Reuni贸n Cient铆fica Internacional "Leptospirosis 2001" 17-18 de mayo del 2001

[摘要] de líneas genéticas con las que se han mejorado los parámetros reproductivos y productivos, se han mejorado las instalaciones con una marcada tendencia a la explotación intensiva. La situación anterior ha llevado a establecer una vigilancia de algunas enfermedades infecciosas como síndrome reproductivo y respiratorio porcino, peste porcina clásica, enfermedad de Aujesky Parvovirosis, leptospirosis y brucelosis. Los estudios serológicos utilizados para el diagnóstico y control sanitario son de gran utilidad en los casos siguientes: conocimiento del estado sanitario de la granja, vigilancia serológica de diferentes áreas, elección de los esquemas de vacunación y conocimiento de niveles de protección. Entre julio y septiembre del año 2000, se muestrearon para estudio serológico de leptospira, 27 granjas correspomdientes a 12 municipios, de los departamentos de Caldas, Quindio y Risaralda. De cada una de las explotaciones se seleccionaron 20 hembras multíparas para un total de 540 sueros, a los que se les realizó la prueba de microaglutinación, en dilución inicial de 1:100 frente a los serovares siguientes: bratislava, pomona, canicola, grippotyfosa e icteroharmorrhagiae. De los sueros examinados, 102 fueron reactores, pero con muy bajo título , principalmente a los serovares canicola e icterohaemorrhagiae, por lo tanto no se consideraron representativos de infección, salvo en una granja donde un animal presentó títulos superiores a 1:800. Vale la pena anotar que todos los animales habían sido vacunados contra leptospirosis antes del primer servivio y revacunadas semestral o anualmente. De lo anterior podemos concluir que la MAT sólo mide exposición reciente al antígeno de campo o al vacunal y en poblaciones vacunadas es difícil la interpretación. Comportamiento de la circulación de Leptospira interrogans en humanos en diferentes regiones de Cuba (1996-2000) Islay Rodríguez, Ana M. Obregón, José Rodríguez, Carmen Fernández, Berta [e-mail] Nacional de Referencia de Leptospiras. IPK. Cuba Con el objetivo de conocer los principales serogrupos de Leptospira interrogans causantes de leptospirosis humana en Cuba fueron estudiadas 228 cepas, obtenidas a partir de hemocultivos provenientes de diferentes regiones del país. Estas cepas fueron caracterizadas serológicamente, utilizando la microaglutinación de serogrupos con 13 sueros policlonales correspondientes a los serogrupos de mayor circulación nacional (según estudios epizootiológicos). Según este trabajo se detectó que, los de mayor circulación eran Ballum (55,70 %), Pomona (21,03 %) y Canicola (14,03 %), encontrándose además Icterohaemorrhagiae, Tarassovi, Australis, Hebdomadis, Sejroe y por primera vez los serogrupos Pyrogenes, Autumnalis y Bataviae, no aislados con anterioridad de humanos en Cuba. Estos resultados relacionan a los ratones, cerdos y perros como principales reservorios de esta entidad y contribuyen al perfeccionamiento de la vacuna cubana contra la leptospirosis humana. New microscopic device for direct reading of the microscopic agglutination test (MAT) for leptospirosis A. SchönbergBundesinstitut für gesundheitlichen Verbraucherschutz und Veterinärmedizin,Diedersdorfer Weg 1, D-12277 Berlin, Germany The microscopic agglutination test (MAT) is the reference test for the diagnosis of leptospirosis against which all other serological tests are evaluated and is the best specified method for import/export testing (WHO leptoguidelines, 2001, O.I.E. Manual of Standards, 1996). It has been reported that the results of the MAT performed in microtitre plates (F form) may be read directly with the aid of a special microscopy equipment using a magnification of 100. In a number of laboratories this method has been in use for some time. Since, however, difficulties arose in respect of availability of appropriate optical equipment we reported about a modification of microscopy equipment from the companies Leitz, Wetzlar, and Carl Zeiss, Oberkochen, in 1980. According to the information of Leitz and several Veterinary Investigation Centres, they got into trouble to provide the long working distance objective: (Leitz, No. 559 141, magnification 10x). The Carl Zeiss company demonstrated a new microscope with an objective of 10x magnification in our laboratory showing a complete equipment for direct reading of the results of MAT. The fact, that several Veterinary Investigation Centres have difficulties to establish the direct reading of the results of the MAT, it is of advantage to introduce this new microscope of the Carl Zeiss Jena GmbH company. This description differs from the description previously given during the “First Meeting of the International Leptospirosis Society” in Nantes, France, 1996:   Microscope “Axioskop”, No. 1051-078,  Objective revolver, No. 1059-202,  Object stage “Axiovert”, No. 451335  Modification of the object stage, No. 0239-500  Object holder M for “Axiovert”, No. 451230  Holder frame M for microtitre plates, No. 471746  Halogenlight 12 V 100 W, No. 380079-9540  Objective “Epiplan” 10X/0,20, No. 442930  Ocular W-Pl 10X/23 No. 455043  Dry-dark-field condenser, No. 465506 The microscopic picture of leptospires was identical and also better in comparison to that which was achieved by the combined equipment of Leitz and Carl Zeiss in 1980. The pathogenic and saprophytic leptospires: differential characteristics and how to distinguish them (Lecture) Marina Cinco, Rossella [e-mail] Scienze Biomediche, Leptospira Laboratory, University of Trieste. Italy The genus Leptospira includes micro-organisms which induce illness in humans and animals, the pathogenic leptospires,as well as free living spirochetes which though non pathogenic, must be known and taken into consideration because they can contaminate the media and can be occasionally isolated from mammals or from the environment, leading to misdiagnosis. In fact the non pathogenic leptospires live in fresh waters, brackish waters and also sea waters, sharing a large adaptability to the environment. Though morphologically indistinguishable the pathogenic and saprophytic leptospires can be distinguished on the basis of some-not many- phenotypic characteristics and on genetic basis. The pathogenic leptospires are able to develop infection-and disease- because they resist to the complement killing: the expression of outer membrane lipoproteins, enzymes and peptidoglican with proinflammatory power contribute to the leptospira pathogenicity; conversely saprophytic leptospires are rapidly lysed by Complement and this stops their survival and multiplication in the hosts. The genotype of pathogenic leptospires shows one gene for the 5S rRNA instead of two; even if there are not clear cut distinctions metabolic differences include differential sensitivity to 1 M NaCl, incubation temperature, chemicals, Lipase activity and different LPS patterns between the two groups. Using a mAb able to recognise only the pathogenic leptospires can do a better distinction. Moreover we developed a seminested PCR based methodology which permit us to distinguish pathogenic leptospires not only in culture but also to detect as few as 5 pathogenic spirochetes from the natural water environment. Some leptospires anyway have been reported of intermediate behaviour. The LPS of Leptospira: its significance for immunity, taxonomy and vaccine development (Lecture) Ben Adler, Dieter Bulach, Alejandro de la Pena Moctezuma [e-mail] University, Department of Microbiology. AustraliaLipopolysaccharide (LPS) is the principle surface component of Leptospira. It is the target for agglutinating, opsonic antibodies and is a major protective antigen. LPS is the key antigen involved in the serological typing and classification scheme, resulting in the recognition of over 200 serovars. Significantly, immunity to infection generally correlates with serovar related antigens, believed to be mainly LPS. Despite this importance, the structure of leptospiral LPS remains unknown and there is limited knowledge of its biosynthesis and consequently of the molecular basis for immunity and serovar specificity. We have taken a genetic approach to attempt to understand the basis of LPS specificity, beginning with a comparison of the subtypes of serovar Hardjo, namely Hardjobovis and Hardjoprajitno which are antigenically indistinguishable but classified in different species (L. borgpetersenii and L. interrogans respectively). The LPS biosynthetic loci for both types were similar and contained genes involved in sugar biosynthesis, subunit assembly and transport. IS elements were found only in the Hardjobovis locus. However, sequence analysis indicated that the Hardjoprajitno locus comprised four segments, two of which appear to have originated from L. borgpetersenii and two from L. interrogans, allowing a hypothesis of the origin of the Hardjoprajitno LPS locus. A comparison of 12 loci from serovars within L. interrogans revealed a different organisation, with five genes, not present in the Hardjo loci, encoding two glycosyl transferases, an epimerase and two membrane proteins. Allelic differences and mutations in these genes may be sufficient to account for the genetic basis of serovar specificity. Crecimiento, virulencia y antigenicidad de Leptospira mozdok en medio emjh modificado Andrés González, Niurka Batista, Yolanda Valdés y Marta Gonzá[e-mail] “Carlos J. Finlay”. Cuba A pesar del alto costo de sus componentes, el medio sintético EMJH es actualmente el más utilizado para el aislamiento y cultivo de cepas de Leptospira, en ocasiones en grandes volúmenes durante la obtención de inóculos para la planta de producción. El presente trabajo evaluó la influencia de concentraciones crecientes de Tween 80 sobre el crecimiento, virulencia y antigenicidad de la cepa vacunal L. mozdok, sin incrementar la concentración de albúmina reportada. El crecimiento se evaluó espectrofotométricamente y se determinó la variación de otros parámetros como rendimiento celular, velocidad de crecimiento y tiempo de duplicación. La estabilidad de la virulencia fue estimada en el modelo hámster sirio y la antigenicidad fue evaluada mediante aglutinación microscópica utilizando un antisuero policlonal de conejo. Bajo condiciones de cultivo controladas la adición de Tween 80 desde 1,25 hasta 3,25 mg/mL incrementó notablemente la velocidad de crecimiento y los rendimientos celulares finales con un consumo total de la fuente de carbono, pero el crecimiento se afectó notablemente a concentraciones superiores. En este rango de concentraciones no se observaron decrecimientos de la virulencia y antigenicidad durante numerosos subcultivos sucesivos. Luego de tres subcultivos en el medio modificado la cepa evidenció una tolerancia adaptativa a las altas concentraciones de ácidos grasos mostrando excelente crecimiento y antigenicidad a concentraciones de Tween 80 extremadamente líticas, aunque la virulencia se afectó notablemente. Los resultados sugieren que la cepa adaptada alcanza una mayor asimilación de la fuente de carbono y energía que la no adaptada bajo las mismas condiciones de crecimiento. Leptospirosis infection by sport and other leisure activities. Report on cases of leptospirosis derived from outdoor leisure activities A. SchönbergBundesinstitut für gesundheitlichen Verbraucherschutz und Veterinärmedizin,Diedersdorfer Weg 1, D-12277 Berlin, Germany Leisure or hobby activities of people take them into risk situations for leptospirosis. These are camping, water skiing, canoeing, swimming, hunting, fishing, farming and gardening, special sport events (for example “iron man contest”), travel/tourism etc. Cultural and social factors modify the effects of leisure activities. A schoolboy fell ill with fever, headache and pain in the leg after camping near the Isar River. The MAT showed a titre against L. interrogans serovar icterohaemorrhagiae of 1:6400. Leptospires were isolated from the urine.A patient, who had been fishing during holidays in Southern Germany became ill after coming home. Renal involvement was the main complication.A family spent holidays in the Netherlands. Their favorite sport was surfing. Several weeks later the two children showed fever, enteritis, hepatitis, conjunctival suffusion, cardiac complications and neurological symptoms. Both children had a MAT-titre of >1:1600 against several L. interrogans serovars.Three male persons aged between 34 and 38, who had been part of a group whose boat had capsized on a river during an outing 18 days previously, presented a variety of symptoms, including high fever, chills, headache with meningism or facial paralysis, mild hepatitis and renal involvement. The diagnosis of leptospirosis was serologically confirmed.A participant (one of 330 competitors) at an iron man contest (triathlon like event), 38 years old, was hospitalized with kidney problems. The patient returned from the Philippines. The MAT showed titres of <1:1600 against 4 L. interrogans serovars.Identification of leptospiral serovars by randomly amplified polymorphic DNA fingerprinting I.F. Veloso; C.E. Salas; E.C. [e-mail] de Veterinária - UFMG. Brazil. Leptospirosis is an important disease with worldwide distribution affecting human as well as wild and domestic animals. Current diagnosis methods for leptospirosis lack sensibility and are time consuming. The Polymerase Chain Reaction (PCR), a rapid and sensitive assay, was been used to detect leptospires in clinical samples. Recently, Random Amplified Polymorphic DNA (RAPD-PCR) wich produces fewer DNA fragments has been used for rapid identification and characterization different serovars. RAPD fingerprinting of 7 laboratory strains of leptospiral serovars (L. hardjo genotype Hardjoprajitno (OMS), L. hardjo genotype Hardjoprajitno (CTG), L. mini, L. szwajizak, L. tarassovi, L. bratislava, L. pomona) was carried out by using B11/B12 primers described previously. RAPD-PCR was found to be a simple rapid method for serovars identification giving reproducible and serovar-specific banding patterns. For most of the serovars, differentiation bands were observed between 100 and 1000 bp. Small differences between strains of serovar hardjo genotype Hardjoprajitno were found in RAPD-PCR and confirmed by transmission densitometre in 563 nm. This result suggest that occur heterogenicity among genotypes isolated of different geographical areas. DNA’s from E. coli, M. Bovis, human and bovine DNA strains did not exhibit any amplification confirmed the specificity of the pair of primers. RAPD-PCR is a simple and rapid method suitable for the identification of some Leptospira serovars. Leptospirosis vaccines. (Lecture) Ben AdlerAustralia Tratamiento inmunoterapéutico en 50 pacientes con leptospirosis crónica refractaria a antibióticosVelasco-Castrejón O1, Rivas-Sánchez B,1 Rivera-Reyes [e-mail] , [e-mail]1Clínica de Medicina Tropical. Unidad de Medicina Experimental. Fac. de Med, UNAM-Hospital General de México, 2Terapia Médica Intensiva. Hospital General de México, SSA En general se acepta que los sobrevivientes espontáneos o por tratamiento médico de leptospirosis aguda, se curan y recuperan ad integrum; sin embargo, en nuestra experiencia es común la observación de numerosos pacientes con molestias compatibles con leptospirosis crónica, que no sufrieron una fase aguda sintomática e incluso en muchos que la sufrieron, a pesar de su “tratamiento correcto” y pasaron a la fase crónica o persistente. En este tipo de pacientes es común que, debido a sus variadas molestias, sean estudiados por médicos de múltiples especialidades sin llegar a ningún diagnóstico, por lo que es frecuente que sean enviados al psiquiatra, e incluso, algunos de ellos suspenden su vida productiva al dejar su empleo o ser despedidos por incapacidad laboral. También tratamos otros pacientes que padecen cirrosis avanzada, aparentemente secundaria a virus hepatitis C, que no respondieron a interferón y, en algunos casos, por no poder realizar transplantes hepáticos, fueron deshauciados. Prácticamente en todos estos pacientes, los títulos serológicos por MAT u otros

[学科分类] 传染病学