression of Rank was also reduced in 5-LO(-/-) and MT-treated mice. Accordingly, LTB4 and LTD4 in association with RANKL promoted osteoclast differentiation and increased TNF-α release in vitro. These data demonstrate that the absence of 5-LO metabolites, LTB4 and CysLTs reduces osteoclast recruitment and differentiation, consequently diminishing bone resorption induced by mechanical loading. Thus, 5-LO might be a potential target for controlling bone resorption in physiological and pathological conditions.

Evaluation of 18S rDNA PCR assay using skin fragments as a diagnostic test for Trypanosoma caninum.

Trypanosoma caninum is a new species that has been recently identified in Brazil and infects domestic dogs. To date, no accurate diagnostic assays for this parasite have been established; thus, our aim was to evaluate more than one type of PCR for the diagnosis and molecular screening of T. caninum in 229 dogs living in Rio de Janeiro state. The tests were based on the amplification and sequencing of the 18S ribosomal DNA (rDNA) gene using healthy skin fragments. Additionally, PCR amplification of the kDNA minicircles region specific to the Leishmania genus was performed. The PCR results were compared with those of culture-based analysis performed with the same specimen. Using cultures, T. caninum and Leishmania chagasi were isolated from 11 and 12 dogs, respectively, whereas the 18S rDNA PCR assay detected parasitic infection in 35 dogs. Among these, 25 dogs showed an amplification pattern similar to T. caninum and 10 showed a pattern similar to L. chagasi; these results were confirmed by sequencing analysis. The kDNA PCR analysis showed that 14 dogs were positive for Leishmania infection. Of these, 2 dogs showed negative culture results and 12 were positive for L. chagasi, including 4 with negative 18S rDNA PCR results. Thus far, culture-based testing has been the only tool used successfully for T. caninum diagnosis. Our results demonstrate that 18S rDNA PCR-based test should be a useful diagnostic tool, particularly for distinguishing between T. caninum and L. chagasi infections in areas where these 2 parasites co-exist.

MyD88 is essential for alveolar bone loss induced by Aggregatibacter actinomycetemcomitans lipopolysaccharide in mice.

Aggregatibacter actinomycetemcomitans is a Gram-negative bacteria highly associated with localized aggressive periodontitis. The recognition of microbial factors, such as lipopolysaccharide from A. actinomycetemcomitans ((Aa)LPS), in the oral environment is made mainly by surface receptors known as Toll-like receptors (TLR). TLR4 is the major LPS receptor. This interaction leads to the production of inflammatory cytokines by myeloid differentiation primary-response protein 88 (MyD88) -dependent and -independent pathways, which may involve the adaptor Toll/interleukin-1 receptor-domain-containing adaptor inducing interferon-ß (TRIF). The aim of this study was to assess the involvement of MyD88 in alveolar bone loss induced by (Aa)LPS in mice. C57BL6/J wild-type (WT) mice, MyD88, TRIF or TRIF/MyD88 knockout mice received 10 injections of Aa LPS strain FDC Y4 (5 µg in 3 µl), in the palatal gingival tissue of the right first molar, every 48 h. Phosphate-buffered saline was injected in the opposite side and used as control. Animals were sacrificed 24 h after the 10th injection and the maxillae were removed for macroscopic and biochemical analyses. The injections of Aa LPS induced significant alveolar bone loss in WT mice. In the absence of MyD88 or TRIF/MyD88 no bone loss induced by (Aa)LPS was observed. In contrast, responses in TRIF(-/-) mice were similar to those in WT mice. Diminished bone loss in the absence of MyD88 was associated with fewer TRAP-positive cells and increased expression of osteoblast markers, RUNX2 and osteopontin. There was also reduced tumor necrosis factor-α production in MyD88(-/-) mice. There was less osteoclast differentiation of hematopoietic bone marrow cells from MyD88(-/-) mice after (Aa)LPS stimulation. Hence, the signaling through MyD88 is pivotal for (Aa)LPS-induced osteoclast formation and alveolar bone loss.

Evaluation of serological cross-reactivity between canine visceral leishmaniasis and natural infection by Trypanosoma caninum.

In order to evaluate if the presence of Trypanosoma caninum can lead to a confuse diagnosis of canine visceral leishmaniasis (CVL), we investigated the serological status of dogs infected by T. caninum and assessed the serological cross-reactivity with CVL. A set of 117 serum samples from dogs infected by T. caninum, Leishmania chagasi and not infected dogs (n=39 in each group) was tested using commercial kits--indirect immunofluorescence (IFI-LVC), ELISA (EIE-LVC) and immunochromatographic test (DPP)--and in house tests with T. caninum (IIF-Tc and ELISA-Tc) and L. chagasi antigens (IIF-Lc and ELISA-Lc). IIF-Tc and ELISA-Tc presented sensitivity of 64.1% and 94.9% and specificity of 23.1% and 35.9%, respectively. The sensitivity of the IFI-LVC, EIE-LVC and DPP tests was 100% and the specificity was 70.5%, 68% and 97.5% respectively. The concordance between the tests was considered as satisfactory. The specificities of IFI-LVC, EIE-LVC and DPP were higher when the group Tc was excluded, with significant values for IFI-LVC (χ2=4.36, P-value=0.036), thus suggesting that the infection by T. caninum can confuse the diagnosis of CVL.

Occurrence of Trypanosoma caninum in areas overlapping with leishmaniasis in Brazil: what is the real impact of canine leishmaniasis control?

Trypanosoma caninum is a parasite of the Trypanosoma genus recently described in the natural infection of dogs in the municipality of Rio de Janeiro, Brazil. Suspecting the existence of a natural cycle and the circulation of this new species, the objective of this study was the taxonomic identification of samples of Trypanosoma spp. isolated from dogs in different Brazilian regions. Parasites were solely obtained from skin fragments culture and characterized by nested-PCR targeting the partial sequence of 18S rRNA gene and PCR products were sequenced. Thirty-three samples, obtained in São Paulo, Minas Gerais, Goiás, Mato Grosso and Rio de Janeiro states were analyzed. PCR and sequencing showed that the isolates were genetically identical or closely similar and confirmed T. caninum identity. This report broadens the geographical distribution of T. caninum in Brazil and discusses the impact of the presence of this parasite in areas of canine leishmaniasis occurrence.

Low dose of propranolol down-modulates bone resorption by inhibiting inflammation and osteoclast differentiation.

BACKGROUND AND PURPOSE: Bones are widely innervated, suggesting an important role for the sympathetic regulation of bone metabolism, although there are controversial studies. We investigated the effects of propranolol in a model of experimental periodontal disease. EXPERIMENTAL APPROACH: Rats were assigned as follows: animals without ligature; ligated animals receiving vehicle and ligated animals receiving 0.1, 5 or 20 mg·kg(-1) propranolol. After 30 days, haemodynamic parameters were measured by cardiac catheterization. Gingival tissues were removed and assessed for IL-1ß, TNF-α and cross-linked carboxyterminal telopeptides of type I collagen (CTX) by elisa, or intercellular adhesion molecule 1 (ICAM-1), receptor activator of NF-κ B ligand (RANKL) and osteoprotegerin (OPG) by Western blot analysis. Sections from the mandibles were evaluated for bone resorption. Also, we analysed the ability of propranolol to inhibit osteoclastogenesis in vitro. RESULTS: Propranolol at 0.1 and 5 mg·kg(-1) reduced the bone resorption as well as ICAM-1 and RANKL expression. However, only 0.1 mg·kg(-1) reduced IL-1ß, TNF-α and CTX levels as well as increased the expression of OPG, but did not alter any of the haemodynamic parameters. Propranolol also suppressed in vitro osteoclast differentiation and resorptive activity by inhibiting the nuclear factor of activated T cells (NFATc)1 pathway and the expression of tartrate-resistant acid phosphatase (TRAP), cathepsin K and MMP-9. CONCLUSIONS AND IMPLICATIONS: Low doses of propranolol suppress bone resorption by inhibiting RANKL-mediated osteoclastogenesis as well as inflammatory markers without affecting haemodynamic parameters.

Laboratory tests performed on Leishmania seroreactive dogs euthanized by the leishmaniasis control program.

In 2008, in the west zone of Rio de Janeiro municipality-Brazil, the leishmaniasis control program identified 155 dogs with titers ≥ 40 by Indirect ImmunoFluorescence (IIF) on blood collected onto filter paper. The objective of this study was to describe the laboratory test findings performed in dogs euthanized by the leishmaniasis program control of Rio de Janeiro municipality. Dogs were examined, subjected to euthanasia and collection of clinical specimens. Parasite isolation was obtained in 29 animals: Leishmania chagasi was isolated in 14 dogs; Leishmania braziliensis was isolated in five dogs; Trypanosoma caninum was obtained in seven animals and one dog had mixed infection (L. braziliensis and L. chagasi). By Polymerase Chain Reaction, seventeen animals were positive in intact skin fragments. In the serological reassessment of serum samples, 28% and 22% were positive for IIF and enzyme immunoassay, respectively. Ninety-one (59%) dogs were negative for all tests performed in this study. The findings indicate that the visceral leishmaniasis control program needs to be adjusted in order to avoid non-infected dogs from being removed or permit that dogs infected with L. chagasi to remain undetected in endemic areas.

Isolation of Trypanosoma caninum in domestic dogs in Rio de Janeiro, Brazil.

SUMMARY: The domestic dog's involvement with different members of the Trypanosomatidae family has been the focus of several studies due to this animal's close proximity to man. Recently this animal has been infected by a new Trypanosoma species (T. caninum), described in Rio de Janeiro and 19 similar isolates were later obtained. The objective of this study was to identify these isolates. All samples were isolated from intact skin cultures and analysed morphologically, by biochemical isoenzyme electrophoresis assays and by several molecular PCR assays. Additionally, anti-Leishmania sp. antibodies were assessed using the indirect Immunofluorescence Antibody Test (IFAT) in all animals. The methodologies employed to identify the isolates, including partial nucleotide sequences of 18S rRNA gene, indicated patterns identical to T. caninum and patterns different from the other species, including T. cruzi and T. rangeli samples. A phylogenetic tree constructed with the partial 18S ribosomal sequence shows that T. caninum is clustered with T. pestanai. Ten (52.6%) animals presented anti-Leishmania sp. antibodies with titres varying from 1:40 to 1:320. Thus, the hypothesis that this protozoan has disseminated among the dogs in Rio de Janeiro must be considered. The importance of a correct diagnosis in those animals and the possible consequences in the areas where visceral leishmaniasis is found are discussed here.

Efficacy of an indirect immunofluorescence test in the diagnosis of canine leishmaniosis.

Of 146 dogs from a visceral leishmaniosis-endemic area that tested seronegative by indirect immunofluorescence (IIF) on blood samples collected on filter paper (IIFp), 51 (34.9%) and 10 (6.8%) tested positive by IIF on serum samples (IIFs) and enzyme immunoassay, respectively. Three samples (2.0%) tested positive by PCR. Leishmania chagasi was isolated from the skin of five (3.4%) dogs. Amastigote forms were identified in two of these five animals following histopathological and immunohistochemical examination. The findings highlight that detection methods such as IIFp can permit dogs infected with L. chagasi to remain undetected in endemic areas with attendant consequences for the epidemiology of infection both in the canine and human populations.

Parasitological diagnosis of canine visceral leishmaniasis: is intact skin a good target?

The objective of this study was to evaluate intact skin of seroreactive dogs as a possible target for the parasitological confirmation of canine visceral leishmaniasis (CVL). For this purpose, 394 dogs identified in serological surveys carried out in the metropolitan region of Belo Horizonte were studied. Blood was collected from all animals for serology and a tissue sample was obtained from two sites for parasitological diagnosis. Skin obtained from the ear and scapular region was simultaneously analyzed in 247 animals and lesion samples and ear skin were analyzed in 147 dogs. Leishmania parasites were isolated from 310 (78.7%) animals, and all isolates were identified as Leishmania chagasi. Simultaneous isolation from two sites was possible in 240 of the 310 animals, including ear and scapular skin in 151/247 (61.1%) and ear skin and skin lesions in 89/147 (60.5%). Ours results suggest that intact skin is one of the main target sites for the parasitological confirmation of CVL in seroreactive dogs.

Trypanosoma caninum n. sp. (Protozoa: Kinetoplastida) isolated from intact skin of a domestic dog ( Canis familiaris) captured in Rio de Janeiro, Brazil.

An unknown Trypanosoma species was isolated from an axenic culture of intact skin from a domestic dog captured in Rio de Janeiro, Brazil, which was co-infected with Leishmania (Viannia) braziliensis. Giemsa-stained smears of cultures grown in different media revealed the presence of epimastigotes, trypomastigotes, spheromastigotes, transitional stages, and dividing forms (epimastigotes or spheromastigotes). The highest frequency of trypomastigotes was observed in RPMI (15.2%) and DMEM (9.2%) media containing 5% FCS, with a mean length of these forms of 43.0 and 36.0 mum, respectively. Molecular analysis by sequential application of PCR assays indicated that this trypanosome differs from Trypanosoma cruzi and T. rangeli when specific primers were applied. On the other hand, a PCR strategy targeted to the D7 domain of 24salpha rDNA, using primers D75/D76, amplified products of about 250 bp in that isolate (stock A-27), different from the amplification products obtained with T. cruzi and T. rangeli. This organism differs from T. cruzi mainly by the size of its trypomastigote forms and kinetoplasts and the absence of infectivity for macrophages and triatomine bugs. It is also morphologically distinct from salivarian trypanosomes reported in Brazil. Isoenzyme analysis at 8 loci demonstrated a very peculiar banding pattern clearly distinct from those of T. rangeli and T. cruzi. We conclude that this isolate is a new Trypanosoma species. The name T. caninum is suggested.

Accuracy of an ELISA and indirect immunofluorescence for the laboratory diagnosis of American tegumentary leishmaniasis.

We compared the accuracy of ELISA and indirect immunofluorescence (IIF) using Leishmania braziliensis and L. major-like antigens and antigens from the Bio-Manguinhos kit for serological diagnosis of American tegumentary leishmaniasis (ATL). Cut-off values were defined by the area under the receiver-operating characteristic curve. For ELISA, statistical analyses revealed better accuracy [95.7% sensitivity, 100% specificity, 100% positive predictive value (PPV), 97.5% negative predictive value (NPV)] and reliability [intraclass correlation coefficient (ICC): 0.940] for L. braziliensis antigen compared with L. major-like antigen (78.7% sensitivity, 82.8% specificity, 73.3% PPV, 86.6% NPV, ICC: 0.833). ELISA optical density values obtained for both antigens were higher in mucosal forms of ATL. For IIF, sensitivity and specificity were 81.5 and 86.2%, respectively, for the L. braziliensis antigen, compared with 95.4 and 77.7% for the L. major-like antigen and 75.4 and 89.2% for the Bio-Manguinhos kit. No difference in the specificity of the IIF test was observed between antigens, whereas sensitivity differed between the L. braziliensis and L. major-like antigens and the Bio-Manguinhos kit. Parallel ELISA and IIF testing increased sensitivity, irrespective of the antigen employed, and serial testing increased overall specificity. These results support the recommendation that ELISA employing L. braziliensis antigen be used as a diagnostic tool for suspected cases of ATL in L. braziliensis-endemic areas.

Leishmania (Viannia) braziliensis genotypes identified in lesions of patients with atypical or typical manifestations of tegumentary leishmaniasis: evaluation by two molecular markers.

Analyses of MLEE, RAPD and LSSP-PCR were used to compare the panel of american tegumentary leishmaniasis (ATL) isolates obtained from lesions of patients with rare clinical manifestations of the disease and typical lesions. All of the 34 samples analyzed by MLEE demonstrated similar electromorphic profiles with Leishmania (Viannia) braziliensis reference strain. Through the RAPD analysis, nine genetic profiles (genotypes) were identified. LSSP-PCR corroborates the initial screening and phenetic analysis has grouped the isolates into two major clusters comprising the nine different genotypes. Prevalent genotype defined as LbmtDNAgen1 was detected in the largest number of isolates. There was no association between genotypes and clinical symptoms. However, two different genotypes could be identified in the initial (LbmtDNAGen9) and reactivated lesion (LbmtDNAGen3) of the same patient. Our results support the idea of a less pronounced genotypic diversity among L. (V.) braziliensis circulating in the State of Rio de Janeiro and demonstrate the useful application of these molecular markers in genetics variability studies.

First report of natural infection of a bush dog (Speothos venaticus) with Leishmania (Leishmania) chagasi in Brazil.

We report here the first known case of natural infection of a bush dog with Leishmania (Leishmania) chagasi in Brazil. The specimen was captured in the wild in the State of Mato Grosso and is currently being held in captivity at Fundação Jardim Zoológico, Rio de Janeiro, Brazil. The leishmaniasis was diagnosed by culture of promastigote forms in intact skin fragments and their characterization by isoenzyme electrophoresis. This report calls attention to the parasitological and etiological control of certain zoonoses, such as leishmaniasis, in wild animals kept in captivity, especially when animals are exchanged between zoos in Brazil.