Improved Canine and Human Visceral Leishmaniasis Immunodiagnosis Using Combinations of Synthetic Peptides in Enzyme-Linked Immunosorbent Assay

Visceral leishmaniasis is endemic in many areas of tropical and subtropical America where it constitutes a significant public health problem. It is usually diagnosed by enzyme-linked immunosorbent assays (ELISA) using crude Leishmania antigens, but a variety of other immunological methods may also be applied. Although these approaches are useful, historically their sensitivity and specificity have often been compromised by the use of complex mixtures of antigens. In this context, the use of combinations of purified, well-characterized antigens appears preferable and may yield better results. In the present study, combinations of peptides derived from the previously described Leishmania diagnostic antigens A2, NH, LACK and K39 were used in ELISA against sera from 106 dogs and 44 human patients. Improved sensitivities and specificities, close to 100%, for both sera of patients and dogs was observed for ELISA using some combinations of the peptides, including the detection of VL in dogs with low anti-Leishmania antibody titers and asymptomatic infection. So, the use of combinations of B cell predicted synthetic peptides derived from antigens A2, NH, LACK and K39 may provide an alternative for improved sensitivities and specificities for immunodiagnostic assays of VL

Histopathology and immunocytochemical study of type 3 and type 4 complement receptors in the liver and spleen of dogs naturally and experimentally infected with Leishmania (Leishmania) chagasi

Os objetivos deste trabalho visaram uma análise comparativa das alterações histopatológicas e da expressão de CR3 e CR4 no fígado e baço de cães natural e experimentalmente infectados com L. chagasi. As lesões histopatológicas fundamentais observadas principalmente nos cães naturalmente infectados foram: os granulomas epitelióides hepáticos, a hiperplasia e a hipertrofia das células de Kupffer, dos folículos de Malpighi e das células mononucleadas da polpa vermelha do baço. Os cortes de fígado e baço corados pela técnica de imunocitoquímica mostraram a presença dos antígenos CD11b,c\CD18 nos animais controles e infectados, sem diferenças qualitativas e quantitativas no fígado. Entretanto, no baço dos cães natural e experimentalmente infectados a expressão de CD 18 (subunidade beta2 da molécula comum aos leucócitos) foi sempre aumentada. Em leishmaniose a resposta imune celular é considerada a mais importante na resolução da doença. A expressão de CD11 b,c\CD 18 pelos macrófagos deve representar um papel central na resposta celular. Estes resultados indicam que tanto na leishmaniose visceral canina experimental ou natural existe uma diferença na ativação de CD 18, o qual deve exercer uma importante função na resposta imunológica na infecção por Leishmania chagasi.The objective of this study was to compare the histopathological changes and expression of CR3 and CR4 in the liver and spleen of dogs naturally and experimentally infected with L. chagasi. The basic histopathological lesions observed mainly in naturally infected dogs were: epithelioid hepatic granulomas, hyperplasia and hypertrophy of Kupffer cells, Malpigui follicles and mononucleated cells of the red pulp of the spleen. Sections from the liver and spleen by immunocytochemistry technique showed the presence of CD11b,c\CD 18 antigens in the control and infected animals and no qualitative or quantitative differences in the liver. Nevertheless, CD18 was always increased in the spleen of naturally and experimentally infected dogs. These results indicate that there is a difference in the activaton of CD 18 in both experimental and natural cases of canine visceral leishmaniasis that should play an important role in the immunological response to Leishmania chagasi infection

Vibrational spectroscopic characterization of the phosphate mineral althausite Mg2(PO4)(OH,F,O) - Implications for the molecular structure

Natural single-crystal specimens of althausite from Brazil, with general formula Mg2(PO4)(OH,F,O) were investigated by Raman and infrared spectroscopy. The mineral occurs as a secondary product in granitic pegmatites. The Raman spectrum of althausite is characterized by bands at 1020, 1033 and 1044 cm-1, assigned to ν1 symmetric stretching modes of the HOPO33- and PO43- units. Raman bands at around 1067, 1083 and 1138 cm-1 are attributed to both the HOP and PO antisymmetric stretching vibrations. The set of Raman bands observed at 575, 589 and 606 cm-1 are assigned to the ν4 out of plane bending modes of the PO4 and H2PO4 units. Raman bands at 439, 461, 475 and 503 cm-1 are attributed to the ν2 PO4 and H2PO4 bending modes. Strong Raman bands observed at 312, 346 cm-1 with shoulder bands at 361, 381 and 398 cm-1 are assigned to MgO stretching vibrations. No bands which are attributable to water were found. Vibrational spectroscopy enables aspects of the molecular structure of althausite to be assessed

Reactivity of sera from infected dogs displaying different antibody titers with combined peptides.

<p>Detection of anti-leishmania total IgG antibodies with synthetic peptides by ELISA assay using canine samples classified according to IFAT reactivity as low (<1∶320 dilutions) and intermediate (>1∶320 <1∶640) titers and 250 nanograms/per well from each peptide. Peptides were combined in pairs in the same reaction as following: peptides 13 and 47 (panel <b>A</b>), 13 and 19 (panel <b>B</b>), 18 and 19 (panel <b>C</b>), 47 and 17 (panel <b>D</b>) and 47 and 18 (panel <b>E</b>).</p

Detection of IgG-antibodies in sera of human patients with visceral leishmaniasis with combined peptides.

<p>Detection of anti-leishmania total IgG antibodies with synthetic peptides by ELISA assay using sera from patients with active visceral leishmaniasis (<i>n</i> = 28) and healthy individuals with previous negative results in ELISA to <i>Leishmania</i> (<i>n</i> = 16) and 2 µg/per well from each peptide. Peptides were combined in pairs in the same reaction as following: peptides 13 and 19 (panel <b>A</b>), 18 and 19 (panel <b>B</b>), 47 and 13 (panel <b>C</b>), 47 and 17 (panel <b>D</b>) and 47 and 19 (panel <b>E</b>).</p

Detection of IgG-antibodies in sera of human patients with visceral leishmaniasis with individual peptides.

<p>Detection of anti-leishmania total IgG antibodies with synthetic peptides by ELISA assay using sera from patients with active visceral leishmaniasis (<i>n</i> = 28) and healthy individuals with previous negative results in ELISA to <i>Leishmania</i> (<i>n</i> = 16) and 4 µg/per well peptides 13 (panel <b>A</b>), 17 (panel <b>B</b>), 18 (panel <b>C</b>), 19 (panel <b>D</b>) and 47 (panel <b>E</b>).</p

Reactivity of sera from infected dogs displaying different IFAT antibody titers with peptides.

<p>Detection of anti-leishmanial total IgG antibodies with synthetic peptides by ELISA assay using canine samples classified according to IFAT reactivity as low (<1∶320 dilutions), intermediate (>1∶320 <1∶640) and high (>1∶640) titers and 500 nanograms/per well of peptides 13 (panel <b>A</b>), 17 (panel <b>B</b>), 18 (panel <b>C</b>), 19 (panel <b>D</b>) and 47 (panel <b>E</b>).</p

ELISA with individual synthetic peptides for detection of IgG-antibodies in dogs with <i>L. infantum</i> infection.

<p>Detection of anti-leishmanial total IgG antibodies with synthetic peptides by ELISA assay using sera from asymptomatic (<i>n</i> = 13) and symptomatic (<i>n</i> = 17) dogs and control group (<i>n</i> = 14). It was used 500 nanograms/well of peptides 13 (panel <b>A</b>), 17 (panel <b>B</b>), 18 (panel <b>C</b>), 19 (panel <b>D</b>) and 47 (panel <b>E</b>). The sensitivity and specificity of asymptomatic and symptomatic dogs ranged between 100 and 90%, respectively, for both.</p

Performance of ELISA employing synthetic peptides and canine sera classified according to IFAT reactivity.

<p>Se: sensitivity, Sp: specificity.</p>*<p>canine sera samples were classified according to IFAT reactivity as low (<1∶320 dilutions) and intermediate (>1∶320 <1∶640) titers and high (>1∶640) antibody titers.</p

Performance of ELISA employing synthetic peptides and sera of patients with visceral leishmaniasis.

<p>Se: sensitivity, Sp: specificity.</p