Search for agglutinating antibodies to Leptospira and Leptospira and Leptonema in horses, Sao Paulo, Brazil

Soros de 922 equinos aparentemente sadios, mantidos na Fazenda do Instituto Butantan (Säo Roque, SP) para produçäo de soros hiperimunes, foram analisados quanto à presença de anticorpos para sorovares de Leptospira interrogans e para Leptonema illini, através da reaçäo de aglutinaçäo microscópica (MA). Entre os 807 (87,5 por cento) animais positivos, 659 (81,7 por cento) reagiram com mais de um sorovar, com títulos entre 1:100 e 1:6.400, havendo predomínio de títulos baixos (<= 1:400), 84 por cento dos soros positivos reagiram com representantes do sorogrupo Icterohaemorrhagiae e 79,2 por cento com Leptonema illini. Dos 23 sorovares utilizados, apenas o tarassovi näo reagiu

Leptospiral antigens in the liver of experimentally infected guinea pig and their relation to the morphogenesis of liver damage.

In order to investigate the morphogenes of experimental leptospirosis by morphologic and immunohistologic methods, 24 guinea-pigs were inoculated intraperitoneally with L. interrogans serogroup Icterohaemorrhagiae. They were divided in 6 groups, sacrificed from the 1st to the 6th day of infection. Semiquantitative analyses of histopathological liver lesions were performed in 1 micron sections of tissue embedded in glycol-methacrylate. The distribution of leptospiral antigen (L. Ag) and its glycolipoprotein (GLP) was demonstrated by peroxidase-antiperoxidase on paraffin embedded tissue. Significant lesions appeared at the 4th day of infection, progressing to a peak on the 6th day. Inflammation was associated with injury of the portal triad. Liver cells showed either swelling or acidophilic degeneration and necrosis, together with loss of cell cohesion, leading to disarray of liver cell plates. Mitochondria were found progressively enlarged and irregularly distributed. L. Ag expression was parallel to the morphological changes. Portal distribution was significant at the 4th day and on later stages centrilobular localization became predominant. Spiral forms suggestive of intact leptospires were initially found but, chiefly at the 6th day, L. Ag was seen in granules, probably resulting from phagocytosis. GLP staining was similar to granular L. Ag in morphology, and distribution. Cytokeratin condensation was seen in liver cells with acidophilic necrosis and was marked in areas of disorganization of cell plates. Our findings lead us to hypothesize a direct leptospiral cytotoxic effect on endothelial and on liver-cell membranes. At first, leptospires themselves would induce subcellular changes acting mainly on membrane permeability. Afterwards, their granular forms, including GLP, would act as adjuvant factors. These findings demonstrate that the disarray of liver cell plates at the late phase of the disease is genuine.

Detection of leptospiral antigen (L. interrogans serovar copenhageni serogroup Icterohaemorrhagiae) by immunoelectron microscopy in the liver and kidney of experimentally infected guinea-pigs.

Guinea-pigs were experimentally infected with L. interrogans serovar copenhageni serogroup Icterohaemorrhagiae and their liver and kidney were studied by immunoelectron microscopy using the post embedding indirect immunogold labelling technique. Primary antibody was a purified rabbit anti-serum produced against the same leptospiral strain used in the inoculum. Gold-labelled leptospiral antigen (LAg) was found close to cell membranes of hepatocytes, kidney tubular cells and endothelial cells of the interstitial capillaries of the kidney. Afterwards it was internalized by hepatic and tubular cells, and eventually found in lysosomes. Phagolysosomes of Kupffer cells were also found to contain remnants of degraded leptospires and gold-labelled LAg. Gold-labelled intact leptospires were detected at the enlarged intercellular spaces between hepatocytes at the areas of hepatic cell plate disarray, showing the potential for leptospiral migration during the septicaemic phase of the disease potentially contributing to the pathogenesis of the lesions. The affinity of leptospiral antigenic material for cell membranes suggests an initial interaction with cell surface proteins followed by its internalization and cell damage. The nature of antigenic material detected, however, remains undefined; it may be a toxin, an enzyme or any other factor/s involved in leptospiral virulence.

Antigens involved in the human antibody response to natural infections with Leptospira interrogans serovar copenhageni.

Serum samples from patients with leptospirosis were screened by the microscopic agglutination test (MAT), ELISA and by immunoblotting. The latter two tests were performed with L. interrogans serovar copenhageni isolated from human blood culture. Immunoblotting with patients' sera revealed antibodies recognizing several leptospiral components in the molecular weight range 14.5-105 kDa of both IgM and IgG response. All patients' serum samples presented IgM antibodies reacting with a diffuse band of mol. wt of 14.8-22 kDa proteinase-K resistant and most reacted with bands of 26.5-28.7, 38-39 and 43-43.5 kDa. The IgG response appeared to be at variance. Examination of sequential serum samples obtained over a 4-8-month period revealed little change in the profile of antigen recognized after the 40th day of infection. Sera from healthy individuals presented IgM antibodies reacting with several leptospiral antigens, but lacked response against those of diffuse band of 14.8-22 kDa.

[An evaluation of the ELISA-IgM test in the early diagnosis of human leptospirosis]. / Avaliação do teste ELISA-IgM no diagnóstico precoce da leptospirose humana.

Thirty-seven sera samples from patients with leptospirosis icterohaemorrhagic form were studied with a time interval of 2 to 12 days between the beginning of the symptoms and the collection blood samples. It was isolated leptospira of 5 patients' hemocultures (13.5%) and from 4 of these the etiological agent pertained to the serogroup Icterohaemorrhagiae serovar copenhageni. Thirty-five of them (94.6%), including the four patients whose the etiological agent was isolated, showed reactivity in the enzyme linked immunosorbent (ELISA) IgM test. By this way, it was demonstrated that this test is important for a rapid diagnosis of human leptospirosis, even in the beginning of the disease, when there is still leptospiraemic phase.

[The serovars of Leptospira interrogans isolated from cases of human leptospirosis in São Paulo, Brazil]. / Sorovares de Leptospira interrogans isolados de casos de leptospirose humana em São Paulo, Brasil.

Eighteen strains of L. interrogans isolated from human cases were serotyped by the agglutinin-absorption test at Instituto Adolfo Lutz in São Paulo, Brazil. Fourteen were identified as serovar copenhageni (icterohaemorrhagiae serogroup), 2 as canicola (canicola serogroup), 1 as castellonis (Ballum serogroup) and 1 as pomona serogroup (serovar not yet defined). The frequency of serovar copenhageni in 100% of the isolates in icterohaemorrhagiae serogroup is emphasized and more studies to verify the real serovars prevalence as subsidy to the epidemiology of this infection are suggested by the authors.

[Evaluation of counterimmunoelectrophoresis with antigens of icterohaemorrhagiae and patoc serovars in the serodiagnosis of human leptospirosis]. / Avaliação da contraimunoeletroforese com antígenos dos sorovars icterohaemorrhagiae E patoc no diagnóstico sorológico da leptospirose humana.

Counterimmunoelectrophoresis (CIE) was applied on paired sera from 135 patients with leptospirosis and on 69 sera from a control group. The sera from patients were subdivided in 4 groups according to the results obtained by the Microscopic Agglutination Test (MAT). The first samples sera from 58 patients were non reagent by MAT. Six monthly samples of sera were taken from 7 patients to follow-up and to determine the level of agglutinin and precipitin antibodies present using MAT and CIE. Serovars icterohaemorrhagiae and patoc were used as antigens. Three types of antigens were compared, 1) Triton-X-100 extracted; 2) heat extracted and 3) a pool of them. The CIE using icterohaemorrhagiae derived antigens types agreed with MAT in 92.64, 92.64 and 94.11% of the leptospirosis sera. The patoc antigens types reacted with the control group in 7.24, 86.95 and 84.05% of the samples, and consequently were eliminated from the present study. The icterohaemorrhagiae CIE reaction become positive earlier than MAT negative sera, and reverted to negative earlier in the follow-up samples from the patients. The CIE was sensitive and specific, gave rapid results and was easy to perform.

[Outbreak of human leptospirosis by recreational activity in the municipality of São José dos Campos, São Paulo. Seroepidemiological study]. / Surto de leptospirose humana por atividade recreacional no município de São José dos Campos, São Paulo. Estudo soroepidemiológico.

An outbreak of human leptospirosis due to recreational activities occurred at São José dos Campos, São Paulo, Brazil in November 1987. It involved a group of persons who had participated in a gathering in a suburb club which had a swimming pool fed with natural water. Epidemiological investigation was carried out and laboratory tests from the patients were done. It was observed that a high prevalence of the pomona serotype (91%) was found in the serological analyses, while the presence of the agent of the infection could not be found in the water club swimming pool.

Imunodiagnostico da leptospirose humana atraves do teste ELISA-IgM, empregando-se diferentes preparacoes antigenicas a partir de sorotipos prevalentes de Leptospira interrogans / Immunodiagnostic of human leptospirosis by ELISA-IgM, employing different antigenic preparations as from prevalent serovars of Leptospira interrogans

Realizou-se estudo comparativo de diferentes sorotipos de Leptospira interrogans utilizados na preparacao de antigenos empregados no teste ELISA, para a deteccao de anticorpos da classe IgM, em amostras de soro na fase precoce e tardia da leptospirose humana. Foram utilizados dez sorotipos, escolhidos entre os que apresentaram maior reatividade na soroaglutinacao microscopica (SAM), na cidade de Sao Paulo. Os cinco sorotipos que apresentaram melhores resultados individualmente no teste ELISA-IgM (canicola, hebdomadis, icterohaemorrhagiae, cynopteri e brasiliensis), foram tambem estudados em mistura antigenica. Os antigenos nao tratados apresentaram maior reatividade do que os antigenos tratados com Triton X-100 (4 por cento) a temperatura de 50§C, durante 4 horas. O teste ELISA-IgM empregando os sorotipos nao tratados, isoladamente, e em mistura antigenica, mostrou-se altamente sensivel, podendo ser empregado como teste de triagem para o diagnostico precoce da leptospirose humana. Outra aplicacao do teste e permitir a deteccao do inicio de situacoes epidemicas ou de surtos, possibilitando acionar medidas de vigilancia epidemiologica

[Immunodiagnosis of human leptospirosis by ELISA-IgM, employing different antigenic preparations from prevalent serovars of Leptospira interrogans]. / Imunodiagnóstico da leptospirose humana através do teste ELISA-IgM, empregando-se diferentes preparações antigênicas a partir de sorotipos prevalentes de Leptospira interrogans.

A comparative study among different serovars of Leptospira interrogans was performed in order to prepare antigens to detect IgM antibodies by ELISA in early and late phase of human leptospirosis. Ten serovars were chosen among the most prevalent detected by microscopic seroagglutination (SAM) in São Paulo city. Using ELISA-IgM five of them showed better results (canicola, hebdomadis, icterohaemorrhagiae, cynopteri and brasiliensis). These ones were also studied in a pool. The non-treated antigens showed higher reactivity than the Triton X-100 (4%/50 degrees C/4h). ELISA-IgM using individually or pool of non-treated antigens proved to be reliable with high sensitivity and should be used for an earlier diagnosis of leptospirosis, as a trial test. Faster diagnostic elucidation can be useful to detect epidemic situations, so, allowing epidemiological surveillance interventions.

[High resolution light microscopy: adaptation of the method and its use in the study of experimental leptospirosis in guinea pigs]. / Microscopia óptica de alta resolução: adaptação do método e sua aplicação ao estudo da leptospirose experimental em cobaias.

Morphological lesions in parenchimal and mesenchimal structures of liver and kidney were studied in guinea-pigs experimentally infected with Leptospira interrogans serogroup icterohaemorrhagiae in comparison with a group of non-infected guinea-pigs. All specimens were submitted to conventional light microscopy as well as to high resolution light microscopy, in one micrometer sections of tissue embedded in glycolmethacrylate. High resolution light microscopy, applied for the first time in leptospirosis, was proved very useful, since it enabled us to visualize cellular structures in the same slide used for panoramic view. Cell cohesion, brush borders, pynocytotic vesicles and organellae distributions were parameters especially suitable for analysis at this low-cost, highly precise procedure in microscopy.

Glicolipoproteína de Leptospira interrogans sorogrupo icterohaemorrhagiae: distribuiçäo em fígado e rim de cobaias experimentalmente infectadas / Glycoproteins from Leptospira interrogans serogroup icterohaemorrhagiae: distribution in the liver and kidney of experimentally infected guinea pigs

Acredtita-se que as lesöes teciduais na leptospirose possam decorrer da açäo direta das leptospiras, de toxinas sintetizadas ou liberadas durante sua lise. O presente estudo visou a extraçäo química da glicolipoproteína (GLP) da aleptospira, a produçäo de anti-soro anti-GLP e a avaliaçäo de sua distribuiçäo em cortes de fígado e rim de cobaias inoculadas e sacrificadas em estudo sequencial diário até o 6§ dia de infecçäo, correspondente ao pico da doença. Procurou-se também correlacionar a expressäo tecidual da GLP com o grau de lesöes locais, em busca de novos subsídios para a compreensäo da patogenia da leptospiros. A GLP foi detectada em fígado e rim de 2 dentre 6 cobaias no 5§ dia e em todas as 6 no 6§ dia de infecçäo, sob a forma de grânulos no citoplasma de macrófagos, livres no interstício ou acolados à membrana de células endoteliais e parenquimatosas, especialmente nas regiöes mais lesadas. A cronologia do aparecimento da GLP e sua distribuiçäo sugerem tratar-se de produto de lise de leptospiras fagocitadas por macrófagos e que esta substância, conquanto näo comprovada como iniciadora das lesöes, asocia-se a seu agravamento nas etapas mais avançadas da leptospirose

[Glycoproteins from Leptospira interrogans serogroup icterohaemorrhagiae: distribution in the liver and kidney of experimentally infected guinea pigs]. / Glicolipoproteína de Leptospira interrogans sorogrupo icterohaemorrhagiae: distribuição em fígado e rim de cobaias experimentalmente infectadas.

Tissue damage in leptospirosis has been ascribed to direct effect of the microorganisms and/or their virulence, including products synthetized by leptospires or released during their lysis. This study aimed at chemical extraction of the glycolipoprotein (GLP) from virulent leptospires, production of a rabbit anti-GLP and analysis of its distribution in liver and kidney of inoculated guinea-pigs, sacrificed sequentially from the 1st to 6th day of infection, covering the whole, spectrum of acute leptospirosis. The comparison of GLP expression to local injuries aimed at new pathogenetic data. GLP was detected in liver and kidney in 2 out of 6 guinea-pigs on the 5th day and in all 6 animals on the 6th day of infection. Granular forms were seen in the cytoplasm of macrophages, free in interstitium or adhered to endothelial and parenchymal cell membranes, especially in the most damaged sites. These findings lead us to the hypothesis of GLP as a toxic factor resulting from leptospiral lysis by macrophages. Although it was not proved as a promoter of initial lesions, it seems to be related to the enhancement of tissue damage late in the course of the disease.