Einleitung: Die Leptospirose ist eine der weltweit bedeutendsten Zoonosen. Durch den häufig asymptomatischen oder unspezifischen Krankheitsverlauf, wird von einer hohen Dunkelziffer ausgegangen. Menschen können sich über direkten Tierkontakt oder indirekten Kontakt mit dem Urin infizierter Tiere, zum Beispiel über kontaminierte Gewässer oder Erde, anstecken. Die Infektion erfolgt dabei über den Eintritt des Erregers in Schleimhäute oder Hautwunden. Ziel der Untersuchungen: Obwohl die Überlebenszeit von Leptospiren in der Umwelt einen entscheidenden Einfluss auf das Infektionsrisiko des Menschen hat, wurden bisher nur sehr wenige Untersuchungen dazu durchgeführt. Diese konzentrierten sich dabei vor allem auf das Überleben der Erreger in Erde oder Wasser. Daher war es Ziel dieser Arbeit die Umweltstabilität eines häufig in Deutschland gefundenen Leptospirenserovars unter verschiedenen Umweltbedingungen zu untersuchen. Zusätzlich zu diesen Untersuchungen war es Ziel dieser Arbeit, durch die Veröffentlichung eines Übersichtsartikels über die Leptospirose Ärzte in Deutschland für diese häufig unerkannt bleibende Erkrankung zu sensibilisieren. Material und Methoden: Ein jahrelang an Kulturmedium adaptierter Labor- und ein erst vor 3 Jahren isolierter Feldstamm von Leptospira kirschneri Serovar Grippotyphosa wurden auf ihr Überleben in der Umwelt untersucht. Es wurde ihre Widerstandsfähigkeit gegenüber verschiedenen Umwelteinflüssen, wie z.B. Tierurin als umgebendes Medium bei verschiedenen Temperaturen oder auch der Einfluss einer Trocknung, untersucht. Nachdem die Leptospiren den Umwelteinflüssen für unterschiedliche Zeitspannen ausgesetzt waren, wurde versucht sie in EMJH-Medium wieder zu kultivieren. Während einer Inkubationszeit von mindestens 28 Tagen bei 29 °C wurden diese Kulturen wöchentlich unter dem Dunkelfeldmikroskop auf das Vorhandensein motiler Leptospiren untersucht. Zusätzlich wurde durch Kultivierungsversuche in EMJH-Medium das Überleben der Leptospiren in einem Wasserstrom mit einer definierten Fließgeschwindigkeit und ihre Verbreitung in diesem Strom mittels real-time PCR untersucht. Alle Versuche wurden im Dreifachansatz durchgeführt. Statistische Untersuchungen wurden mittels eines zweiseitigen Mann-Whitney-U Tests (Fehler 1. Art α = 0,05) durchgeführt. Ergebnisse: Die beiden untersuchten Stämme von L. grippotyphosa überlebten nicht in unverdünntem Tierurin. In verdünntem Tierurin überlebten die Stämme, je nach Temperatur und Verdünnungsmedium, zwischen 1 - 72 Stunden (Laborstamm) und 4 - 24 Stunden (Feldstamm). Beide Stämme überlebten signifikant länger bei 15 °C als bei 37 °C (p < 0,001, bzw. p = 0,041). Der Laborstamm überlebte signifikant länger in verdünntem Rindereurin (max. 72 h bei 15 °C) als in verdünntem Hundeurin (max. 4 h; p = 0,027). Im Gegensatz dazu, überlebte der Feldstamm signifikant länger in Hundeurin (max. 24 h bei 15 °C) als in Rinderurin (max. 4 h; p = 0,028). Das vollständige Trocknen auf einer festen Oberfläche war bei Temperaturen zwischen 15 °C und 37 °C für beide Stämme letal. Jedoch war, unabhängig von der untersuchten Temperatur, eine halbe Stunde vor der vollständigen Trocknung eine Kultivierung der Leptospira spp. noch möglich. In einem Wasserstrom konnten sich die Leptospiren aufgrund ihrer Eigenbewegung schneller und langsamer als die Durchschnittsgeschwindigkeit (0,01 m / s) des Wassers bewegen, überlebten jedoch die mechanischen Schäden während des Schlauchdurchflusses nicht. Schlussfolgerungen: Das Überleben von Leptospira spp. ist offensichtlich von vielen Faktoren abhängig. Eine schnelle Verdünnung nach der Ausscheidung mit dem Urin scheint dabei essentiell zu sein. Niedrigere Temperaturen sowie eine feuchte Umgebung verbessern ihre Widerstandsfähigkeit gegen schädliche Einflüsse, während Trockenheit oder mechanische Schädigung ihr Überleben nicht ermöglichen. Wegen der großen Bedeutung der leptospiralen Überlebenszeit in der Umwelt für das Infektionsrisiko von Mensch und Tier sind weitere Untersuchungen auf diesem Forschungsgebiet in der Zukunft nötig.:1. Einleitung 2. Literaturübersicht 2.1 Die Geschichte der Leptospirose 2.2 Morphologie und Übertragungswege der Erreger 2.3 Taxonomie 2.4 Haupt- und Nebenwirte 2.5 Die Erkrankung beim Menschen 2.5.1 Vorkommen 2.5.2 Klinik und Therapie 2.6 Die Erkrankung beim Tier 2.6.1 Bei Nutztieren 2.6.2 Bei Haustieren 2.6.3 Bei Wildtieren 2.7 Diagnostik 2.8 Die Umweltstabilität der Erreger 2.8.1 In Erde 2.8.2 In Wasser 2.8.3 In Urin 3. Publikationen 3.1 Publikation Nr. 1 3.2 Publikation Nr. 2 4. Diskussion und Schlussfolgerung 5. Zusammenfassung 6. Summary 7. Literaturverzeichnis 8. Danksagung / Introduction: Leptospirosis is one of the most important zoonosis worldwide. Due to the often asymptomatic or non-specific course of the disease, a high number of unreported cases is assumed. Humans can get infected through direct contact with animals or indirect contact with the urine of infected animals, for example through contaminated water or soil. The infection occurs via entry of the pathogen through mucous membranes or skin wounds. Aims of the study: Although the survival time of Leptospira spp. in the environment has a crucial influence on the human infection risk, very few studies have been carried out so far. These studies focused primarily on the survival of the pathogens in soil or water. It was therefore the aim of this study to investigate the environmental stability of a leptospiral serovar that is frequently found in Germany under different environmental conditions. In addition to these investigations, the aim of this work was to raise the awareness of physicians in Germany for this often undetected and neglected disease by publishing a review article on leptospirosis. Material and Methods: The survival in the environment of both a laboratory strain, which has been adapted to culture medium for years and a field strain, which was isolated only 3 years ago, of Leptospira kirschneri Serovar Grippotyphosa was studied. Their resistance to various environmental influences, such as animal urine as surrounding medium at different temperatures or the influence of drying, were examined. After the leptospires were exposed to these influences for various time periods, an attempt was made to cultivate them in EMJH medium. During an incubation period of at least 28 days at 29 °C, the cultures were examined weekly for the presence of motile leptospires under the darkfield microscope. In addition, the survival of the leptospires in a water stream with a defined velocity and their distribution in this stream were examined by real-time PCR and cultivation experiments in EMJH medium. All experiments were carried out in triplicate. The statistical analysis was done using a two-tailed Mann-Whitney U test (type-1-error α = 0.05). Results: Both examined strains of L. grippotyphosa did not survive in undiluted animal urine. In diluted animal urine, the strains survived between 1-72 hours (laboratory strain) and 4-24 hours (field strain), depending on the temperature and dilution medium. Both strains survived significantly longer at 15 °C than at 37 °C (p < 0.001 or p = 0.041). The laboratory strain survived significantly longer in diluted cattle urine (max. 72 h at 15 °C) than in diluted dog urine (max. 4 h) (p = 0.027) while the field strain survived significantly longer in dog urine (max. 24 h at 15 °C) than in cattle urine (max. 4 h) (p = 0.028). Complete drying on a solid surface at temperatures between 15 °C and 37 °C was lethal for both strains. However, regardless of the temperature examined, Leptospira spp. were still cultivatable half an hour before the time point of complete drying. In a water stream, leptospires were able to move faster or slower than the average velocity of the water (0.01 m / s) due to their intrinsic mobility but were not able to survive the mechanical damage caused by running water in the hose system. Conclusions: Overall, it can be concluded that the survival of Leptospira spp. depends on many factors. Rapid dilution after urine excretion appears to be essential. Lower temperatures and a humid environment improve their survival time, while drought or mechanical damage is lethal to them. Because of the great importance of leptospiral survival in the environment for the infection risk of humans and animals, further studies in this research area will be necessary in the future.:1. Einleitung 2. Literaturübersicht 2.1 Die Geschichte der Leptospirose 2.2 Morphologie und Übertragungswege der Erreger 2.3 Taxonomie 2.4 Haupt- und Nebenwirte 2.5 Die Erkrankung beim Menschen 2.5.1 Vorkommen 2.5.2 Klinik und Therapie 2.6 Die Erkrankung beim Tier 2.6.1 Bei Nutztieren 2.6.2 Bei Haustieren 2.6.3 Bei Wildtieren 2.7 Diagnostik 2.8 Die Umweltstabilität der Erreger 2.8.1 In Erde 2.8.2 In Wasser 2.8.3 In Urin 3. Publikationen 3.1 Publikation Nr. 1 3.2 Publikation Nr. 2 4. Diskussion und Schlussfolgerung 5. Zusammenfassung 6. Summary 7. Literaturverzeichnis 8. Danksagung

Orientador: Luis Antonio Mathias / Banca: José Jurandir Fagliari / Banca: Fernanda Senter Magajevski / Resumo: Foram investigados a prevalência e os fatores de risco da leptospirose bovina no Estado do Maranhão. O Estado foi dividido em quatro circuitos amostrais com base em parâmetros de produção distintos que variam conforme os diferentes sistemas de produção, as práticas de manejo, a finalidade de exploração, o tamanho médio dos rebanhos e os sistemas de comercialização. Objetivou-se estudar as características epidemiológicas da leptospirose bovina no Estado do Maranhão, de modo a determinar a prevalência em bovinos e em rebanhos, detectar as sorovariedades de Leptospira spp. presentes, identificar os fatores de risco eventualmente associados à leptospirose em bovinos e diferenciar os circuitos pecuários entre si no que se refere à prevalência de leptospirose. A pesquisa foi realizada em 136 propriedades rurais pertencentes ao circuito I, no qual 841 fêmeas bovinas com idade igual ou maior que 24 meses foram analisadas; 238 do circuito II, com 2.582 fêmeas analisadas; 122 do circuito III, com 869 fêmeas analisadas; e 77 do circuito IV, com 540 fêmeas analisadas; no total, 573 propriedades e 4.832 fêmeas foram estudadas. A presença de anticorpos contra Leptospira spp. foi verificada pela técnica de soroaglutinação microscópica (SAM). Das 4.832 fêmeas bovinas analisadas, 1.904 (35,94%; IC 95% = 33,01% - 38,98%) foram reagentes. Das 573 propriedades analisadas, 380 (64,81%; IC 95% = 61,10% - 68,35%) foram consideradas positivas. As sorovariedades Hardjo e Wolffi foram as mais frequentes em todo o Estado. O circuito III foi o que apresentou menor prevalência de leptospirose em todas as comparações. As variáveis identificadas como fatores de risco de leptospirose foram: presença de equinos (p = 0,000), presença de capivaras (p = 0,034) e rebanhos bovinos com 32 ou mais fêmeas adultas (p = 0,002) / Abstract: Prevalence and risk factors of bovine leptospirosis in the state of Maranhão were investigated. Based on production parameters that vary across different production systems, management practices, the purpose of exploitation, the average size of herds and market systems, the state was divided in four sampling circuits. The study aimed to investigate the epidemiological features of bovine leptospirosis in the state of Maranhão, in order to determine the prevalence of the infection in cattle and herds, to determine the occurrence of serovars of Leptospira spp., to identify risk factors associated with leptospirosis in cattle and to differentiate the livestock circuits itself regarding the prevalence of leptospirosis. The survey was conducted in 136 herds in the circuit I, in which 841 ≥ 24 months old females were analyzed; 238 in the circuit II and 2,582 females were analyzed; 122 in the circuit III and 869 females were analyzed; 77 in the circuit IV and 540 females were analyzed; a total of 573 herds and 4,832 females were analyzed. The presence of antibodies against Leptospira spp. was verified by microscopic agglutination test (MAT). Of the 4,832 cows examined, 1,904 (35.94%, CI 95% = 33.01% - 38.98%) were positive. Of the 573 herds, 380 (64.81%, CI 95% = 61.10% - 68.35%) were positive. Serovars Hardjo and Wolffi were the most frequent in the state. The circuit III showed the lowest prevalence of leptospirosis in all comparisons. The variables presence of horses (p = 0.000), presence of capybaras (p = 0.034) and herds with up to 32 adult females (p = 0.002) were identified as risk factors for leptospirosis / Mestre

A leptospirose é uma zoonose de ampla distribuição mundial, causada pela infecção por sorovares patogênicos do gênero Leptospira. Assim como outras espécies de animais acometidos, os cães que sobreviverem à fase aguda da doença podem se tornar portadores, excretando a bactéria através da urina. Algumas alterações no hemograma, bioquímica sérica e urinálise, juntamente com o histórico do paciente e fatores de risco, auxiliam na suspeita da doença; porém, o diagnóstico definitivo é realizado através de testes mais específicos. Estes testes incluem sorologia, técnicas moleculares como a reação em cadeia da polimerase (PCR) e cultura para isolamento do sorovar. O presente trabalho caracterizou e comparou a leptospirose em três populações caninas de Porto Alegre. Foram avaliados 33 cães com suspeita da doença, atendidos no Hospital de Clínicas Veterinárias (HCV) da Universidade Federal do Rio Grande do Sul (UFRGS); 65 cães provenientes do Centro de Controle de Zoonoses (CCZ) de Porto Alegre; e 155 cães residentes no bairro Arquipélago na zona urbana de Porto Alegre, onde existe uma alta incidência de leptospirose humana. O diagnóstico da leptospirose canina foi baseado na sorologia e PCR no soro e urina. Um total de 14,6% (37/253) dos cães apresentaram resultado positivo na PCR no sangue (leptospiremia) e 14,2 % (36/253) na urina (leptospirúria). Em relação à sorologia, 48,2% (122/253) foram positivos para um ou mais sorovares. Os sorovares mais prevalentes foram canicola, icterohaemorrhagiae e copenhageni. A presença de ratos no ambiente foi associada a leptospirúria (P=0,02). Na população do HCV, o aumento da creatinina sérica (P=0,009), icterícia (P=0,004) e glicosúria (P=0,04) foram associados com leptospirúria. Apesar destas associações encontradas, observou-se que a ausência de sinais clínicos ou de alterações no hemograma, bioquímica sérica ou urinálise não excluíram a infecção (P>0.05). Em um segundo estudo, foi investigada a eficácia da doxiciclina na eliminação do estado de portador renal em quatro cães assintomáticos. Destes, três estavam infectados com o sorogrupo Canicola e um com o sorogrupo Icterohaemorrhagiae. Os cães foram acompanhados por 30 dias após o início do tratamento, e a ausência de leptospiras na urina foi confirmada através de três resultados seriados de PCR negativa em cada cão. Finalmente, valor preditivo da proteína C reativa (C-RP) na leptospirose foi investigado em 62 cães, comparando sua concentração sérica e urinária com os resultados de sorologia e PCR. Sorologia positiva foi associada com C-RP urinária (P= 0,038). Houve apenas associação fraca entre proteína C-reativa sérica e PCR no sangue (área sob a curva= 0,68), e não foi observada associação entre C-RP urinária e PCR na urina. Não foram observadas vantagens de se incluir a C-RP como um teste de triagem para leptospirose em cães. As informações obtidas com os estudos aqui citados mostra a importância do diagnóstico definitivo, preferencialmente realizado através de PCR; a necessidade de se testar cães expostos a fatores de risco, independente de seu estado aparente de saúde; a importância de medidas sanitárias para a prevenção da doença e o tratamento adequado para que se elimine o possível estado de portador renal dos cães. / Leptospirosis is a zoonosis of worldwide distribution, caused by infection with pathogenic serovars of the genus Leptospira. As other affected species of animals, the dogs that survive the acute phase of the disease can become carriers, excreting the bacteria in the urine. Some alterations in blood count, serum biochemistry and urinalysis, along with patient history and risk factors, may contribute to a presumptive diagnosis, but the definitive diagnosis is made using more specific tests. These tests include serology, molecular techniques like polymerase chain reaction (PCR) and culture for the serovar isolation. The present study characterized and compared leptospirosis in three canine populations of Porto Alegre, RS. Thirty three dogs with suspected disease were evaluated at the Veterinary Hospital of the Federal University of Rio Grande do Sul, 65 dogs from the Control Center of Zoonoses from Porto Alegre and 155 dogs from Archipelago neighborhood, where there is a high incidence of human leptospirosis. The diagnosis of canine leptospirosis was based on serology and PCR in serum and urine. A total of 14.6% (37/253) of dogs tested positive in PCR in blood (leptospiremia) and 14.2% (36/253) in the urine (leptospiruria). With regard to serology, 48.2% (122/253) were positive for one or more serovars. The most prevalent serovars were canicola, icterohaemorrhagiae and copenhageni. The presence of rats in the environment was associated with leptospiruria (P = 0.02). In the Veterinary Hospital population, increased serum creatinine (P = 0.009), jaundice (P = 0.004) and glucosuria (P = 0.04) were associated with leptospiruria. Despite these associations, it was observed that the absence of clinical signs or changes in blood count, serum biochemistry and urinalysis did not exclude infection (P> 0.05). In a second study, we investigated the effectiveness of doxycycline to eliminate the carrier state in four asymptomatic dogs. Three were infected with serogroup Canicola and one with serogroup Icterohaemorrhagiae. The dogs were followed for 30 days after starting treatment, and the absence of leptospires in urine was confirmed by three serial results of negative PCR in each dog. Finally, the predictive value of C-reactive protein (C-RP) in leptospirosis was investigated in 62 dogs, comparing its serum and urinary concentrations with serology and PCR. Seropositivity was associated with urinary C-RP (P = 0.038). There was only a weak association between C-RP and serum PCR in blood (AUC = 0.68), and no association was found between urinary C-RP and PCR in urine. There were no advantages to include the C-RP as a screening test for leptospirosis in dogs. The information gathered from the studies cited here shows the importance of the definitive diagnosis, preferably performed by PCR; the need to test dogs exposed to risk factors, regardless of their apparent health condition, the importance of sanitary surveillance for the prevention of the disease, and adequate treatment to eliminate the carrier state.

El trabajo de tesis consta de tres bloques: 1) la caracterización de dos proteínas (BatA y BatB) codificadas en el genoma de Leptospira interrogans; 2) el estudio del rol de los neutrófilos durante la infección con Leptospira interrogans, utilizando un modelo de leptospirosis murina experimental; 3) el análisis de la relación entre el polimorfismo Arg753Gln del TLR2 humano y la leptospirosis.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2002. / Härtill 4 uppsatser.

A leptospirose é uma zoonose de ampla distribuição mundial, causada pela infecção por sorovares patogênicos do gênero Leptospira. Assim como outras espécies de animais acometidos, os cães que sobreviverem à fase aguda da doença podem se tornar portadores, excretando a bactéria através da urina. Algumas alterações no hemograma, bioquímica sérica e urinálise, juntamente com o histórico do paciente e fatores de risco, auxiliam na suspeita da doença; porém, o diagnóstico definitivo é realizado através de testes mais específicos. Estes testes incluem sorologia, técnicas moleculares como a reação em cadeia da polimerase (PCR) e cultura para isolamento do sorovar. O presente trabalho caracterizou e comparou a leptospirose em três populações caninas de Porto Alegre. Foram avaliados 33 cães com suspeita da doença, atendidos no Hospital de Clínicas Veterinárias (HCV) da Universidade Federal do Rio Grande do Sul (UFRGS); 65 cães provenientes do Centro de Controle de Zoonoses (CCZ) de Porto Alegre; e 155 cães residentes no bairro Arquipélago na zona urbana de Porto Alegre, onde existe uma alta incidência de leptospirose humana. O diagnóstico da leptospirose canina foi baseado na sorologia e PCR no soro e urina. Um total de 14,6% (37/253) dos cães apresentaram resultado positivo na PCR no sangue (leptospiremia) e 14,2 % (36/253) na urina (leptospirúria). Em relação à sorologia, 48,2% (122/253) foram positivos para um ou mais sorovares. Os sorovares mais prevalentes foram canicola, icterohaemorrhagiae e copenhageni. A presença de ratos no ambiente foi associada a leptospirúria (P=0,02). Na população do HCV, o aumento da creatinina sérica (P=0,009), icterícia (P=0,004) e glicosúria (P=0,04) foram associados com leptospirúria. Apesar destas associações encontradas, observou-se que a ausência de sinais clínicos ou de alterações no hemograma, bioquímica sérica ou urinálise não excluíram a infecção (P>0.05). Em um segundo estudo, foi investigada a eficácia da doxiciclina na eliminação do estado de portador renal em quatro cães assintomáticos. Destes, três estavam infectados com o sorogrupo Canicola e um com o sorogrupo Icterohaemorrhagiae. Os cães foram acompanhados por 30 dias após o início do tratamento, e a ausência de leptospiras na urina foi confirmada através de três resultados seriados de PCR negativa em cada cão. Finalmente, valor preditivo da proteína C reativa (C-RP) na leptospirose foi investigado em 62 cães, comparando sua concentração sérica e urinária com os resultados de sorologia e PCR. Sorologia positiva foi associada com C-RP urinária (P= 0,038). Houve apenas associação fraca entre proteína C-reativa sérica e PCR no sangue (área sob a curva= 0,68), e não foi observada associação entre C-RP urinária e PCR na urina. Não foram observadas vantagens de se incluir a C-RP como um teste de triagem para leptospirose em cães. As informações obtidas com os estudos aqui citados mostra a importância do diagnóstico definitivo, preferencialmente realizado através de PCR; a necessidade de se testar cães expostos a fatores de risco, independente de seu estado aparente de saúde; a importância de medidas sanitárias para a prevenção da doença e o tratamento adequado para que se elimine o possível estado de portador renal dos cães. / Leptospirosis is a zoonosis of worldwide distribution, caused by infection with pathogenic serovars of the genus Leptospira. As other affected species of animals, the dogs that survive the acute phase of the disease can become carriers, excreting the bacteria in the urine. Some alterations in blood count, serum biochemistry and urinalysis, along with patient history and risk factors, may contribute to a presumptive diagnosis, but the definitive diagnosis is made using more specific tests. These tests include serology, molecular techniques like polymerase chain reaction (PCR) and culture for the serovar isolation. The present study characterized and compared leptospirosis in three canine populations of Porto Alegre, RS. Thirty three dogs with suspected disease were evaluated at the Veterinary Hospital of the Federal University of Rio Grande do Sul, 65 dogs from the Control Center of Zoonoses from Porto Alegre and 155 dogs from Archipelago neighborhood, where there is a high incidence of human leptospirosis. The diagnosis of canine leptospirosis was based on serology and PCR in serum and urine. A total of 14.6% (37/253) of dogs tested positive in PCR in blood (leptospiremia) and 14.2% (36/253) in the urine (leptospiruria). With regard to serology, 48.2% (122/253) were positive for one or more serovars. The most prevalent serovars were canicola, icterohaemorrhagiae and copenhageni. The presence of rats in the environment was associated with leptospiruria (P = 0.02). In the Veterinary Hospital population, increased serum creatinine (P = 0.009), jaundice (P = 0.004) and glucosuria (P = 0.04) were associated with leptospiruria. Despite these associations, it was observed that the absence of clinical signs or changes in blood count, serum biochemistry and urinalysis did not exclude infection (P> 0.05). In a second study, we investigated the effectiveness of doxycycline to eliminate the carrier state in four asymptomatic dogs. Three were infected with serogroup Canicola and one with serogroup Icterohaemorrhagiae. The dogs were followed for 30 days after starting treatment, and the absence of leptospires in urine was confirmed by three serial results of negative PCR in each dog. Finally, the predictive value of C-reactive protein (C-RP) in leptospirosis was investigated in 62 dogs, comparing its serum and urinary concentrations with serology and PCR. Seropositivity was associated with urinary C-RP (P = 0.038). There was only a weak association between C-RP and serum PCR in blood (AUC = 0.68), and no association was found between urinary C-RP and PCR in urine. There were no advantages to include the C-RP as a screening test for leptospirosis in dogs. The information gathered from the studies cited here shows the importance of the definitive diagnosis, preferably performed by PCR; the need to test dogs exposed to risk factors, regardless of their apparent health condition, the importance of sanitary surveillance for the prevention of the disease, and adequate treatment to eliminate the carrier state.

A leptospirose é uma zoonose de ampla distribuição mundial, causada pela infecção por sorovares patogênicos do gênero Leptospira. Assim como outras espécies de animais acometidos, os cães que sobreviverem à fase aguda da doença podem se tornar portadores, excretando a bactéria através da urina. Algumas alterações no hemograma, bioquímica sérica e urinálise, juntamente com o histórico do paciente e fatores de risco, auxiliam na suspeita da doença; porém, o diagnóstico definitivo é realizado através de testes mais específicos. Estes testes incluem sorologia, técnicas moleculares como a reação em cadeia da polimerase (PCR) e cultura para isolamento do sorovar. O presente trabalho caracterizou e comparou a leptospirose em três populações caninas de Porto Alegre. Foram avaliados 33 cães com suspeita da doença, atendidos no Hospital de Clínicas Veterinárias (HCV) da Universidade Federal do Rio Grande do Sul (UFRGS); 65 cães provenientes do Centro de Controle de Zoonoses (CCZ) de Porto Alegre; e 155 cães residentes no bairro Arquipélago na zona urbana de Porto Alegre, onde existe uma alta incidência de leptospirose humana. O diagnóstico da leptospirose canina foi baseado na sorologia e PCR no soro e urina. Um total de 14,6% (37/253) dos cães apresentaram resultado positivo na PCR no sangue (leptospiremia) e 14,2 % (36/253) na urina (leptospirúria). Em relação à sorologia, 48,2% (122/253) foram positivos para um ou mais sorovares. Os sorovares mais prevalentes foram canicola, icterohaemorrhagiae e copenhageni. A presença de ratos no ambiente foi associada a leptospirúria (P=0,02). Na população do HCV, o aumento da creatinina sérica (P=0,009), icterícia (P=0,004) e glicosúria (P=0,04) foram associados com leptospirúria. Apesar destas associações encontradas, observou-se que a ausência de sinais clínicos ou de alterações no hemograma, bioquímica sérica ou urinálise não excluíram a infecção (P>0.05). Em um segundo estudo, foi investigada a eficácia da doxiciclina na eliminação do estado de portador renal em quatro cães assintomáticos. Destes, três estavam infectados com o sorogrupo Canicola e um com o sorogrupo Icterohaemorrhagiae. Os cães foram acompanhados por 30 dias após o início do tratamento, e a ausência de leptospiras na urina foi confirmada através de três resultados seriados de PCR negativa em cada cão. Finalmente, valor preditivo da proteína C reativa (C-RP) na leptospirose foi investigado em 62 cães, comparando sua concentração sérica e urinária com os resultados de sorologia e PCR. Sorologia positiva foi associada com C-RP urinária (P= 0,038). Houve apenas associação fraca entre proteína C-reativa sérica e PCR no sangue (área sob a curva= 0,68), e não foi observada associação entre C-RP urinária e PCR na urina. Não foram observadas vantagens de se incluir a C-RP como um teste de triagem para leptospirose em cães. As informações obtidas com os estudos aqui citados mostra a importância do diagnóstico definitivo, preferencialmente realizado através de PCR; a necessidade de se testar cães expostos a fatores de risco, independente de seu estado aparente de saúde; a importância de medidas sanitárias para a prevenção da doença e o tratamento adequado para que se elimine o possível estado de portador renal dos cães. / Leptospirosis is a zoonosis of worldwide distribution, caused by infection with pathogenic serovars of the genus Leptospira. As other affected species of animals, the dogs that survive the acute phase of the disease can become carriers, excreting the bacteria in the urine. Some alterations in blood count, serum biochemistry and urinalysis, along with patient history and risk factors, may contribute to a presumptive diagnosis, but the definitive diagnosis is made using more specific tests. These tests include serology, molecular techniques like polymerase chain reaction (PCR) and culture for the serovar isolation. The present study characterized and compared leptospirosis in three canine populations of Porto Alegre, RS. Thirty three dogs with suspected disease were evaluated at the Veterinary Hospital of the Federal University of Rio Grande do Sul, 65 dogs from the Control Center of Zoonoses from Porto Alegre and 155 dogs from Archipelago neighborhood, where there is a high incidence of human leptospirosis. The diagnosis of canine leptospirosis was based on serology and PCR in serum and urine. A total of 14.6% (37/253) of dogs tested positive in PCR in blood (leptospiremia) and 14.2% (36/253) in the urine (leptospiruria). With regard to serology, 48.2% (122/253) were positive for one or more serovars. The most prevalent serovars were canicola, icterohaemorrhagiae and copenhageni. The presence of rats in the environment was associated with leptospiruria (P = 0.02). In the Veterinary Hospital population, increased serum creatinine (P = 0.009), jaundice (P = 0.004) and glucosuria (P = 0.04) were associated with leptospiruria. Despite these associations, it was observed that the absence of clinical signs or changes in blood count, serum biochemistry and urinalysis did not exclude infection (P> 0.05). In a second study, we investigated the effectiveness of doxycycline to eliminate the carrier state in four asymptomatic dogs. Three were infected with serogroup Canicola and one with serogroup Icterohaemorrhagiae. The dogs were followed for 30 days after starting treatment, and the absence of leptospires in urine was confirmed by three serial results of negative PCR in each dog. Finally, the predictive value of C-reactive protein (C-RP) in leptospirosis was investigated in 62 dogs, comparing its serum and urinary concentrations with serology and PCR. Seropositivity was associated with urinary C-RP (P = 0.038). There was only a weak association between C-RP and serum PCR in blood (AUC = 0.68), and no association was found between urinary C-RP and PCR in urine. There were no advantages to include the C-RP as a screening test for leptospirosis in dogs. The information gathered from the studies cited here shows the importance of the definitive diagnosis, preferably performed by PCR; the need to test dogs exposed to risk factors, regardless of their apparent health condition, the importance of sanitary surveillance for the prevention of the disease, and adequate treatment to eliminate the carrier state.

Orientador: Raul José Silva Gírio / Banca: Vicente Borelli / Banca: Silvio Arruda Vasconcellos / Banca: Luís Antônio Mathias / Banca: Samir Issa Samara / Resumo: O trabalho presente objetivou pesquisar a presença de Leptospira interrogans sorovar Canicola em 32 cães, dos quais 20 infectados e 12 controle, em 4 grupos de estudo com 5 infectados e 3 de controle. No G1 os animais foram eutanasiados 7 dias após a inoculação, no G2, 15 dias, no G3, 30 dias e no G4 45 dias. Colheu-se sêmen, urina e sangue nos dias 0, 3, 5, 7, 10 e após, a cada 5 dias, com realização de exames andrológicos do sêmen e bioquímicos da urina e do sangue, e reação em cadeia da polimerase (PCR) dessas amostras. Avaliou-se o estado clínico, e o soro sangüíneo foi submetido à soro-aglutinação microscópica (SAM). Os títulos sorológicos dos infectados chegaram a 2.560. Na PCR, detectou-se DNA de leptospiras primeiro no sêmen e depois na urina, e não se detectou nos testículos, epidídimos, próstata, rins e fígado. Na Levaditi, foi encontrada leptospira em um fígado, e no histopatológico, 5 animais com prostatite, evidenciada também pelo aumento do número de espermatozóides com a patologia cabeça isolada. O exame andrológico mostrou declínio na motilidade, no vigor, na concentração espermática e defeitos maiores acima de 10%. No hemograma, observou-se alterações a partir do dia 3 pós-infecção, e ao exame bioquímico a partir do dia 30, indicando possíveis alterações hepáticas e renais. Conclui-se que cães infectados com Leptospira interrogans sorovar Canicola apresentam queda na qualidade espermática, sendo importantes fontes de infecção para o homem e animais, podendo inclusive transmitir a leptospirose por meio de sêmen nos primeiros dias pós-infecção, antes da transmissão urinária, sendo detectada pela PCR anteriormente a SAM. / Abstract: The present study aimed to investigate the presence of Leptospira interrogans serovar Canicola. in 32 dogs, of which 20 are infected and 12 control, 4 study groups with 5 infected and 3 of control. In G1 animals were euthanasing 7 days after inoculation, the G2, 15 days, the G3, 30 days in the G4 and 45 days. It is collected semen, urine and blood on days 0, 3, 5, 7, 10 and after, every 5 days, with laboratory tests of semen andrological and biochemical of urine and blood, and polymerase chain reaction (PCR ) Of these samples. Evaluated the clinical status, and serum was submitted to serum-microscopic agglutination (SAM). The serological evidence of infection arrived in 2560. In PCR, DNA was found in semen of leptospires first and then in the urine, and is not detected in testis, epididymis, prostate, kidney and liver. In Levaditi, was found leptospira in a liver, and the histopathology, 5 animals with prostatitis, evidenced also by increasing the number of sperm with the pathology head alone. The examination showed andrologic decline in motility, in effect, defects in sperm concentration and higher above 10%. In the blood, it was observed changes from day 3 post-infection, and the biochemical examination on the day 30, indicating possible kidney and liver abnormalities. It follows that dogs infected with Leptospira interrogans serovar Canicola present decrease in sperm quality, and important sources of infection for humans and animals, which may include forward to leptospirosis through semen during the first day post-infection, before urinary transmission, and PCR detected earlier by the SAM. / Doutor

Submitted by (edna.saturno@ufrpe.br) on 2016-11-03T12:56:44Z No. of bitstreams: 1 Romulo Menna Barreto Valenca.pdf: 688661 bytes, checksum: d80c675dd68a0dfbf5ff078ecc666613 (MD5) / Made available in DSpace on 2016-11-03T12:56:44Z (GMT). No. of bitstreams: 1 Romulo Menna Barreto Valenca.pdf: 688661 bytes, checksum: d80c675dd68a0dfbf5ff078ecc666613 (MD5) Previous issue date: 2009-10-05 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The objective of this study was to determine the prevalence and identify the risk factors associated with infection by Leptospira spp. in commercial pig farms in the state of Alagoas, Brazil. To compose the study, were used 342 pigs (312 sows and 30 boars) from at seven swine herds distributed in five districts of the state of Alagoas. The serological diagnosis of infection was done by Microscopic Agglutination Test (SAM). The risk factors analysis was performed by the application of research questionnaires, consisting of objective questions relating to the designer, the general characteristics of the property, the production management, and reproductive health. Detected a prevalence of 16.10% (55/352) of pigs positive. The risk factors associated were: The not perform quarantine (p = 0.003, OR = 5.43, CI = 1.79 -16.41) and artificial insemination utilization (p = 0.023, OR = 3.38, CI = 1.18 - 9, 66). Was foundsignificant association of sows infection with the increased number of stillborn and mummified, and with the increase frequency of estrus recurrence and increased the numbers of days from weaning to service of seropositive sows. It is concluded that infection with Leptospira spp. is disseminated in commercial pig farms in the state of Alagoas, favoring the reproductive failures and decrease of zootechnical performance. The risk factors identified in this study are facilitators in the dissemination of infection and should be adjusted to control the disease in the herds studied. / Objetivou-se com este estudo calcular a prevalência e identificar os fatores de risco associados à infecção por Leptospira spp. em suínos criados em granjas tecnificadas no Estado de Alagoas, Brasil. Para compor a amostra do estudo de prevalência foram utilizados 342 suínos, sendo 312 matrizes e 30 varrões, oriundos de sete granjas de ciclo completo e distribuídas em cinco municípios do Estado de Alagoas. O diagnóstico sorológico da infecção foi realizado pela técnica de Soroaglutinação Microscópica (SAM). A análise dos fatores de risco foi realizada por meio da aplicação de questionários constituídos por perguntas objetivas referentes ao criador, às características gerais da propriedade, ao manejo produtivo, reprodutivo e sanitário. Obteve-se uma prevalência de 16,10% (55/342) de suínos soropositivos. Os fatores associados foram: não realização de quarentena (p = 0,003; OR = 5,43; IC = 1,79 – 16,41) e a utilização da inseminação artificial (p = 0,023; OR = 3,38; IC = 1,18 – 9,66). Detectou-se associação entre a infecção dasmatrizes e o aumento do número de natimortos e mumificados, assim como com a maior freqüência de repetição de cio e aumento do intervalo desmama/estro nas matrizes soropositivas. Conclui-se que a infecção por Leptospira spp. encontra-se disseminada nas granjas suinícolas tecnificadas no Estado de Alagoas e contribui com a ocorrência de falhas reprodutivas e queda nos índices zootécnicos nessas propriedades. Os fatores de risco identificados nesse estudo são facilitadores na disseminação do agente e devem ser corrigidos para controlar a doença nos rebanhos estudados.

La leptospirose est une zoonose de distribution mondiale provoquant plus d'un million de cas sévères chez l'Homme et causant des infections chez les animaux. Les agents responsables de cette maladie sont les espèces pathogènes du genre Leptospira, bactéries atypiques dont les mécanismes de virulence sont largement méconnus. Des vaccins humains et vétérinaires existent mais leur action est restreinte à certains sérovars qui ne reflètent que partiellement l'épidémiologie actuelle, complexe et fluctuante dans l'espace et le temps. Les objectifs de la thèse présentée dans ce manuscrit ont été d'une part de caractériser de nouveaux facteurs de virulence de Leptospira et d'autre part d'identifier des candidats vaccins capables d'induire une protection croisée dans le cadre du projet collaboratif COVALEPT mené par Merial, regroupant des partenaires académiques et industriels. Dans un premier temps, un outil de numération par cytométrie en flux, plus rapide et performant que la méthode standard, a été développé. Par la suite, l'étude de mutants générés par transposition aléatoire ou spontanés a permis d'identifier trois facteurs de virulence : LIC12144, requis pour la synthèse des lipopolysaccharides, FliM, essentiel à la motilité, et LIC11221, potentiellement impliqué dans le chimiotactisme. De plus, deux candidats vaccins, une protéine recombinante et une souche vivante atténuée, ont été conçus et ont démontré une efficacité partielle contre la maladie en modèle rongeur. En conclusion, ce travail apporte des connaissances inédites sur les facteurs de virulence de Leptospira et permet d'envisager le développement de vaccins à large spectre de protection contre la leptospirose / Leptospirosis is a worldwide zoonosis responsible for more than 1 million human cases and severe infections in animals. The causative agents are pathogenic species of Leptospira. The virulence mechanisms of these atypical bacteria remain largely unknown. Human and animal vaccines exist but the protection is restricted to a few serovars, representing only partially the complex and fluctuating epidemiology of the disease. The aims of the thesis presented in this manuscript were to characterize new virulence factors on Leptospira on the one hand and to identify new cross-protective vaccine candidates on the other hand, in the context of the COVALEPT collaborative project led by Merial, gathering industrial and academic partners. First, a flow cytometry based enumeration method for leptospires was developed, more rapid and efficient than the current standard technique. Then, the study of mutants generated by random transposition and spontaneously avirulent strains led to the identification of three virulence factors: LIC12144, required for lipopolysaccharides biosynthesis, FliM, essential for motility, and LIC11221 which may be involved in chemotaxis. Finally, two vaccine candidates conferring partial protection against the disease in a rodent model were conceived, a recombinant protein and a live attenuated strain. Altogether, this work brings new knowledge on Leptospira virulence and supports the development of broad spectrum vaccines against leptospirosis