Auswahl der wissenschaftlichen Literatur zum Thema „Leptospira kirschneri Serovar Grippotyphosa“

Leptospirosis is still a global health problem because it affects human health in rural and urban areas, both in industrialized and developing countries. The aim of the study was to detect Leptospira spp. bacteria in kidney tissues isolated from rats in the Napu and Bada Highlands of Poso District, Central Sulawesi Province. Kidneys sample from 63 rats were collected from Napu and Bada Highlands of Poso District, Central Sulawesi Province in MayJune 2018. Polymerase Chain Reaction (PCR) was used to detect Leptospira. The molecular characterizations were conducted based on the 16SrRNA and LipL32 genes. Data were analyzed descriptively to describe the presence of pathogenic Leptospira DNA. Analysis phylogenetic was performed using MEGA 6.2 software. A total of 63 rats was successfullycaught during the study consisting of males and female for 36 (57.1%) and 27 (42.9%), respectively. The species of rats were R. exulans, R. tanezumi, R. argentiventer, R. norvegicus, M. Musculus, Paruromys dominator, Maxomys sp., and Rattus sp. The pathogenic of Leptospira DNA was detected in rats with R. argentiventer and Paruromys dominatorspecies using the 16S rRNA and LipL32 gene. Sample sequences using LipL32 target gene is a close similarity with L. interrogans serovar Hardjo, serovar Autumnalis, Lai, Icterohaemorrhagiae, Balico, Grippotyphosa, Mini, Canicola, Hebdomadis; L. noguchii serovar Pomona and L. kirschneri whereas the sample sequence using 16S rRNA targetgene showed similarity with L. interrogans serovar Canicola, Copenhagen, Autumnalis, Pyrogenes, Javanica, Icterohaemorrhagiae, Manilae, Bratislava, Linhae, Hebdomadis, and L. kirschneri serovar Grippotyphosa. The PCR method with the target gene 16SrRNA and LipL32 are able to detect Leptospira spp. in rats R. argentiventer and P. dominator species Keywords: Leptospira, 16S rRNA, LipL32, PCR, Kidney’s Rat Leptospirosis masih merupakan masalah kesehatan global karena mempengaruhikesehatan manusia di daerah pedesaan dan perkotaan, baik di negara industri maupun mnegara berkembang. Tujuan penelitian adalah untuk mendeteksi bakteri Leptospira spp di jaringan ginjal dari tikus di Dataran Tingi Napu dan Bada Kabupaten Poso, Provinsi Sulawesi Tengah. Ginjal tikus sebanyak 63 sampel dikoleksi dari Dataran Tinggi Napu dan Bada Kabupaten Poso, Provinsi Sulawesi Tengah pada bulan Mei – Juni 2018. PCR digunakan untuk mendeteksi Leptospira. Karakterisasi molekuler dilakukan berdasarkan gen 16SrRNA dan LipL32. Data dianalisis secara deskriptif untuk menggambarkan keberadaaN Leptospira yang patogenik. Analisis filogenetik dilakukan dengan menggunakan perangkat lunak Mega 6.2. Sebanyak 63 tikus berhasil ditangkap selama penelitian yang terdiri dari jantan dan betina, masing masing 36 ekor (75,1%) dan 27 ekor (42,9%). Spesies tikus adalah R. exulans, R. tanezumi, R. argentiventer, R. norvegicus, M. Musculus, Paruromys dominator, Maxomys sp, dan Rattus sp. DNA Leptospira patogenik terdeteksi pada tikus dengan spesies R. argentiventer dan Paruromys dominator menggunakan gen 16SrRNA dan LipL32 Sekuen sampel dengan target gen LipL32 menunjukkan kesamaan dengan L. interrogans serovar Hardjo, serovar Autumnalis, Lai, Icterohaemorrhagiae, Balico, Grippotyphosa, Mini, Canicola, Hebdomadis; L. noguchii serovar Pomona dan L. kirschneri. Sedangkan sekuen sampel dengan target gen 16S rRNA menunjukkan kesamaan dengan L. interrogans serovar Canicola,Copenhagen, Autumnalis, Pyrogenes, Javanica, Icterohaemorrhagiae, Manilae, Bratislava, Linhae, Hebdomadis, dan L. kirschneri serovar Grippotyphosa. Metode PCR dengan target gen 16SrRNA dan LipL32 mampu mendeteksi Leptospira spp. pada tikus dengan spesies R. argentiventer dan P. dominator. Kata kunci: Leptospira, 16S rRNA, LipL32, PCR, Ginjal Tikus

Leptospirosis is the most neglected widespread zoonosis worldwide. In Spain, leptospirosis reports in people and animals have increased lately. Cats can become infected with Leptospira, as well as be chronic carriers. The aim of this study was to determine serological antibody prevalence against Leptospira sp., blood DNA, and shedding of DNA from pathogenic Leptospira species in the urine of cats in Spain. Microagglutination tests (MAT) and blood and urine TaqMan real-time polymerase chain reaction (PCR) were performed. Leptospira antibodies were detected in 10/244 cats; with 4.1% positive results (95% confidence interval (CI): 2.1–7.18%). Titers ranged from 1:20 to 1:320 (serovars Ballum; Bataviae; Bratislava; Cynopteri; Grippotyphosa Mandemakers; Grippotyphosa Moskva; Pomona; and Proechimys). The most common serovar was Cynopteri. Blood samples from 1/89 cats amplified for Leptospira DNA (1.12%; 95% CI: 0.05–5.41%). Urine samples from 4/232 cats amplified for Leptospira DNA (1.72%; 95% CI: 0.55–4.10%). In conclusion free-roaming cats in Spain can shed pathogenic Leptospira DNA in their urine and may be a source of human infection. Serovars not previously described in cats in Spain were detected; suggesting the presence of at least 4 different species of pathogenic leptospires in the country (L. borgpetersenii; L. interrogans; L. kirschneri; and L. noguchii).

La leptospirosis es una enfermedad infecciosa de carácter zoonótico y de distribución mundial, es caudada por espiroquetas del género Leptospira, especies: L. interrogans y L. biflexa (según clasificación fenotípica) o L. interrogans, L. biflexa y L. kirschneri (según clasificación genotípica). Los factores de virulencia asociados a Leptospiras patógenas son endotoxinas, hemolisinas esfingomielinasa, fosfolipasa y proteínas superficiales de adherencia. En los bovinos se ha encontrado Leptospira borgpetersenii Serovar hardjo, tipo hardjo bovis como un serovar adaptado, y Leptospira interrogans Serovar hardjo, tipo hardjo prajitmo de la especie interrogans, L. icterohaemorrhagiae, L. canicola y L. grippotyphosa como un serovares no adaptados. A nivel mundial Leptospira hardjo no es el único agente patógeno asociado a las fallas reproductivas y tampoco es la única serovariedad de Leptospira encontrada en bovinos. La inmunidad presentada frente a la leptospirosis es de tipo humoral, con un periodo de incubación de 4 a 10 días, se disemina en hígado, riñones, pulmones, tracto reproductor (placenta) y líquido cefalorraquídeo, produciendo daño al endotelio de los vasos sanguíneos, isquemia localizada en los órganos, necrosis tubular renal, daño hepatocelular y pulmonar, meningitis, miositis y placentitis. Las sustancias toxicas causan lisis de los eritrocitos y atraviesan la barrera placentaria produciendo la muerte fetal por anoxia terminando en aborto 1 o 2 días después.

Brucella canis is a cause of canine infertility and abortion. Veterinarians and veterinary laboratorians screen for antibodies to B. canis with serologic tests including a rapid slide agglutination test (RSAT; D-Tec CB, Zoetis, San Diego, CA). False-positive results are possible because of cross-reactivity to antibodies to some gram-negative bacteria. Cross-reactivity has been reported between antibodies of Brucella abortus and Leptospira spp. with serologic tests for bovine brucellosis; however, this has not been documented with serologic tests for canine brucellosis, to the author’s knowledge. The RSAT was evaluated with the sera from dogs experimentally challenged with 1 of 4 serovars of Leptospira spp.: L. kirschneri serovar Grippotyphosa, or L. interrogans serovars Canicola, Icterohaemorrhagiae, or Pomona. Experimental infections were confirmed through results of microscopic agglutination testing and/or lateral flow immunochromatography testing. The sera of 32 dogs collected at day 0 and days 7, 10, and 14 yielded negative results with the RSAT. Antibodies produced through experimental infections to these 4 serovars of Leptospira spp. did not cross-react with Brucella antigen with the RSAT; therefore, cross-reactivity of anti-leptospiral antibodies may not be of concern for B. canis rapid slide agglutination testing of dogs.

Forty-four specific pathogen-free beagles, median age 65 days, received two subcutaneous doses of either a commercially available, five-way combination vaccine or the same vaccine in combination with a tetravalent Leptospira bacterin (Canicola, Grippotyphosa, Icterohaemorrhagiae, Pomona). They were subsequently challenged with a pathogenic strain L kirschneri serovar Grippotyphosa 470 days following completion of the vaccination protocol. Titres of agglutinating serum antibodies were determined at various time points before and after both vaccination and challenge, along with postchallenge reisolation of the challenge organisms from blood and urine, and evaluation of renal histopathology. Clinical signs of generalised leptospirosis were not observed in any of the dogs after challenge. In order to demonstrate efficacy, leptospirosis was defined as having at least one positive urine sample and a positive renal histopathology score; or, in the absence of renal pathology, multiple positive urine samples. Leptospiremia was not demonstrated in any of the vaccinated dogs versus 27 per cent of the controls; leptospiruria was noted in 5 per cent of the vaccinates compared with 76 per cent of controls; and renal lesions were observed in 15 per cent of the vaccinates and 65 per cent controls. Using these criteria, the vaccine was able to significantly prevent leptospirosis (P=0.0001) in the vaccinated animals. This study establishes duration of immunity of at least 15 months for the prevention of disease and renal excretion of leptospires for the Leptospira serovar Grippotyphosa fraction of a quadrivalent Leptospira vaccine.

We determined the seropositive percentage and the determinants of Leptospira exposure in domestic donkeys presented for slaughter in the State of Durango, Mexico. We examined 194 donkeys in 4 gathering premises. Sera of donkeys were analyzed for anti- Leptospira IgG antibodies using a microscopic agglutination assay with a panel of 7 Leptospira antigens. The association between Leptospira seropositivity and general characteristics of donkeys was assessed by bivariate and multivariate analyses. Antibodies against Leptospira (for at least 1 of the 7 Leptospira serovars examined) were found in 151 of 194 (77.8%) donkeys. Seropositive donkeys were found in all regions, municipalities, and premises examined. The predominant serovar was L. interrogans Icterohaemorrhagiae ( n = 117; 60.3%), followed by L. interrogans Sejroe ( n = 96; 49.5%), L. interrogans Canicola ( n = 10; 5.2%), L. kirschneri Grippotyphosa ( n = 6; 3.1%), L. borgpetersenii Ballum ( n = 5; 2.6 %), L. biflexa Semaranga ( n = 3; 1.5%), and L. noguchii Panama ( n = 2; 1%). Leptospira seropositivity was associated with gathering premises (OR = 2.64; 95% CI: 1.27–5.46; p = 0.009) and municipalities (OR = 0.11; 95% CI: 0.01–0.78; p = 0.02). Our results demonstrate an apparently high seropositive percentage of Leptospira infection in the donkeys studied in Mexico.

ABSTRACT Leptospiral protein antigens are of interest as potential virulence factors and as candidate serodiagnostic and immunoprotective reagents. We identified leptospiral protein antigens by screening a genomic expression library with serum from a rabbit hyperimmunized with formalin-killed, virulent Leptospira kirschneri serovar grippotyphosa. Genes expressing known outer membrane lipoproteins LipL32 and LipL41, the heat shock protein GroEL, and the α, β, and β′ subunits of RNA polymerase were isolated from the library. In addition, a new leptospiral gene that in Escherichia coli expressed a 45-kDa antigen with an amino-terminal signal peptide followed by the spirochetal lipobox Val−4-Phe−3-Asn−2-Ala−1↓Cys+1 was isolated. We designated this putative lipoprotein LipL45. Immunoblot analysis of a panel of Leptospira strains probed with LipL45 antiserum demonstrated that many low-passage strains expressed LipL45. In contrast, LipL45 was not detected in high-passage, culture-attenuated strains, suggesting that LipL45 is a virulence-associated protein. In addition, all leptospiral strains tested, irrespective of culture passage, expressed a 31-kDa antigen that was recognized by LipL45 antiserum. Southern blot and peptide mapping studies indicated that this 31-kDa antigen was derived from the carboxy terminus of LipL45; therefore, it was designated P31LipL45. Membrane fractionation studies demonstrated that P31LipL45 is a peripheral membrane protein. Finally, we found that P31LipL45 levels increased as Leptospira entered the stationary phase, indicating that P31LipL45 levels were regulated. Hamsters infected with L. kirschneri formed an antibody response to LipL45, indicating that LipL45 was expressed during infection. Furthermore, the immunohistochemistry of kidneys from infected hamsters indicated that LipL45 was expressed by L. kirschneri that colonized the renal tubule. These observations suggest that expression of LipL45 responds to environmental cues, including those encountered during infection of a mammalian host.

Abstract Blood serum samples collected from randomly selected groups of 32 pigs and 41 cows reared in farms belonging to the rural community “A” located in eastern Poland and exposed to the Vistula river floods, and serum samples from groups of 41 pigs and 40 cows from farms belonging to the rural community “B” located also in eastern Poland but not in the area exposed to floods, were examined by the microscopic agglutination test for the presence of antibodies against 18 Leptospira serovars. The percentage of serum samples presenting positive results with at least one serovar were higher in pigs and cows from community “A” comparing to community “B” (34.4% vs. 4.9% and 26.8% vs. 15.0%, respectively). In the case of pigs, the difference was statistically significant (P=0.0015). The reactions with 12 Leptospira serovars (Australis, Bataviae, Bratislava, Canicola, Hardjo, Hebdomadis, Icterohaemorrhagiae, Pomona, Poi, Cynopteri, Grippotyphosa, Celledoni,), belonging to four species (L. interrogans, L.borgpetersenii, L. kirschneri, L. weili) were found in the examined animals. In community “B”, six reactions with one serovar and two reactions with two serovars were noted whereas in community “A” - 19 reactions with one serovar, one reaction with two serovars and two reactions with six serovars were observed. The titres in animals reared in community “A” were significantly higher (up to 25,600) compared to community “B” (up to 200, P=0.0094). The obtained results suggest that the exposure to flooding may increase the infection rate in pigs and cows from afflicted areas to some extent.

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