ESTATUS DE PAÍS LIBRE DE FIEBRE AFTOSA Julián Castro M. 2012

Consecución y mantenimiento del estatus de libre de fiebre aftosa Julián Castro Marrero Que un país consiga el estatus de libre de fiebre aftosa, con o sin vacunación, tiene importancia económica y política por la influencia que tiene en el comercio internacional de animales y sus productos, en especial de carnes y sus derivados. El mercado mundial demanda alimentos inocuos y procedentes de zonas exentas de enfermedades transmisibles de los animales, mientras la producción de alimentos se ve afectada por los programas y perfiles de sanidad animal de los países. Episodios críticos como los de fiebre aftosa, influenza aviar y encefalitis espongiforme bovina o enfermedad de las vacas locas acontecidos en diferentes regiones del mundo, dieron lugar, en su momento, a bajas en las ofertas y a colocar diferenciales de precios entre los diversos países. Por otra parte, las desmejoras del perfil sanitario de los rebaños trae consigo, concomitantemente, bajas en su productividad. En este escenario, la condición de haber logrado el estatus de libre de fiebre aftosa, con o sin vacunación, tiene importancia económica y política por influenciar en el comercio internacional de animales y sus productos, en especial de carnes y sus derivados. En el caso de la carne al incrementar la oferta en un mercado signado por barreras sanitarias, que lo limitan y afectan en el volumen del producto a negociar, como en el vaivén de los precios que se negocian. La Organización Mundial de Sanidad Animal (OIE) ha definido y establecido, con la participación de los países miembros, un procedimiento sobre bases científicas, transparente e imparcial, para el reconocimiento del estatus sanitario sobre fiebre aftosa, con las categorías de libre sin vacunación y libre con vacunación, para países o zonas. El reconocimiento oficial se realiza a través de una resolución adoptada por la Asamblea Mundial de Delegados de la OIE, que se realiza anualmente, en el mes de mayo. Los países miembros que tienen el estatus sanitario libre de la enfermedad deben presentar cada año, a finales de noviembre, un formulario de reconfirmación. La condición sanitaria de libre se podrá retirar, en caso de ocurrencia de la enfermedad, o sí a criterio de la Comisión Científica de la OIE el país o zona ya no cumplen con los requisitos establecidos en el código zoosanitario terrestre. Del mismo modo, esta puede decidir la restitución al presentarse una solicitud y comprobar que nuevamente se alcanzan las exigencias establecidas, en ambos casos el Director General de la OIE realiza la comunicación. Condiciones que debe cumplir un país ser oficialmente libre de fiebre aftosa Para la consecución y mantenimiento del estatus de libre de fiebre aftosa de una zona o país, el país debe disponer de unas condiciones mínimas necesarias, y de acuerdo a lo observado en el funcionamiento de los servicios de sanidad animal de las naciones, estas son algunas de ellas: Que exista voluntad política para erradicar la enfermedad y mantener un programa de prevención, y se contemple dentro de los planes y políticas de la nación esta intención. Que el país posea una organización técnico-administrativa que asegure buena cobertura de atención en campo y laboratorio, acorde con los objetivos y metas programáticas contempladas. Disponer de un sistema de vigilancia epidemiológica que, partiendo de la caracterización de los perfiles ganaderos y la industria animal, de los ecosistemas de la enfermedad, del modelo epidemiológico de las vías de la introducción del virus, de las fuentes de infección y difusión de la enfermedad, sirva para orientar las acciones y la toma de decisión oportuna. Que el tránsito externo e interno de animales de especies susceptibles a la fiebre aftosa sea perfectamente controlable. Disponer de un marco de legislación sanitaria y poder adecuarlo a los propósitos y a los cambios del programa. Mantener una monitorización que oriente acciones para ejercer un estricto control de la oferta del virus. Mantener un sistema periódico de auditoría del programa, ejecutada con una modalidad que permita la participación de agentes externos al servicio oficial. Utilizar la decisión política de erradicar para obtener respaldo a las decisiones técnico-científicas por encima de las de carácter político-económicas, en lo que concierne al programa. En un trabajo de PANAFTOSA, titulado Fundamentos básicos para la creación y mantenimiento de áreas libres de fiebre aftosa, en el que intervine con el Dr. Augé de Mello, se consideran fases del proceso de liberación y, por las experiencias recogidas en el desarrollo de los programas en América del Sur, se sugiere que el proceso se establezca en cuatro fases: Eliminar el endemismo o presencia ocasional del virus de la fiebre aftosa, limitando al máximo las condiciones que favorezcan su permanencia y difusión. Conseguida la ausencia de casos clínicos y pasado un periodo estimado de dos años, en una segunda fase se instrumentarán acciones para demostrar la ausencia de la infección en las especies susceptibles y se acrecentarán las acciones para prevenir que ocurran nuevos casos. Consolidación de mecanismos de integración nacional o subregional que garanticen la protección sanitaria del territorio comprometido, ya que la existencia de riesgo en las zonas limítrofes trae consigo el constante peligro de retrotraer situaciones superadas. Del mismo modo conviene ampliar cada vez más las fronteras del radio de acción del territorio libre. Declarar internamente el territorio libre, mientras se tramita ante el organismo de referencia el reconocimiento del estatus de libre de la enfermedad, con o sin vacunación, así como trabajar para mantener el estado de no infección.En esta coyuntura o momento programático cobran mayor valor las actividades siguientes: Incrementar controles activos para restringir el ingreso de animales susceptibles o productos según los riesgos determinados por región de origen, frecuencia y cantidades. Suspender la vacunación sistemática y masiva. Si las condiciones de riesgo evaluadas no garantizan esta posibilidad, se mantendrán los esquemas utilizados o se establecerá una vacunación selectiva en zonas y/o predios de mayor riesgo. Realizar muestreos serológicos orientados a monitorizar el estado inmunitario de la población animal sujeta de vacunación. Realizar muestreos serológicos orientados a conocer de la actividad viral existente. Mantener un sistema de vigilancia epidemiológica de alerta, basado en la caracterización del riesgo de introducción de la enfermedad. Ejecución continua y permanente de un programa de vigilancia epidemiológica, conjunta entre países, para las zonas fronterizas. Mantener mecanismos de vigilancia con la utilización de centinelas en áreas que potencialmente pudieran ser inicio de episodios o focos. Disponer de un plan de emergencia sanitaria utilizando conceptos de regionalización con zonas de contención, protección y vigilancia, y adiestrando al personal en su instrumentación, y dando a conocer a la comunidad de su existencia, propósito, actividades y forma de participar. La zona o país mantendrá en forma permanente la evaluación de los factores de peligro de una posible reintroducción del agente infeccioso, a través de las relaciones comerciales internacionales y/o el turismo, utilizando la metodología del análisis de riesgo. Controlar estrictamente la sanidad del sistema de ingreso de cargas, pasajeros, desechos de bordo de naves y aeronaves y vehículos que puedan traer el agente de fiebre aftosa al país o zona. Instrumentar de forma constante procedimientos de educación, divulgación y concientización destinados a ganaderos, industriales, profesionales del agro y la comunidad en general para crear hábitos acordes con el objeto de evitar la introducción de la enfermedad. Disponer de una legislación que permita facilidades de operación cónsonas con la condición sanitaria lograda. Mejora de la situación de la fiebre aftosa en Suramérica Desde mediados de la década de los noventa en Suramérica se registran importantes avances en el Plan Hemisférico de Erradicación de la Fiebre Aftosa, especialmente en el cono sur del continente, como consecuencia de tres variables introducidas en la estrategia sanitaria: La utilización masiva de la vacuna de adyuvante oleoso. La participación social. La instrumentación de proyectos subregionales. El esfuerzo logró impactos que se traducen en el reconocimiento de zonas y países libres. No obstante, se presentaron registros de reintroducción de la enfermedad en el 2001, 2003, 2005, 2006 y 2011, situaciones que fueron superadas y los países lograron recuperar su anterior estatus sanitario. Según los datos aportados por los países, en sus informes anuales a la Comisión Sudamericana de Lucha contra la Fiebre Aftosa, para su 38 reunión ordinaria en 2011, la situación sanitaria era la que se recoge en la siguiente tabla. Situación sanitaria Superficie km2 (%) Rebaños con bovinos y/o bubalinos (%) Total bovinos y/o bubalinos (%) Libre sin vacunación 3.808.129 (21,4) 854.912 (18,9) 11.694.110 (3,5) Libre con vacunación 8.743.526 (49,2) 2.662.945 (52,7) 272.851.766 (81,5) Zona tampón 88.190 (0,5) 16.869 (0,3) 479.199 (0,1) No libre 5.124.056 (28,8) 1.522.726 (30,1) 49.557.982 (14,8) TOTAL 17.663.901 (100) 5.057.452 (100) 334.583.057 (100) Situación sanitaria de fiebre aftosa de Sudamérica, año 2011, según Informes de los países a la 39ª COSALFA. (Elaboración: Unidad de Epidemiología, PANAFTOSA. Venezuela no se incluye por ausencia de información actualizada) Para el logro de esta situación ha habido grandes esfuerzos e inversiones del sector oficial y privado, en especial de los servicios veterinarios (públicos y privados) y las asociaciones de ganaderos. Mas la lectura del momento político-económico señala que existen en el sector pecuario involucrado exigencias técnico-científicas para que se mantenga este nivel de desarrollo. Y, sobre todo, se avance a otra etapa del proceso que acerque y consiga la definitiva erradicación de la enfermedad, y a su vez contenga operaciones de menor costo. Ocurre en estos procesos de largo alcance y duración que se corre el riesgo de que la maquinaria creada se desgaste en la motivación y se vea exigida financieramente para mantener las vacunaciones masivas y periódicas. De esta forma, se producen escenarios no deseables, en donde se pueden registrar asuntos de importancia, que de ocurrir causarían verdaderos problemas para los necesarios avances y su consolidación, como: Fallas en las inmunizaciones. Reacciones tardías en asuntos de importancia como el que se detecten en pruebas de campo (Elisa 3 ABC- EITB), en una zona reconocida como libre con vacunación, porcentajes positivos mayores al 20%, y no se de a conocer ninguna acción inmediata para analizar y corregir la situación detectada. Se conozca de la sospecha u ocurrencia de un episodio y no haya la pronta comunicación entre las partes involucradas y zonas circunvecinas. Cómo conseguir la erradicación completa Avanzar a otra etapa del proceso implica demostrar científicamente la ausencia de la circulación viral y sustituir la vacunación masiva por una estrategia de prevención. Esta ultima sustentada en una sensible vigilancia epidemiológica. Con mecanismos de alarma y detección precoz y capacidad de respuesta inmediata apoyada en un plan de emergencia subregional, caracterizado por su alta divulgación y total dotación de recursos. Ante un paso como el planteado en el párrafo anterior, y con las experiencias desarrolladas por las naciones en cooperación con las agencias internacionales, toman atención prioritaria líneas de acción como las siguientes: Perfeccionar la utilización de las zonas de alta vigilancia creadas por acuerdo entre la OIE y el comité veterinario permanente, donde en una franja de 15 kilómetros hacia el interior y a cada margen de la frontera de los países se activa una especial acción conjunta de catastros y seguimiento continuo del funcionamiento de los sistemas de producción y la situación epidemiológica. Mantener actualizada la caracterización de riesgo en las subregiones, categorizando el diferencial de riesgo de las zonas tanto al interior de los países como en las fronteras. Mantener actualizada la caracterización de tránsito de ganado interno a los países, en las fronteras y en las subregiones, a la luz de sus sistemas de producción, el comercio internacional y la situación epidemiológica existente. Así como mantener mecanismos que detecten cambios previsibles por avances o deterioros que la dinámica del proceso produzca. Revisar y perfeccionar los mecanismos de detección precoz de focos de infección o enfermedad clínica, los procedimientos de control de focos y adecuar los planes de emergencia para evitar la difusión de la enfermedad en caso de ser reintroducida y utilizar simulacros a manera de adiestramiento continuo. Complementar el uso de pruebas serológicas en las actividades de vigilancia epidemiológica activa con pruebas de aislamiento viral por Probang, en los casos con resultados sospechosos o positivos. Optimizar los mecanismos de vigilancia internacional, que reciban información inmediata sobre alarmas, la comuniquen a los países, garantizando transparencia de la información sobre la ocurrencia de la fiebre aftosa. Incrementar las acciones de bioseguridad, con un marco legal que permita actuaciones acordes al proceso de erradicación. Institucionalizar un grupo multinacional de atención de emergencias, consolidar su constitución y permanencia, con disposición de un documento guía para facilitar su operación. Se avanza en la dirección adecuada La consecución y mantenimiento del estatus de libre de fiebre aftosa tiene su real cimiento, bases de soporte y continuidad en sus servicios veterinarios, las federaciones de ganaderos, agroindustriales y transportistas comprometidos. Es el meollo del asunto. De ahí, que la mejor estrategia es invertir para que estos servicios sean cada vez más eficaces y los ganaderos más participativos, aunado esto a contar con una comunidad enterada de la importancia económica del asunto, para que coopere en esa dirección de la política de desarrollo, al ser el rubro carne un puntal de gran peso especifico en la economía. La eficacia de los servicios y la participación de los productores deben permitir disponer de acciones de vigilancia y/o inmunización tipo elite. Las metodologías existen y la inversión tiene una alta tasa de retorno, dada por aumentar la productividad y proveer acceso a mayores y mejores mercados. Claro, este planteamiento demanda mayor capacitación, concientización y cobertura de los servicios. Del mismo modo una mayor conciencia en el conglomerado del gremio ganadero y la comunidad en general. Hay un trabajo arduo que desarrollar para demostrar y dar a conocer que se transita, en lo económico y científico, por el camino correcto. Objeto de lograr la motivación que permita aminorar el desgaste de la maquinaria, y evitar que descuidos institucionales oficiales o privados den cabida a hechos que produzcan retrocesos en esa larga jornada hacia la erradicación. Jornada que para los gremios de productores, profesionales e industriales y agencias de cooperación internacional hasta ahora ha sido exitosa y de aportes importantes en las luchas sanitarias. Bibliografía consultada Castro, J. Estrategias sanitarias en base al análisis de riesgo. Portal Albéitar. Zaragoza. 2010. Castro, J. Integración geopolítica en las estrategias de sanidad animal. Veterinaria.org. Málaga. 2011. Castro, J. Promoción de salud publica veterinaria. Portal Albéitar. Zaragoza. 2012. Castro, J. Riesgo sanitario y la importación de animales y sus productos. Agrytec.com. Quito. 2012. Muzio, F. Perspectivas para la erradicación de la fiebre aftosa en el Cono Surde América. Rio grande do Sul. 2010. Organización Mundial de Sanidad Animal. Código Sanitario de Animales Terrestres. Paris. 2010. OCDE-FAO. Perspectivas agrícolas 2005-2014. Roma. 2005. Pan-aftosa. Fundamentos básicos para la creación y mantenimiento de áreas libres de fiebre aftosa. Rio de Janeiro. 2000. Pan-aftosa. Informe de situación de programas de fiebre aftosa. América del Sur. Rio de Janeiro.2002. Pan-aftosa. Informe de situación de programas de fiebre aftosa. América del Sur. Rio de Janeiro.2011. [ (Foto: Sxc.hu)]

BIG BANG

Cuando apenas eramos unos átomos: La Química del Universo Sabemos que vivimos en un planeta. Sabemos que este planeta gira alrededor de una estrella. Sabemos también que esta estrella, el Sol, gira alrededor del centro de la Vía Láctea, y que lo mismo hacen las miles de millones de estrellas que pueblan nuestra acogedora galaxia. Sabemos de que están hechas las estrellas, y que se formaron en un pasado muy muy lejano de gigantescas nubes de Hidrógeno que colapsaron para crearlas. Sabemos que estas nubes de hidrógeno se crearon de… Mmmm, ¿De qué se crearon estas nubes de hidrógeno? Ciertamente podemos darle una explicación racional y lógica a varios de los objetos que observamos en el cielo. Sin embargo, todo esto que observamos ¿¡de donde rayos salió!? ¿Apareció de pronto todo lo que observamos, como por arte de magia? ¿Dónde y cómo se creo todo lo que observamos? La respuesta hace 400 años probablemente incluía dentro de sus ecuaciones la intervención divina y no nos hacíamos este tipo de preguntas simplemente porque la respuesta a todo lo que conocíamos recibía el nombre de Dios. Todo lo que veíamos y observábamos en el cielo debía su existencia a las bondades divinas. Bien lo sabe Galileo, que fue obligado por la Iglesia Católica a retractarse de que la Tierra giraba en torno al Sol a cambio de ser quemado en la hoguera. El tiempo y el desarrollo de las ciencias sin embargo nos contaron otra historia. El problema de la creación del universo comenzó a ser explorado desde otras aristas, y después de una existencia entera como raza, hemos llegado a una explicación razonable a cómo y por qué vemos todo lo que vemos. Esta teoría tiene nombre y apellido, y se llama Teoría del Big Bang o Teoría de la Gran Explosión. Todos tienen como la visión cuando leen u oyen sobre el Big Bang de esta gigantesca explosión que de pronto estalló y que dio origen al Universo, y en realidad ocurrió más o menos de esa manera. Se han hecho observaciones y estas indican que en algún instante todo tuvo que haber comenzado con un evento de proporciones como el Big Bang. Tan solo unos instantes luego del Big Bang, mientras el Universo se expandía rápidamente desde un estado inimaginablemente denso, a temperaturas imposiblemente altas, algo maravilloso ocurrió. Luego de aproximadamente 3 minutos de ocurrida esta explosión comenzaron a formarse los primeros átomos. Lo mas sorprendente de todo es que la evidencia de que el universo comenzó como una gigantesca bola de fuego la podemos encontrar en cada uno de los átomos que componen las estrellas, planetas e incluso nosotros mismos. La Teoría Nucleosíntesis del Big Bang (BBN) es la rama de la astrofísica encargada de relacionar las abundancias químicas observadas con las predicciones teóricas de la Teoría del Big Bang y es uno de los pilares fundamentales de la cosmología moderna. De hecho estudiando solo las abundancias de los elementos más livianos como el Hidrógeno y el Helio, BBN entrega un cuadro detallado del origen cósmico de los átomos. Hasta el momento podemos distinguir más de 116 elementos químicos diferentes, cada uno con sus propias particularidades. El cobre, por ejemplo, es un metal y posee un color rojizo mientras que el oxígeno se encuentra presente en estado gaseoso y muchas veces se hace invisible a nuestra vista. La diferencia entre uno y otro reside principalmente en la cantidad de átomos que componen cada elemento químico de la tabla periódica. Gracias a los descubrimientos realizados durante los primeros decenios del siglo pasado, podemos conocer y comprender la realidad del átomo y su estructura interna. Cada átomo contiene un núcleo central que a su vez esta compuesto por uno o más protones con carga positiva. El Hidrógeno, el átomo más simple y abundante en el Universo, tiene tan solo un protón en su núcleo. De este modo es el número de protones en el núcleo lo que marca la diferencia entre un elemento y otro. Al interior del núcleo podemos encontrar también otra partícula, el Neutrón. El Neutrón es ligeramente mas pesado que el protón y carece de carga eléctrica. Tal y como lo indica su nombre, es eléctricamente neutro. La presencia o ausencia de neutrones en el núcleo es lo que hace la distinción entre variaciones de un mismo elemento. A estas variaciones en el número de neutrones les llamamos Isótopos. Un tercer tipo de partícula participa también de la estructura del átomo y recibe el nombre de Electrón. Este se encuentra fuera del núcleo orbitándolo, tal y como lo hace la Tierra con respecto al Sol. Esta partícula es mucho más pequeña y liviana que el protón y el neutrón, y posee carga eléctrica negativa. Esta descripción de la estructura interna de los átomos fue un gran acierto en el desarrollo de las ciencias naturales pues nos entrego información fundamental para entender y comprender la naturaleza de la materia. Fue durante un viaje en tren realizado el año 1937 que el físico Hans Bethe (1906 – 2005), mientras regresaba a su casa en Ithaca, Nueva York de una conferencia de física nuclear, dio las primeras luces al misterio de los interiores estelares. Basándose en las altas temperaturas y densidades inferidas por los astrónomos para los interiores estelares, Bethe demostró como es posible que elementos simples se junten para formar elementos de mayor complejidad, liberando energía en el proceso de fusión. Esto permitió comprender como la fusión nuclear sirve de combustible para una estrella y como los núcleos de estas son verdaderas fábricas de elementos químicos pesados. De esta manera podíamos entonces explicar la existencia de todos los elementos químicos y sus abundancias respectivas en el Universo. Se elaboró un modelo para estas abundancias basados en la nucleosíntesis estelar y se calculó teóricamente la abundancia química de los principales elementos presentes en el Universo. La teoría predecía abundancias de Hidrógeno y Helio en cantidades completamente diferentes a lo que se observaba. Las abundancias para átomos como Carbono, Oxígeno y Hierro predichas por la teoría eran observables en las estrellas, sin embargo había un gran problema pues la nucleosíntesis estelar entregaba un Universo con muy poquito Helio, cuando eso no era precisamente lo que observábamos. Aproximadamente un 24% de la materia del Universo se encuentra en forma de Helio molecular, menos de un 2% en elementos mas pesados que este y todo el resto corresponde a Hidrógeno molecular. La gran discrepancia entre teoría y observación causo muchos problemas pues nadie sabía de donde estaba el error y durante muchos años los astrónomos trabajaron duro para resolver esta aparente paradoja. No fue sino hasta el año 1948 que comenzaron a aparecer nuevas ideas sobre como resolver el problema de las abundancias químicas. Ralph Alpher, físico norteamericano, con la ayuda de George Gamow, físico ruso y reconocido ebrio, comenzaron a pensar en los mismos procesos de nucleosíntesis pero no ya en estrellas, sino en el comienzo de los tiempos cuando el Universo estaba recién comenzando su expansión. Gamow le preguntó a Alpher, que ocurriría en un Universo que comienza infinitamente pequeño, denso y caliente pero que se expande hasta enfriarse alcanzando el tamaño actual. Más específicamente, Gamow le pidió a su pupilo que trabajara en las reacciones nucleares que podrían ocurrir en los estados tempranos del Universo, cuando este era caliente y denso. Si bien los primeros cálculos no fueron del todo precisos, fueron los primeros pasos en la construcción de la teoría de Nucleosíntesis del Big Bang. Si bien esta teoría no fue bien aceptada los primeros años, a mediados de los 60s nuevos datos le dieron la razón a Alpher y Gamow, y la teoría del Big Bang y de como reaccionaban los átomos los primeros instantes fue finalmente aceptada socialmente. Cuando miramos en el cielo profundo vemos a las galaxias alejarse aceleradamente de nosotros. El premio Nobel de Ciencias del año recién pasado de hecho lo recibió un grupo de astrónomos que demostraron que nuestro Universo se encuentra en un estado de expansión acelerada. De modo que si damos vuelta el sentido en el que giran los relojes hacía el tiempo del Big Bang esta expansión se invierte, y el cosmos cada vez se comienza a volver más denso y caliente. Estructuras como estrellas, planetas y galaxias se derriten todas en una espesa sopa de gas primordial. Mas atrás en el tiempo aún, este gas primordial se descompone en un océano infinitamente caliente de protones, neutrones y otras partículas subatómicas. En este punto el Universo tiene una temperatura de miles de millones de grados Celsius y una densidad tal que una cucharadita de este Universo temprano pesaría más de 100.000 toneladas. Es aquí donde la Nucleosíntesis del Big Bang toma las riendas del asunto. Tenemos entonces un mar ultra denso y caliente de protones y neutrones y otras partículas, entonces ¿cómo se forman los átomos? Un núcleo de Hidrógeno está compuesto por 1 protón y el Helio tiene dos protones y dos neutrones. ¿Cómo es que se forma entonces desde un átomo de Hidrógeno uno de Helio? La fusión de Hidrógeno en Helio es una batalla campal entre la fuerza Electromagnética y la fuerza Nuclear Fuerte, dos de las fuerzas que gobiernan el comportamiento y la interacción entre las componentes del cosmos. La fuerza Electromagnética es de repulsión entre dos partículas que poseen la misma carga y de atracción si es que las cargas son opuestas. De este modo si queremos unir dos protones (ambos poseen la misma carga) necesitamos superar la fuerza electromagnética de repulsión. Por otro lado la fuerza Nuclear Fuerte es aquella que permite que los protones y los neutrones permanezcan unidos al interior del núcleo atómico, y esta actúa solo a distancias cortas. A gran escala la fuerza electromagnética manda y la fuerza Nuclear Fuerte es despreciable, sin embargo si acercamos el protón lo suficientemente a otro protón la fuerza EM se hace despreciable con respecto a la nuclear Fuerte, produciéndose la fusión entre los dos protones. Esto es exactamente lo que ocurrió los primeros instantes del Big Bang. En esta sopa de partículas elementales a alta temperatura y alta densidad, las partículas poseen la energía necesaria para chocar unas con otras superando la barrera electromagnética quedando fusionadas, formándose en el proceso los elementos químicos primordiales. Sin embargo el Universo se expandía y enfriaba rápidamente provocando la disminución de la densidad y temperatura, por lo que el proceso de fusión nuclear en el Universo temprano tenía sus segundos contados. Lo que la nucleosíntesis estelar no pudo explicar la nucleosíntesis del Big Bang hizo con lujo de detalles y con un nivel de precisión hermosamente alto. El modelo de BBN predecía el 24% de Helio molecular que observábamos, y lo que fue más hermoso aún, la precisión del modelo nos gritaba que efectivamente en algún momento en un pasado remoto efectivamente ocurrió un Big Bang. Todo aquello que no se pudo fusionar debido al enfriamiento por la expansión del Universo, quedo en forma de átomos del hidrógeno. Estos posteriormente formaron las primeras nubes de Hidrógeno, que sirvieron de material para la construcción de las primeras estrellas, que luego de formadas comenzaron a organizarse para formar las galaxias. Del material circundante a las estrellas se comenzaron luego a formar los planetas, y luego de unos cuantos miles de años, en estos planetas comenzaron a aparecer las primeras formas de vida, las cuales evolucionaron, desarrollaron su intelecto, y comenzaron a mirar al cielo buscando las respuestas a las preguntas que hoy por hoy nos estamos respondiendo.

Jean-Marie Camille Guérin

Jean-Marie Camille Guérin nació en Poitiers (Vienne, Francia) el 22 de diciembre de 1872. Su padre fue director de una empresa de obras públicas y murió de tuberculosis cuando Camille tenía diez años, en 1882. Su madre se volvió a casar más tarde con A. Venien, un veterinario de Châtellerault. Estudió en el Lycée Descartes de esta ciudad y en 1892 ingresó en la Escuela de Veterinaria de Maisons-Alfort. Allí permaneció hasta 1896, mostrando gran interés en el tema de las enfermedades infecciosas. Uno de sus profesores fue el veterinario y biólogo Edmond Nocard (1850-1903). Ese mismo año obtuvo, según los datos del Instituto Pasteur, el doctorado en veterinaria. En 1895, a consecuencia de una visita del Consejo municipal de Higiene de Lille, se confió a Calmette la misión de organizar un instituto de sueroterapia y de investigación microbiológica en la industriosa ciudad de Lille. El centro se inauguró en 1899 y fue nombrado primer director. Necesitaban a un veterinario y Guérin fue aceptado para el cargo. Participó en la producción de sueros antivenenosos y en la preparación de vacuna antivariólica. Utilizando el conejo como huesped intermediario para asegurar la perennidad de la actividad de las cepas, mejoró considerablemente la producción de esta última. En 1900 fue nombrado jefe de laboratorio del Instituto. Ese mismo año contrajo matrimonio con Marie Lavergne, con la que tuvo dos hijos. En 1905 puso a punto un método de control de las vacunas jennerianas (antivariólicas). Este trabajo fue recompensado con la medalla de oro de la comisión de la vacuna de la Academia de medicina. Entre 1905 y 1915 publicó con Calmette una conjunto de trabajos sobre el mecanismo de infección de la tuberculosis. Al principio la virulencia de las cepas usadas era tan grande que la inoculación de 3 mg a un ternero le producía la muerte a las cinco semanas. Mediante artificios de laboratorio trataron de volver avirulenta esta cepa. Después de numerosos pases en el medio de patata biliada glicerinada, comprobaron que los caracteres del bacilo no se modificaban más. Se trataba de un bacilo fijo, de virulencia conocida, inofensivo para los animales de laboratorio, aunque se inyectara a dosis considerables a los cobayas, tan sensibles a la tuberculosis, y a los conejos –sensibles al bacilo bovino– que confería una resistencia a los bóvidos contra la infección tuberculosa. Los trabajos quedaron interrumpidos a consecuencia de la invasión de la ciudad por los alemanes en 1915. Entre ese año y 1918, junto con el resto de componentes del Instituto, trató de proteger lo mejor posible a la población civil de Lille a pesar de que una buena parte del material científico fue destruido o robado. En 1918 su mujer murió de una meningitis tuberculosa. Guerin continuó con los trabajos sobre la vacunación antituberculosa. En 1919 fue nombrado jefe de servicio del Instituto Pastur de Lille, cargo que ocupó hasta e1928. En 1921, como hemos dicho, Calmette y Guérin llegaron a obtener una cepa de bacilos atenuados capaz de conferir la inmunidad. El pediatra Bernard B. Weil-Hallé (1875-1958) realizó la primera aplicación de la vacuna al suministrarla por vía oral a un recién nacido cuya madre había fallecido de tuberculosis. El niñó sobrevivió, lo que animó a Hallé y a Raymond Turpin a vacunar durante los tres años siguientes a 317 niños de la Maternidad de la Charité de Berlín. El éxito obtenido fue la causa de que el uso de la vacuna se extendiera tanto que, siete años después, ya se habían vacunado en Francia 116.000 niños, cifra que se elevó a 242.250 dos años más tarde. La primera comunicación oficial de Calmette y Guerin sobre el BCG se presentó el 29 de junio de 1924 en la Academia de Medicina de París y fue firmada por Calmette, Guerin, Weill-Halle, Turpin y Leger. Desde entonces las vacunaciones se sucedieron con rapidez. No obstante, la polémica sobre la eficacia de la vacuna BCG continuó; tuvo tantos defensores como detractores. El asunto de Lübeck (1930-32) significó un duro revés. Murieron más de sententa niños de un total de 230 vacunados, lo que fue achacado injustamente a la vacuna. En realidad ésta había sufrido una contaminación por bacilo tuberculoso virulento procedente del laboratorio de Bruno Lange, del Instituto Robert Koch; ambos se habían almacenado en la misma habitación. La luz se hizo al final de dieciséis largos meses. Se descubrió la verdad y los culpables de la negligencia fueron condenados. La comisión de la vacuna contra la viurela, de la sección de higiene de la Sociedad de Naciones, reunida en Berlín, adoptó el método de Guérin como método internacional de control de las vacunas jennerianas. En 1928 C. Guérin dejó Lille para hacerse cargo de la dirección del servicio de tuberculosis del Instituto Pasteur de París. En 1930 la Conferencia internacional contra la tuberculosis, reunida en Oslo, manifiestó su confianza plena en la BCG, a pesar del drama de Lübeck. En 1933 murió Calmette. Hasta 1934, más de 800.000 vacunas habían sido distribuidas en Francia y, a partir de 1950, la vacunación con BCG fue declarada como obligatoria en ese país. En 1935 Guérin fue elegido miembro de la Academia de medicina. Fue su presidente en 1951 y recibió el premio Boggio en 1907. En 1939 fue nombrado vicepresidente del Comité Nacional de Defensa contra la Tuberculosis (CNDT), siendo A. Honnorat presidente. Entre 1939 y 1945 vivió en el Instituto Pasteur, después de que su vivienda parisina fuera requisada por el ejército alemán. En 1945 fue miembro del Comité Nacional de Higiene Social (Ministerio de Sanidad Pública) y en 1948 dirigió el primer Congreso internacional sobre la BCG en París. Fue Presidente de la Academia de Veterinarios de Francia en 1949. En 1955 la Academia de las Ciencias le concedió el gran premio de investigación científica. El 9 de junio de 1961 murió en el Hospital Pasteur a la edad de 89 años, siendo enterrado en Châtellerault, junto a su esposa. José L. Fresquet. Profesor titular. Instituto de Historia de la Medicina y de la Ciencia (Universidad de Valencia - CSIC). Julio de 2004. Bibliografía —Báguena Cervellera, M.J. La tuberculosi i la seva història. Barcelona, Fundacó Uriach, 1992. —Hawgood, B.J. Doctor Albert Calmette 1863-1933: founder of antivenomous serotherapy and of antituberculous BCG vaccination, Toxicon 37 (1999), 1241-1258. —Repères chronologiques. Camille Guérin (1872-1961). Institut Pasteur. http://www.pasteur.fr/infosci/archives/gue0.html (Consultado en junio de 2004).

ANIMAL MODELS IN VACCINOLOGY

Animal Models in Vaccinology Coenraad F.M. Hendriksen CONTENTS Introduction ..................................................................................................................................... 1 Vaccine Development Technologies ................................................................................................ 3 First-Generation Vaccines....................................................................................................... 4 Second-Generation Vaccines .................................................................................................. 4 Third-Generation Vaccines ..................................................................................................... 4 Adjuvants ............................................................................................................................... 5 The Historical Role of Animal Models in Vaccinology.................................................................. 5 Characteristics of the Use of Laboratory Animals in Vaccinology ................................................ 7 Animal Models in Vaccinology ....................................................................................................... 8 Animal Models in Vaccine Development .............................................................................. 8 Animal Models in Vaccine Production ................................................................................ 10 Animal Models in Vaccine Quality Control ........................................................................ 11 Conclusion..................................................................................................................................... 13 References ..................................................................................................................................... 13 INTRODUCTION Vaccines belong to the category of immunobiologicals — products that are produced by or derived from a living organism. Immunobiologicals include a variety of products, such as vaccines, immunoglobulins, monoclonal antibodies, and antisera. The characteristic feature of vaccines is that these preparations are capable of inducing a specific and active immunity against an infecting agent or its toxin. 1 Vaccination is one of the most powerful and cost-effective tools in modern medicine. The worldwide immunization campaigns against a number of infectious diseases (e.g., diphtheria, tetanus, and measles) have led to substantial decreases in morbidity and mortality rates and, in the cases of smallpox and poliomyelitis, to complete and almost complete eradication, respectively. In the coming years, the importance of vaccines will continue to increase because of the emergence of antibiotic-resistant strains of bacteria such as Mycobacterium tuberculosis , the impact of new viral infections such as human immunodeficiency virus (HIV) and severe acute respiratory syndrome (SARS), increased biological warfare threats (e.g., smallpox and anthrax), 2 HANDBOOK OF LABORATORY ANIMAL SCIENCE global travel and tourism, the high incidence and economic effects of infectious diseases in large livestock industries, and various other factors. 2 Vaccination is not without controversy. Some groups oppose vaccination programs for religious reasons. Others adhere to the speculative concept that infant vaccination may stimulate allergic sensitization. It has been hypothesized that due to today’s comprehensive immunization program, children would be more prone to develop allergic diseases due to a shift of the Th1/Th2 balance in the immune system. 3,4 Although studies have been initiated to scientifically underpin this theory, no proof is yet available. The main reason that parents withhold immunizations from their children, however, is fear of vaccine-associated adverse effects. No vaccine is totally safe and totally effective, and adverse reactions have been reported with all vaccines, although products differ in the extent of their effects. 5 Because public acceptability of immunizing children is inversely related to the extent of adverse reactions, much attention has been given to improving existing vaccines. One of the best examples is the whooping cough (pertussis) whole cell vaccine. Due to campaigns in the U.K. tabloid press in the mid-1970s, which magnified the adverse reactions of whole cell pertussis immunization out of proportion, vaccine uptake dropped sharply from 80 to 30% in the U.K. and also decreased in other countries. Later studies showed that the claimed adverse reactions were highly exaggerated. 6 Table 1.1 summarizes the frequency of adverse reactions from postimmunization surveillance data as well as morbidity data from natural whooping cough infections. It can be seen from this table that whooping cough vaccination is highly cost effective. Nevertheless, public concern about adverse effects stimulated a renewed interest in basic research toward a safer product, 7 ultimately resulting in the first acellular pertussis (acP) vaccine, 8 which only includes the protective epitopes of the pertussis microorganism. The pertussis case is a good example of how public concern can affect the use of laboratory animals. Animal models played a crucial role in the development and screening of new pertussis vaccine candidates, and literally hundreds of thousands of animals were used in the development of the new product. A traditional link exists between laboratory animals and vaccines. As far back as the end of the 19th century, vaccine research provided a major impetus for the development of animal models. Some of the animal models currently used in routine vaccine quality control are in fact slight modifications of the tests developed by Emile von Behring or Paul Ehrlich in the 1890s. A close association between laboratory animals and vaccines still exists. Animals are particularly required for vaccine development and for quality control. Few animals are currently needed for vaccine production. This chapter focuses on the animal models used in vaccine research and testing. It provides information on technologies in vaccine development, on the historical context of animal models in vaccine research, and on characteristics of animal use in current vaccine development, production, and quality control. Table 1.1 Benefits of Vaccination against Natural Whooping Cough Incidence (No. per 100,000 Cases) Following Adverse Reactions Infection Vaccination Ratio of Infection/Vaccination Shock — 15 — Convulsions 4000 45 89 Encephalitis 2000 1.5 1300 Permanent brain damage 1300 0.3 4300 Death 2000 0.2 10,000 Source : Galazka, A.M., Lauer, B.A., Henderson, R.H., and Keja, J., Bull. WHO , 62, 357, 1984. ANIMAL MODELS IN VACCINOLOGY 3 VACCINE DEVELOPMENT TECHNOLOGIES The work of Edward Jenner on smallpox is generally considered to be the first scientific approach to vaccine development. After 25 years of study, Jenner published in 1798 the results of his successful experiment in which an 8-year-old boy, James Phipps, was inoculated with cowpox material and subsequently challenged with smallpox virus. Jenner’s study was entirely based on epidemiology and observation. Animal experiments did not contribute to it in any way. 10 It took almost 100 years before Louis Pasteur discovered several new vaccines, against fowl cholera (1880), anthrax (1881), and rabies (1885). All these vaccines were partly developed by trial and error without full understanding of the pathogenesis of the diseases. Nevertheless, Pasteur was the first to approach vaccine development in a systematic and coherent way. The virulent rabies virus, obtained from the saliva of infected dogs and humans, was attenuated in rabbits by multiple passages of the virus in cerebral and spinal cord tissue and finally by exposure to atmospheric oxygen. After Pasteur’s pioneering work, vaccination as a means of combating infectious diseases was taken up and extended by many researchers. In fact it was Pasteur who came up with the word “vaccination” as a tribute to Edward Jenner (the Latin word vacca means “cow”). The term “vaccinology,” introduced by Jonas Salk in 1977, can be defined as “the study and the application of the requirements for effective immunization,” meaning nothing other than the science of vaccines from A to Z, 11 thus including development, production, quality control, and research on vaccine-related issues such as adjuvants. Figure 1.1 shows the major vaccines for human use that have been developed since Jenner’s smallpox vaccine. They amount to a total of 21 products. Although the number is small, these products all have had tremendous impact on human health care. Several diseases with high morbidity and mortality rates in the 19th century, such as diphtheria and measles, are now almost unknown, * Meningococcal C * Hepatitis A * H. influenzae b * Hepatitis B * Pneumococcus * Meningococcus * Rubella * Mumps * Measles * Polio (Sabin) * Polio (Salk) * Yellow fever * Influenza * Pertussis * Cholera * Tuberculosis * Tetanus * Diphtheria * Typhoid 1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 * Diphtheria and Tetanus IgG * Smallpox * Rabies 1800 1900 Figure 1.1 History of vaccine development. (Only vaccines for human use are shown.) (Adapted from van der Zeist, B.A.M. and Lenstra, J.A., LAB/ACBC , 9, 18, 1988.) 4 HANDBOOK OF LABORATORY ANIMAL SCIENCE at least in Western countries, while smallpox has been eradicated and poliomyelitis is almost eradicated. 12 Roughly, vaccine development from the early days until now can be divided in three time periods, with each era characterized by specific production technologies. First-Generation Vaccines These conventional products consist of attenuated or inactivated (whole) microorganisms. Pasteur’s technique of attenuation was subsequently used for the production of several other vaccines, such as the oral poliomyelitis vaccine developed by Sabin in 1954 and the measles vaccine in 1958. Inactivated vaccines are produced by inactivating the whole microorganism or bacterial products (toxins) using heat or chemicals such as formaldehyde. Examples of inactivated vaccines are the whole cell pertussis vaccine, tetanus toxoid, and the inactivated poliomyelitis vaccine developed by Salk in 1953. Bacterial microorganisms have been grown on culture media, and viruses have been cultured in laboratory animals (e.g., rabies virus in rabbit or suckling mouse brains), in embryonated eggs, and after the 1950s also in cell cultures. In addition, immune response to vaccine antigens is enhanced by addition of an adjuvant product, generally aluminum salts, AlPO 4 and Al(OH) 3 . Conventional vaccines are characterized by high complexity in composition and structure. As a consequence, emphasis has to be given to extensive batch-related quality control. Vaccine batches are produced in volumes of up to 1000 L. Second-Generation Vaccines A rational approach to vaccine development started in the late 1950s. The approach was based on the subunit principle — the isolation and purification of only those antigenic components (antigenic epitopes) of the microorganism that underlie the protective immune response. Examples of subunit viral vaccines are the influenza vaccines based on surface protein and a subviral hepatitis B vaccine. A breakthrough in subunit vaccine development only occurred after the introduction of hybridoma technology to produce monoclonal antibodies, 14 as these antibodies allowed selection and identification of protective epitopes. The acellular pertussis (acP) vaccine, including one or multiple antigenic components of the pertussis microorganism, 8 is an example of a bacterial subunit vaccine, now used in most Western countries. A group of products of particular interest in this context is the bacterial polysaccharide vaccines, derived from capsular polysaccharides of Grampositive or Gram-negative bacteria. Polysaccharides are poorly immunogenic — a problem that can only be overcome by chemical linkage to proteins. Examples of polysaccharide vaccines available are those against Neisseria meningitides, Haemophilus influenzae, and Streptococcus pneumoniae. Third-Generation Vaccines Advances in biotechnology and concomitant developments in the fields of molecular immunology and protein chemistry opened the door to new vaccine production strategies in the 1980s. Recombinant DNA (rDNA) technology allowed for the insertion of one or more genes from microorganisms encoding immunologically relevant proteins into host systems such as viruses (e.g., vaccinia and adenovirus), yeast, or animal cells. Thus, expression of these genes by the vectors induces immunity after administration. An example of a licensed rDNA product is the yeast- and Chinese hamster ovary (CHO)-derived recombinant hepatitis B (rHBsAg) vaccine. However, most experimentally produced rDNA (carrier) vaccines still have not passed official registration. Another approach to rDNA vaccines involves deleting those genes that encode for pathogenicity. One such product has been produced for Aujeszky disease. 15 Another example is the double mutant acP vaccine. 16 ANIMAL MODELS IN VACCINOLOGY 5 Advances in biochemistry have increased interest in polypeptide vaccines produced by chemical synthesis. The appeal of this approach is that it permits the manufacture of chemically well-defined products on an industrial scale. However, it has meanwhile become clear that, though the required antigenic peptides can be synthesized, it is extremely difficult to obtain the spatial configuration (glycosylation) of the peptide chains necessary for immunogenicity. To date, not one peptide vaccine has reached the stage of clinical trials. Finally, the use of plasmid expressing recombinant genes of pathogens encoding for immune responses has become the basis of the DNA vaccine strategy. Intramuscular injection of the plasmid results in uptake by muscle cells, expression of the recombinant gene, and the elicitation of immunity against the protein product of the pathogen. 17 DNA vaccination has been studied for a long list of pathogens such as influenza, rabies, and tuberculosis, but few products have entered clinical trials yet. 18 Adjuvants An integral part of vaccinology is research on adjuvant products. Adjuvants are chemicals or biomolecules which, when given in conjunction with a vaccine, enhance and/or modify immune responses. Currently, no adjuvant products exist that are absolutely safe. Consequently, safety and adjuvanticity must be balanced between obtaining maximum immune stimulation and minimum adverse effects. 5 Since early vaccine development, research has been performed to develop effective formulations. However, the only adjuvants approved by regulatory authorities for use in humans include aluminium phosphate and aluminum hydroxide. As side effects are somewhat less a problem in the livestock industry, the range of products allowed for use in the veterinary clinic is much larger. New vaccine production strategies have resulted in more defined but smaller antigenic structures such as subunits and peptides. These products are less immunogenic compared to whole cell vaccines. In addition, some new vaccines require modulation of immune response toward cellmediated immunity (CMI). Unfortunately, aluminum “alum” is a weak adjuvant and stimulates mostly humoral immunity. Therefore, significant efforts are now underway to develop new generations of adjuvants. Promising new products, such as muramyl-dipeptide (MDP) or immunostimulatory complexes (ISCOM), not only induce strong antibody responses but also induce more balanced Th1/Th2 responses; they therefore might be useful for vaccines that require responses of the Th1 (CMI) type. 19 Furthermore, an urgent need now exists for adjuvants that support new vaccine administration strategies, such as oral vaccines. THE HISTORICAL ROLE OF ANIMAL MODELS IN VACCINOLOGY In 1884, the German microbiologist Robert Koch published his book Die Aethologie und die Bekämpfung der Tuberkulose , which elaborated on the criteria that should be met in relating a microorganism to a given infection. These criteria, also known as Koch’s postulates, have been of historical importance regarding the role of animal models in the study of infectious diseases. Apart from the isolation of the microorganism in pure culture, the postulates stated that these pure cultures, following the introduction into a suitable animal model, should result in the typical clinical signs of the disease in the laboratory animal. Koch’s postulates gained general acceptance in microbiology and thus in vaccine development. When it was difficult to find a suitable animal model, as was the case for poliomyelitis, progress in vaccine development stagnated. By contrast, rapid results were obtained in research on diphtheria and tetanus when it was found that animal models were easily available. 20 As an example, a historical overview is given of the use of animal models in the production and quality control of diphtheria therapy and prophylaxis. In the 1800s, diphtheria was a highly contagious disease in young children with a mortality rate of up to around 40%. The 6 HANDBOOK OF LABORATORY ANIMAL SCIENCE disease was also known as the “strangling angel of children”; infected children suffocated due to a pseudomembranous inflammation of the pharynx. For a number of reasons diphtheria makes an interesting case. It nicely shows the crucial contribution animal models made to a major breakthrough in medical history (Table 1.2). Further, it also played a crucial role in the heated discussions about the moral status of animal experimentation that took place at the end of the 19 th century. Diphtheria antitoxin and vaccine completely changed the previously hopeless treatment of a child with diphtheria. Shortly after the introduction of diphtheria antiserum, morbidity and mortality rates declined significantly. The fact that the use of animal models had made the treatment of this feared disease in young children possible convinced many of the value of this type of experimentation and undermined the criticism leveled at it. As a result, the animal welfare movement lost power for several decades.20 From the end of the 19th century, the animal model played an important role in vaccinology, first in gaining insight into the etiology and course of disease, second in the development of prevention and, in the case of antisera, of treatment, and finally, in the development of quality control of these preparations. As an example, an overview is given of the animal model in the development of diphtheria antitoxin and vaccine quality control (Table 1.3). In fact, Koch’s postulates still do apply to today’s vaccine development, and the role of the animal model remains indisputable. However, new technologies have been or are about to be introduced that will modify the role of the animal model. Information will be provided in the following sections, but first statistics and some characteristics of the use of laboratory animals in vaccinology will be presented. Table 1.2 Animal Models in the Development of Diphtheria Treatment and Prevention Development Year Scientist Animal Speciesa Isolation of the causal microorganism Corynebacterium diphtheriae 1884 Loeffler Pigeon, chicken, rabbit, guinea pig Production of the exotoxin 1884 Roux and Yersin Various animal species, guinea pig Demonstration of the therapeutic value of antitoxin 1890 Behring and Kitasato Guinea pig, dog, mouse, rat, various animal species Large-scale production of antitoxin 1894 Roux and Martin Dog, sheep, goat, cow, horse Toxin-antitoxin mixtures for active immunization 1913 Behring Guinea pig Diphtheria toxoid (vaccine) 1923 Ramon Various animal species aThe animal species finally chosen is in bold type. Source: Adapted from Hendriksen, C.F.M., in Replacement, Reduction, and Refinement of Animal Experiments in the Development and Control of Biological Products, Brown, F., Cussler, K., and Hendriksen, C., Eds., Developments in Biological Standardization 86, Karger, Basel, 1996, 3. Table 1.3 Animal Models in the Quality Control of Diphtheria Antitoxin and Vaccine Development Year Scientist Animal Species Quality control of antitoxin 1892 Behring and Wernicke Guinea pig Introduction of a standard preparation in potency testing 1897 Ehrlich Guinea pig Potency test using parallel-line bioassay 1937 Prigge Guinea pig Multiple intradermal challenge test 1974 Knight Guinea pig Serological potency test 1985 Kreeftenberg Mouse Source: Adapted from Hendriksen, C.F.M., in Replacement, Reduction, and Refinement of Animal Experiments in the Development and Control of Biological Products, Brown, F., Cussler, K., and Hendriksen, C., Eds., Developments in Biological Standardization 86, Karger, Basel, 1996, 3. ANIMAL MODELS IN VACCINOLOGY 7 CHARACTERISTICS OF THE USE OF LABORATORY ANIMALS IN VACCINOLOGY Few specific data are available on the use of laboratory animals in vaccinology. In the Netherlands, yearly statistics on the use of animals in biomedical research and testing have included a specific question on biologicals. Figure 1.2, which specifies the purposes of animal use in the Netherlands in 2002, shows that the category of biologicals accounted for 22.7% of total use. Although the use of animals for biologicals has been reduced in the last 20 years, the number has increased as a relative percentage of total use for biomedical research (Figure 1.3). Figure 1.4 further specifies the category of biologicals. Since this information is not included in the official Dutch statistics, the data given are percentages and are based on personal inquiry. As can be seen, vaccine development and vaccine quality control are particularly animal demanding. Although no data are available, it is assumed that the percentage of animal use for biologicals as compared to total animal use will be about the same in other European countries as well as in the U.S. In nonindustrialized countries, the percentage might even be higher as vaccine production and quality control quite often are major areas of biomedical research in those countries. Some other characteristics of the use of animals for the category of biologicals are given in Table 1.4, which shows that many of the animals are required for regulatory purposes — the registration of a new product and routine batch release testing. Furthermore, animal use is also characterized by a high level of pain and suffering. Particular, vaccine potency testing is based on animal models of a general design dating back to Behring and Ehrlich and often includes an immunization–challenge procedure. Figure 1.2 Purposes of use of laboratory animals in the Netherlands and percentage of total use. (From Zo doende 1978–2002, Annual statistics on the use of laboratory animals in the Netherlands, Keuringsdienst van Waren (KvW), The Hague, 2002.) Figure 1.3 Use of laboratory animals in the Netherlands in the period between 1978 and 2002: total use and use for the purpose of biologicals. (From Zo doende 1978–2002, Annual statistics on the use of laboratory animals in the Netherlands, Keuringsdienst van Waren (KvW), The Hague, 2002.) Fundamental Biologicals Education Diagnostic Pharmaceuticals Toxicity testing 0 200 400 600 800 1000 1200 1400 1600 1978 1980 1984 1988 1992 1996 2000 Year No. animals × 1000 Total Biologicals 8 HANDBOOK OF LABORATORY ANIMAL SCIENCE ANIMAL MODELS IN VACCINOLOGY Laboratory animals are still essential in vaccinology for vaccine development, vaccine production, and finally for vaccine batch release. Although in vitro methods are used and their contribution to vaccinology is becoming more important, many scientific questions still require an integrated immune system and consequently, an intact animal. A broad range of animal models is used, depending on the type of vaccine, the target animal species, and whether animals are used in development activities, in production, or in quality control. A general outline of these models will be given in the following paragraphs. Animal Models in Vaccine Development The first step in vaccine development is the establishment of an infection model to study pathogenesis. Aspects that are dealt with include route of infection, target organs, incubation time, virulence, and clinical disease progress. Well-characterized and relevant infection models are decisive for the success of further studies. For veterinary vaccines, it will be clear that the target animal species is the model of choice, unless there are reasons (e.g., cost or availability) to establish a laboratory animal model. For human vaccines, there is a need for specific laboratory animal models. History shows that when it was difficult to find a suitable animal model, progress stagnated. For instance, this was the case for poliomyelitis vaccine development. Although monkeys could be infected by intracerebral inoculation, for a number of reasons this appeared not to be a particularly suitable experimental animal for this kind of research. Figure 1.4 Use of laboratory animals for the category of biologicals: specification of purposes. The 0.1% of laboratory animals used for vaccine production is too small to indicate in the diagram. Animals are no longer used for monoclonal production. Table 1.4 Characteristics of Use of Animals for the Category of Biologicals and for Total Animal Usea Characteristic Percentage for the Category of Biologicals Percentage for Total Animal Use (All Categories) Percentage of use of animals 23 100 Use of animals for regulatory purposes 69 28 Substantial pain and suffering 21 13 No pain relief 1 5 a Biologicals include antisera, immunoglobulins, hormones, vaccines, blood products, cytokines, etc. Source: Zo doende 2002. Annual statistics on the use of laboratory animals in the Netherlands. Keuringsdienst van Waren (KvW), The Hague, 2002. Vaccine dev. (10%) Vaccine q.c. (49%) Hormones (25%) Polyclonal antibodies (5%) Blood products (5%) Others (5%) ANIMAL MODELS IN VACCINOLOGY 9 Recent examples of infection models to study vaccine development are those for emerging infections such as HIV and SARS. Several papers have discussed the use of a chimpanzee model for HIV research (e.g., reference 22). However, apart from economical and ethical constraints, the model only partly reproduced clinical progress as seen in humans. Another approach focused on studying animal viruses analogous to HIV, particularly the lentiviruses that induce acquired immunodeficiency syndrome (AIDS)-like illness in animals — SIV (simian immunodeficiency virus) infections in cynomolgus monkeys23 and FIV (feline immunodeficiency virus) infection in cats.24 Thus information could be obtained about genetic diversity, infection characteristics, etc. More recent is research on SARS. To date, infection models have been described using monkeys (cynomolgus macaques),25 cats, and ferrets.26 Having an infection model also offers a way to upscale virus production or to attenuate the virus by serial passage. Vaccine development generally starts with obtaining information about immunogenicity. Depending on the type of vaccine, microorganisms will be attenuated/inactivated as is the case for conventional vaccines, or studies will start to identify and select the antigenic structures (epitopes) that are relevant for immunogenicity and could be used as (subunit) vaccine leads. Part of this work will be done in vitro, but particularly information on complex immunological processes such as type of immune response (humoral and/or cytotoxic T-cell), antibody classes produced, duration of immune response, protective activity, and antigenic stability often requires the use of laboratory animals. In the case of conjugate vaccines, the effect on immune response due to protein linkage has to be studied, and in the case of vector vaccines, the best vector has to be identified and characterized. Additionally, adjuvants have to be identified that are optimal in enhancing and/or modifying immune responses. Thus, animal studies are performed to study antigen–adjuvant interactions and to select an adjuvant that is potent and safe. Another focus of study is safety aspects. In the case of conventional vaccines, information has to be provided on loss of virulence, thermal stability, and antigenic stability of attenuated vaccines and on protocols for effective inactivation of inactivated vaccines. In the last few decades safety requirements have increased in diversity, particularly with the introduction of new production technologies. For instance, the safety requirements for registration of rDNA vaccines specify studies on local and systemic toxicity including histopathological effects, virulence of the vector, stability of the integrated sequence, transmission from vaccinated to nonvaccinated animals, reversion of virulence, hypersensitivity, and drug interactions. Apart from the development of new vaccines, continuing efforts are made to improve existing vaccines, generally to reduce adverse effects, such as is the case for the pertussis vaccine, but also to optimize vaccine efficacy, as for tuberculosis and measles, or to overcome problems of antigenic variation (influenza vaccine).19 Another area of research is aimed toward the development of combined vaccines. The number of products in pediatric immunization programs has increased significantly in the last few decades and will further increase in the near future. As the acceptance rate for immunization is inversely correlated with the number of injections, there is a need to combine vaccines and to limit the number of injections. Thus, studies focus on the interaction of the various vaccine components as well as on new administration approaches such as prefilled ampules and gene gun “injection.” Early-phase vaccine development is based on fundamental research. Study protocols, although fulfilling the conditions of Good Laboratory Practice (GLP) and Good Manufacturing Practice (GMP), are not laid down in formal procedures. However, toward the end of development, studies, in particular those on immunogenicity and safety, become part of the registration dossier for obtaining the product license. Registration dossiers for product licenses are approved in Europe by the European Agency for the Evaluation of Medicinal Products (EMEA) and in the U.S. by the Food and Drug Administration (FDA) or the U.S. Department of Agriculture (USDA). Generally, registration is indicative of the animal models that will be specified for batch release testing. 10 HANDBOOK OF LABORATORY ANIMAL SCIENCE Animal Models in Vaccine Production Laboratory animal use in vaccine production is limited in numbers. Bacterial microorganisms are grown in culture media that might require blood products or beef extract. Due to the BSE (bovine spongiform encephalopathy) crisis, however, there now is a strong tendency to use synthetic media only, without any animal products. Virus production needs a cell substrate for propagation. Until the introduction of technologies that allowed for large-scale cell culture, viruses had to be grown in living animals (rodents or nonhuman primates) or in embryonated eggs. For example, the rabies vaccine was produced in brains of suckling mice until cell culture–produced vaccines became available in the seventies.27 Now, rabies vaccines are generally produced in cell culture, but in vivo production is still used, particularly in developing countries, because these vaccines are cheap and easy to produce, despite the safety disadvantages of animal-produced vaccines. Only a few viral vaccines are still based on ex vivo production. One of these is the polio vaccine. Some manufacturers still rely on virus propagation in primary or subcultured monkey kidney cells, although the virus can also be produced in continuous cell lines (CCLs) such as Vero cells or human diploid cells. Table 1.6 presents an overview of cell culture and non–cell culture virus vaccines. Apart from the greater safety of vaccines produced in CCLs, the use of CCL in the manufacture of viral vaccines has other advantages: 1. The cell culture substrate is more consistent and “clean” than primary culture. 2. The use of large-scale tissue culture procedures allows more efficient and more reproducible cell growth. 3. It provides higher yields of virus. 4. It could reduce or even preclude the use of animals.29 Despite these advantages, the acceptability of CCL as a substrate in the production of viral vaccines has been controversial due to the suspected oncogenic potential of CCLs — that is, the potential to induce cancer in humans after vaccine administration. However, it has now been well established that DNA obtained from these cells has no detectable activity in vivo.30 The use of primary cell cultures has continued since then because vaccine safety is not only a scientific issue but a political issue as well. However, there is reason to believe that virus propagation in CCLs will gradually but completely replace the use of animals. Table 1.6 Cell Culture and Non–Cell Culture Virus Vaccines Cell Culture Virus Vaccines Non–Cell Culture Virus Vaccines Polio Smallpox (vaccinia): bovine, lymph, skin Measles Rabies: various brain or spinal cords (rabbit, sheep, goat, mouse), duck embryo Mumps Japanese encephalitis: mouse brain Rubella Yellow fever: mouse brain, hen’s egg Adenovirus Influenza: chick embryo allantoic fluid Rabies Varicella Hepatitis A Rotavirus Cytomegalovirus Source: Katz, S.L., Wilfert, C.M., and Robbins, F.C., in Vaccinia, Vaccination, Vaccinology. Jenner, Pasteur and Their Successors, Plotkin, S.A. and Fantini, B., Eds, Elsevier, Paris, 1986, 213. ANIMAL MODELS IN VACCINOLOGY 11 Animal Models in Vaccine Quality Control Central to the vaccine batch release process is quality control. In particular, conventionally produced vaccines have a tight testing program because these products are complex mixtures that might differ from batch to batch. New-generation vaccines can be produced more consistently, and consequently quality control, and thus the use of laboratory animals, is less extensive. Quality control takes place within a regulatory framework, and guidelines for quality control tests are laid down by (inter)national regulatory bodies such as FDA and USDA for the U.S. and the European Pharmacopoeia for the Member States of the Council of Europe. Broadly, controls are directed almost entirely towards the safety or the lack of toxicity and the efficacy or potency of the vaccine. Control on safety ensures that the vaccine does not contain ingredients that are harmful to humans or animals after administration. The harm can be traceable to the agent (the bacterial or viral strain or toxin), to chemicals added (intentionally or unintentionally), or to the substrate used (culture media, culture cells, or embryonated hen’s eggs). Table 1.7 specifies the tests that, depending on the type of vaccine, can be part of the safety-testing scheme. Next to safety testing, by far the greatest effort in quality control goes into potency testing to demonstrate that the vaccine induces protective immunity after its administration. In the case of live vaccines (e.g., mumps, oral polio, bacille Calmette-Guérin [BCG]), the efficacy of each vaccine batch is related to the number of live particles, determined either by counting or by titration, that is, entirely in vitro. Potency testing of inactivated vaccines, however, generally requires the use of experimental animals. The tests often rely on a limited number of basic principles, quite often relating to procedures already established in the early days of vaccinology. One of the approaches (the “parallel-line” potency test) is to immunize groups of animals with serial dilutions (three or four) of the vaccine under study and a reference preparation with known potency, respectively. Generally, these ranges of dilutions include groups of animals receiving a low, an intermediate, and a high vaccine dose. After a number of weeks, the animals are challenged with the virulent microorganism or toxin, and specific clinical signs or death is recorded for the observation period given. Based on the information obtained from the various groups, a dose–response curve can be plotted, both for the vaccine under study and for the reference preparation, and the dose that protects Table 1.7 Safety Tests in Quality Control of Vaccines and Animal Models Used Safety Aspect Specific Safety Test Animal Model Used The agent Specific toxicity test (bacterial vaccines)a Mice, guinea pigs Identity test (all) (Neuro)virulence test (live vaccines)a Monkeys (intracerebral and intraspinal) Test for residual live virusa (live vaccines) Various animal species Test for endotoxina Rabbits Vaccine additives Abnormal toxicity (= innocuity) testa (all) Mice and guinea pigs Target animal safety test (veterinary vaccines)a Target animals Thimersal content Sterility test (all) Test for endotoxin levelsa Rabbits Chemical tests to characterize components pH (all) Substrate used Tests for extraneous microorganisms* (live) Various animal species, e.g., suckling mice, chickens Test for tumorigenicity Mice a Tests that usually are based on animal models. Source: Adapted from Hendriksen, C.F.M., Laboratory Animals in Vaccine Production and Control. Replacement, Reduction, and Refinement, Kluwer Academic, Dordrecht, the Netherlands, 1988. 12 HANDBOOK OF LABORATORY ANIMAL SCIENCE 50% of the animals is calculated. An alternative strategy for toxoid vaccines is to immunize one group of animals with the vaccine under study, to bleed these animals after a number of weeks, and to estimate levels of protective antibodies by titration of serum samples mixed with fixed doses of toxin in groups of animals. Both approaches require large numbers of animals (e.g., 140 for the parallel-line potency test) and induce substantial levels of suffering. Attention is now being given to the development of methods that could replace, reduce, and refine (the 3Rs) the use of laboratory animals. The reasons for this trend include: • Concern about the extensive use and the substantial levels of pain and distress inflicted on the animals • The questionable relevance of some animal models, such as the rabies vaccine and whole cell pertussis vaccine potency tests, which show very poor reproducibility • The fact that animal tests are time consuming and interfere with the limited shelf life of vaccines • New developments and strategies in vaccine production such as standardization of production processes and the introduction of GMP, Quality Assurance (QA), and in-process control that make extensive quality control less relevant and even superfluous. Extensive overviews of 3R developments have been published.31–33 A summary of some of the achievements is given in Table 1.8. Although these developments had an impact on the numbers of animals used, a breakthrough can only take place if the concept of “demonstration of consistency” is generally accepted. The key issue of consistency has emerged from the new generation of vaccines. These vaccines, which are based on new technologies, are produced in a consistent way and the stress of quality control is on in-process monitoring rather than on final batch testing. In-process testing is almost exclusively based on in vitro biochemical and physicochemical tests. The consistency concept has become state of the art for the new generations of vaccines. Also, in the field of conventional vaccines, continued advances in production technology have resulted in more defined and thus less variable products. This, together with the implementation of GMP and QA, makes people feel that for a conventionally produced vaccine, the extent of batch release testing should reflect the level of consistency obtained with the vaccine. Thus, a vaccine manufacturer should perform extensive testing (including animal testing) during the development phase and on the first few batches of the new product to characterize the vaccine thoroughly. However, if consistency in production is demonstrated, then testing could rely on a battery of easy-to-use in vitro assays to characterize (fingerprint) the vaccine and confirm consistency. If this new approach is applied, the number of animals used for quality control of conventional vaccines will be reduced to an absolute minimum. Table 1.8 Summary of Major 3R Developments in Vaccine Quality Control Vaccine Animal Test 3R Alternative Toxoids Potency test based on challenge procedure Serological-based potency test Erysipelas Potency test based on challenge procedure Serological-based potency test All Abnormal toxicity test Deleted from test specificationsa Hepatitis B Mouse potency test In vitro method (ELISA) Polio (live) Neurovirulence test in monkeys MAPREC assay and transgenic mouse test Relevant vaccines Lethal challenge procedure Use of humane endpoints Note: ELISA = enzyme-linked immunosorbent assay; MAPREC = mutant analysis by polymerase chain reaction (PCR) and restriction enzyme cleavage. a Deleted only from the European Pharmacopoeia monographs but still specified by other regulatory bodies. ANIMAL MODELS IN VACCINOLOGY 13 CONCLUSION Animal models have played and still play an essential role in vaccinology. They have made possible the worldwide immunization of children with pediatric vaccines that are both safe and efficacious. Until recently, laboratory animals were required for development, production (in case of viral vaccines), and particularly, for quality control. This resulted in the use of large numbers of animals for these purposes, often in models that induced severe pain and suffering. Changes are now taking place that will affect this situation. Newer generations of vaccines are more defined and can be produced more consistently than the conventionally produced vaccines were. As a consequence, a shift in the need for laboratory animals will take place. The burden of animal research with the newer generation of vaccines will be on development, while for the conventional vaccines it was on routine batch quality control. Therefore, it can be anticipated that the numbers of animals needed will be reduced in the near future. However, limited numbers will still be needed to evaluate the interaction of the vaccine with the complex immune system in an intact organism. REFERENCES 1. European Pharmacopoeia, Vaccines for human use, Pharmeuropa, 15, 3, 449, 2003. 2. Hendriksen, C., Spieser, J.-M., Akkermans, A., Balls, M., Bruckner, L., Cussler, K., Daas, A., Descamps, J., Dobbelaer, R., Fentem, J., Halder, M., van der Kamp, M., Lucken, R., Milstien, J., Sesardic, D., Straughan, D., and Valadares, A., Validation of alternative methods for the potency testing of vaccines, ATLA, 26, 747, 1998. 3. Holt, P.G., Rowe, J., Loh, R., and Sly, P.D., Development of factors associated with risk for atopic disease: implications for vaccine strategies in early childhood, Vaccine, 21, 3432, 2003. 4. Uter, W., Stock, C., Pfahlberg, A., Guillen-Grima, F., Aguinaga-Ontoso, I., Brun-Sandiumenge, C., and Kramer, A., Association between infections and signs and symptoms of “atopic” hypersensitivity — results of a cross-sectional survey among first-year university students in Germany and Spain, Allergy, 58, 580, 2003. 5. Bussiere, J.L., McCormick, G.C., and Green, J.D., Preclinical safety assessment considerations in vaccine development, in The Subunit and Adjuvant Approach, Powell, M.F. and Newman, M.J., Eds., Plenum Press, New York, 1995, 61. 6. Nicoll, A., Contraindications to whooping cough immunization — myths or realities? Lancet, i, 679, 1985. 7. Granström, M., The history of pertussis vaccination: from whole-cell to subunit vaccines, in Vaccinia, Vaccination, Vaccinology. Jenner, Pasteur and Their Succesors, Plotkin, S.A. and Fantini, B., Eds., Elsevier, Paris, 1996, 107. 8. Sato, Y., Kimura, M., and Fukumi, H., Development of a pertussis component vaccine in Japan, Lancet, i, 122–126, 1984. 9. Galazka, A.M., Lauer, B.A., Henderson, R.H., and Keja, J., Indications and contraindications for vaccines used in the Expanded Programme on Immunization, Bull. WHO, 62, 357, 1984. 10. Parish, H.J., A History of Immunization, E & S Livingstone, London, 1965. 11. Moulin, A.-M., Philosophy of vaccinology, in Vaccinia, Vaccination and Vaccinology: Jenner, Pasteur and Their Successors, Plotkin, S. and Fantini, B., Eds., Elsevier, Paris, 1996, 17. 12. WHO, State of the World’s Vaccines and Immunization, revised ed., World Health Organization, Geneva, 2003, 111. 13. van der Zeijst, B.A.M. and Lenstra, J.A., Vaccinology, LAB/ABC, 9, 18, 1988. 14. Köhler, G. and Milstein, C., Continuous cultures of fused cells secreting antibody of predefined specificity, Nature, 256, 495, 1975. 15. Quint, W., Gielkens, A., Van Oirschot, J., Berns, A., and Cuypers, H.T., Construction and characterization of deletion mutants of pseudo-rabies virus: a new generation of “live” vaccines, J. Gen. Virol., 68, 523, 1987. 14 HANDBOOK OF LABORATORY ANIMAL SCIENCE 16. Rappuoli, R., Podda, A., and Pizza, M., Progress towards the development of new vaccines against whooping cough, Vaccine, 10, 1027, 1992. 17. Ellis, R.W., The applications of rDNA technology to vaccines, in Vaccinia, Vaccination, Vaccinology. Jenner, Pasteur and Their Successors, Plotkin, S.A. and Fantini, B., Eds., Elsevier, Paris, 1996, 303. 18. Chattergoon, M., Boyer, J., and Weiner, D.R., Genetic immunization: a new era in vaccines and immune therapeutics, FASEB J., 11, 753, 1997. 19. Klein, M., Future prospects for vaccination, in Vaccinia, Vaccination, Vaccinology. Jenner, Pasteur and Their Successors, Plotkin, S.A. and Fantini, B., Eds., Elsevier, Paris, 1996, 295. 20. Hendriksen, C.F.M., A short history of the use of animals in vaccine development and quality control, in Replacement, Reduction, and Refinement of Animal Experiments in the Development and Control of Biological Products, Brown, F., Cussler, K., and Hendriksen, C., Eds., Developments in Biological Standardization 86, Karger, Basel, 1996, 3. 21. Zo doende 1978–2002, Annual statistics on the use of laboratory animals in the Netherlands, Keuringsdienst van Waren (KvW). 22. Prince, A.M., Moor-Jankowski, J., Eichberg, J.W., Schellekens, H., Mauler, R.F., Girard, M., and Goodall, J., Chimpanzees and AIDS research, Nature, 333, 513, 1988. 23. Daniel, M.D., Kirchoff, F., Czajak, S., Sehgal, P.K., and Desrosiers, R.C., Protective effects of a live attenuated SIV vaccine with a deletion in the nef gene, Science, 258, 1938, 1992. 24. Siebelink, K.H., Chu, I.H., Rimmelzwaan, G.F., Weijer, K., van Herwijnen, R., Knell, P., Egberink, H.F., Bosch, M.L., and Osterhaus, A.D., Feline immunodeficiency virus (FIV) infection in the cat as a model for HIV infection in man: FIV-induced impairment of immune function, AIDS Res. Hum. Retroviruses, 6, 1373, 1990. 25. Kuiken, T., Fouchier, R.A., Schutten, M., Rimmelzwaan, G.F., van Amerongen, G., van Riel, D., Laman, J.D., de Jong, T., van Doornum, G., Lim, W., Ling, A.E., Chan, P.K., Tam, J.S., Zambon, M.C., Gopal, R., Drosten, C., van der Werf, S., Escriou, N., Manuguerra, J.C., Stohr, K., Peiris, J.S., and Osterhaus, A.D., Newly discovered coronavirus as the primary cause of severe acute respiratory syndrome, Lancet, 362, 263, 2003. 26. Martina, B.E., Haagmans, B.L., Kuiken, T., Fouchier, R.A., Rimmelzwaan, G.F., Van Amerongen, G., Peiris, J.S., Lim, W., and Osterhaus, A.D., Virology: SARS virus infection of cats and ferrets, Nature, 425, 915, 2003. 27. Wiktor, T.J., Plotkin, S.A., and Koprowski, H., Development and clinical trials of the new human rabies vaccine of tissue culture (human diploid cell) origin, Develop. Biol. Stand., 40, 3, 1978. 28. Katz, S.L., Wilfert, C.M., and Robbins, F.C., The role of tissue culture in vaccine development, in Vaccinia, Vaccination, Vaccinology. Jenner, Pasteur and Their Successors, Plotkin, S.A. and Fantini, B., Eds., Elsevier, Paris, 1986, 213. 29. Horaud, F., Viral vaccines and cell substrate: a “historical” debate, in Vaccinia, Vaccination, Vaccinology. Jenner, Pasteur and Their Successors, Plotkin, S.A. and Fantini, B., Eds., Elsevier, Paris, 1996, 217. 30. WHO, Cells, Products, Safety: Background Papers from the WHO Study Group on Biologicals, Developments in Biological Standardization 68, Karger, Basel, 1987. 31. Hendriksen, C.F.M., Laboratory Animals in Vaccine Production and Control. Replacement, Reduction, and Refinement, Kluwer Academic, Dordrecht, the Netherlands, 1988. 32. Weisser, K. and Hechler, U., Animal Welfare Aspects in the Quality Control of Immunobiologicals, Paul Ehrlich Institute, Russell and Burch House, Nottingham, U.K., 1997. 33. Halder, M., Three Rs potential in the development and quality control of immunobiologicals, ALTEX, 18(Suppl. 1), 13, 2001.

Typing of the rabies virus in Chile, 2002–2008 V. YUNG1*, M. FAVI1 AND J. FERNANDEZ2 1 Seccio´n Rabia, Subdepartamento Virologı´a, Instituto de Salud Pu´blica de Chile, Santiago, Chile 2 Subdepartamento Gene´tica Molecular, Instituto de Salud Pu´blica de Chile, Santiago, Chile Received 20 July 2011; Final revision 23 February 2012; Accepted 4 March 2012 SUMMARY In Chile, dog rabies has been controlled and insectivorous bats have been identified as the main rabies reservoir. This study aimed to determine the rabies virus (RABV) variants circulating in the country between 2002 and 2008. A total of 612 RABV isolates were tested using a panel with eight monoclonal antibodies against the viral nucleoprotein (N-mAbs) for antigenic typing, and a product of 320-bp of the nucleoprotein gene was sequenced from 99 isolates. Typing of the isolates revealed six different antigenic variants but phylogenetic analysis identified four clusters associated with four different bat species. Tadarida brasiliensis bats were confirmed as the main reservoir. This methodology identified several independent rabies enzootics maintained by different species of insectivorous bats in Chile. Key words: Antigenic variant, bats, phylogenetic analysis, rabies. Rabies is a fatal viral zoonosis caused by viruses of the genus Lyssavirus in the family Rhabdoviridae. It is transmitted between mammals, including bats, primarily through bite inoculation of the rabies virus (RABV) present in the saliva of infected individuals [1]. Members of the Lyssavirus genus constitute a single monophyletic clade, distinct from other rhabdoviruses. The genus consists of 11 genotypes (seven established genotypes and four newly described lyssaviruses from Eurasia). Genotype 1 (RABV, classical RABV) has worldwide distribution and at present is the only genotype to be isolated in the Americas (South, Central and North) that forms endemic cycles within terrestrial mammals and bats [2]. Rabies occurs in two different epidemiological forms : urban rabies, with dogs and domestic animals as the principal reservoir and transmitter, and sylvatic rabies, with various wild species in the Carnivora and Chiroptera orders acting as reservoirs and transmitters. In Chile, dog rabies has been controlled, and since 1985 insectivorous bats have been identified as the country’s main rabies reservoirs and infection source for sporadic cases of rabies in domestic animals [3, 4]. At least four genera of insectivorous bats (Tadarida, Myotis, Histiotus, Lasiurus) are widely distributed in Chile. The role of these species as reservoirs hosts and transmitters supports the theory that diverse viral variants of rabies are circulating. Recent evidence suggests that all RABV variants affecting terrestrial carnivores may have originated from cross-species transmission events from long-term enzootic batassociated variants. A molecular-clock model based on genetic divergence of RABV variants in bats of different species suggests that in North America, the divergence of extant bat-associated RABVs from a common ancestor took place between 1651 and * Author for correspondence: Dr V. Yung, Marathon 1000, co´ digo postal 7780050, N˜ un˜ oa, Santiago, Chile. 1660 C.E. The bat RABV variants found in Latin America in common vampire bats (Desmodus rotundus) and free-tailed bats (genus Tadarida, family Mollosidae) are the closest ones to the earliest common ancestor [5]. The purpose of this study was to investigate which RABV variants were circulating in Chile in 2002–2008. During this period, 11 342 animals from areas around the country were submitted for rabies testing. Of this number, 653 insectivorous bats, one dog and one cat tested positive using the fluorescent antibody test (FAT). Applying the mouse inoculation test (MIT) [6], 612 samples were successfully isolated and then typed using a panel of eight monoclonal antibodies against the viral nucleoprotein (N-mAbs) provided by the Centers for Disease Control and Prevention (USA). The reaction patterns obtained with different mAbs for determining the antigenic variant have been described in a previous report [4]. Ninety-nine of the RABV isolates were selected for performing nucleotide sequence analyses. Of these, 66 were from T. brasiliensis bats (the most common species submitted for testing), 31 were from the remaining insectivorous bat species (L. cinereus, L. borealis, H. macrotus, M. chiloensis) and two were taken from domestic animals (dog and cat). All 99 were collected in Chile’s central region (Fig. 1). Total RNA extraction was conducted using TRIzol (Invitrogen, USA) in accordance with the manufacturer’s instructions. Complementary DNA (cDNA) was produced by reverse transcription– polymerase chain reaction using primers 10 g and 304 as described previously and a product of 320-bp of the nucleoprotein gene (1157–1476) was sequenced using the BigDye Terminator Cycle Sequence kit v3.1 (Applied Biosystems, USA) [7]. Nucleotide sequences were analysed with an ABI PRISM 3130 genetic analyser (Applied Biosystems). A phylogenetic tree was reconstructed for aligned nucleotide sequences by means of a neighbour-joining (NJ) analysis with 1000 bootstrap replicates using the MEGA 3>1 software tool [8]. Bootstrap resampling analysis of 1000 replicates was employed to estimate the reliability of the prediction tree. For the phylogenetic analysis, sequences from other countries in the Americas were included (GenBank accession numbers are given in Fig. 2). Two non-rabies lyssaviruses, European bat 1 (EBLV Genbank accession no. U22845) and Duvenhage virus (DUVV Genbank accession no. U22848) were used as outgroups [9]. Reaction patterns using a panel of eight mAbs of 613 rabies isolates revealed six different antigenic variants in the Chilean bat species (Table 1). Of this total, 572 isolates were antigenic variant 4 (568 from T. brasiliensis bats, two from M. chiloensis bats and one each from a dog and a cat) and 18 were antigenic variant 6 (14 from L. cinereus bats, four Map of Chile Region Species Year Fig. 1 [colour online]. Geographical distribution of sequenced rabies cases (Chile, 2002–2008). (Email: vyung@ispch.cl) from L. borealis bats). Eleven isolates from H. macrotus bats and one from T. brasiliensis bats were associated with an atypical antigenic variant described previously in Chile that is unrelated to any previously described reaction panel using a panel with eight N-mAbs [7]. Five isolates from M. chiloensis bats were characterized as antigenic variant 3, two from M. chiloensis bats as variant 8, and four from T. brasiliensis bats as variant 9 (associated with T. brasiliensis mexicana). Although it offers a rapid, simple and inexpensive means of typing for epidemiological studies, antigenic analysis with mAbs is lacking in precision. To obtain a more accurate determination of the diversity of the RABV in bat populations, partial sequencing and phylogenetic analyses of 99 Chilean RABV isolates were conducted. Four monophyletic clusters associated with four different bat species were thus identified, each one defined as a group of related sequences that share specific patterns of nucleotide variation and are associated with rabies maintained and transmitted by the same or some other bat species according to taxonomic identification of specimens (Fig. 2). Cluster I contained 66 isolates obtained from 64 T. brasiliensis bats and two domestic animals (a dog and a cat), but due to the large number of isolates with 100% nucleotide similarity we took only representative sequences for the phylogenetics analyses. The overall average identity in these isolates was 95.9%. This variant is distantly related to the genetic variant circulating in the North American T. brasiliensis bat population but is very closely related to the genetic variants in Argentinean and Colombian bats. The RABV found in T. brasiliensis in Chile does not seem to be closely related to rabies in the same species in North America, where the RABV lineage found in T. brasiliensis is related primarily to the vampire viruses [10]. Since RABV circulates in Chile in insectivorous bats only, it is not found in haematophagous bat species. Cluster II was represented by isolates from six M. chiloensis bats (colonial and non-migratory) with an overall average identity of 95.5%. They were antigenically identified as variants 3 and 8 (Table 1), but in the genetic analysis they segregated into a different cluster associated with Argentinean Myotis bats. Cluster III was composed of 10 isolates, nine from H. macrotus bats and one from a T. brasiliensis bat, with an overall average identity of 98.6%. These isolates clustered with viruses associated with H. macrotus in Argentina and a Histiotus-like bat found inMexico [11]. Very little is known about the biology and distribution of this bat species, which may be found in other parts of the Americas in addition to Chile, Argentina and Mexico [10]. Finally, Cluster IV was made up of 16 isolates, of which four were from L. borealis bats, 11 from L. cinereus bats and one from T. brasiliensis. The overall average identity was 99.5%. The Lasiurus genus is solitary and often described as a tree-dweller due to its roosting preference. It is also migratoryand hence has a more southerly range during the winter. All three of these species share the same phylogenetic lineage as Lasiurus bats in North America. Some bat species seem able to maintain the same virus variant in geographically distant territories. The two T. brasiliensis cases observed in this cluster are probably spillovers of an endemic cycle maintained by Lasiurus sp. This spillover transmission mechanism may be due to the fact that solitary bat species such as Lasiurus spp. can develop furious rabies, in which case they may actively attack bats or other animals [12]. One isolate (Mch-3171), obtained from a M. chiloensis bat and antigenically identified as variant 4, segregated into a different cluster, with an insectivorous bat from Colombia. It was more narrowlyrelated to cluster II. However, given the lack of Table 1. Antigenic typing with monoclonal antibodies (mAbs) of rabies isolates from Chile Antigenic variant 2002 2003 2004 2005 2006 2007 2008 Total 4 98Tb 66Tb 74 Tb 92 Tb 99 Tb 71 Tb 68 Tb 572 1 Mch 1 Mch 1 Dog 1 Cat 6 2 Lc 2 Lc 2 Lb 3 Lc 3 Lc 1 Lc 2 Lc 18 1 Lc 2 Lb NT 2 Hm 1 Tb 1 Hm 1 Hm 1 Hm 3 Hm 2 Hm 11 3 1 Mch 1 Mch 1 Mch 1 Mch 1 Mch 5 8 1 Mch 1 Mch 2 9 2 Tb 2 Tb 4 NT, Not typed; Tb, Tadarida brasiliensis ; Lc, Lasiurus cinereus; Lb, Lasiurus borealis; Hm, Histiotus macrotus; Mch, Myotis chiloensis. Rabies isolates are grouped according to patterns of reaction with eight N-mAbs. 4 V. Yung and others statistical support for its potential association with other RABVs so far reported, complete nucleoprotein sequences and a more comprehensive sampling encompassing RABV diversity in the region are needed to help identify whether it is a new variant or the reservoir host associated with it. Although antigenic typing of RABV using mAbs may distinguish diverse variants of the virus, distinguishing different types within a variant may becomedifficult using this method, which could be more easily and accurately done with molecular characterization via nucleotide and amino-acid sequence determinations. These molecular analyses may help to unravel the precise genetic diversity of a RABV and the sequence characteristic of RABVs specifically associated with each host species. The first phylogenetic investigation into bat RABV using partial N gene sequencing established that there were distinctlineages of bat RABV associated with different bat species [13]. RABV is widespread in the Americas and genetic differentiation in RABVs is believed to have occurred in response to their association with particular host species [14]. However, topography may play a less significant role in shaping the phylogeny of bat RABV than it potentially does for terrestrial mammal RABV [15]. When a physical barrier is considerable (e.g. the Andes mountain range) genetic isolation may occur, as demonstrated by the separation of the Chilean strains from isolate samples obtained in other Latin American locations [12]. In Chile, where long-term enzootic canine RABVs have not been detected since 1990, the disease is confined to the wild cycle mainly due to T. brasiliensis bats. Although no human rabies cases have been reported since 1996, rabies remains a public health risk in Chile and other parts of Latin America because of the frequency of contact between humans and bats. The coexistence of an abundant bat population with humans and their domestic animals in the urban centres of these countries poses a new challenge to the understanding of rabies epidemiology in metropolitan areas [16, 17]. The approach adopted in this study enabled the identification of several rabies enzootics maintainedindependently by different species of insectivorous bat through transmission events involving bat-to-bat or bat-to-terrestrial species. The investigation also confirmed T. brasiliensis as the main RABV reservoir and the existence of compartmentalization in Chile in other bat species. Finally, we note that studies of RABV characterization are a valuable asset in supporting epidemiological surveillance systems for the disease andselecting control strategies and monitoring programmes, which can have major impacts on both human health and ecosystems. ACKNOWLEDGEMENTS We thank the laboratory staff who assisted in this study for their excellent technical assistance and also thank Kenneth Rivkin for his valuable contribution in the translation of this paper. The authors are also grateful for funding provided by the Public Health Institute of Chile. DECLARATION OF INTEREST None. REFERENCES 1. Johnson N, et al. Phylogenetics comparison of the genus Lyssavirus using distal coding sequences of the glycoprotein and nucleoprotein genes. Archives of Virology 2002; 147: 2111–2123. 2. Kuzmin IV, et al. The rhabdoviruses: biodiversity, phylogenetics, and evolution. Infection, Genetics and Evolution 2009; 9: 541–53. 3. Nun˜ ez S, et al. Wild rabies in insectivorous bats in Chile. Bulletin of Pan American Health Organization 1987; 103: 140–145. 4. Favi M, et al. Role of insectivorous bats in the transmission of rabies in Chile. Archivos de Medicina Veterinaria 1999; 31: 157–165. 5. Calisher C, et al. Bats: important reservoir hosts of emerging viruses. Clinical Microbiology Reviews 2006; 19: 531–545. 6. Koprowsky H. The mouse inoculation test. In: Kaplan MN, Koprowsky H, eds. Rabies. Laboratory Techniques. Ginebra: OMS, 1976, pp 88–97. 7. Yung V, Ferna´ ndez J, Favi M. Genetic and antigenic typing of rabies virus in Chile. Archives of Virology 2002; 147: 197–205. 8. Kumar S, Tamura K, Nei M. MEGA 3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Briefings in Bioinformatics 2004; 5: 150–63. 9. Kissi B, Tordo N, Bourhy H. Genetics polymorphism in the rabies virus nucleoprotein gene. Virology 1995; 209: 526–537. 10. Velasco-Villa A, et al. Molecular diversity of rabies viruses associated with bats in Mexico and other countries of the Americas. Journal of Clinical Microbiology 2006; 44: 1697–710. Rabies virus in Chile 5 11. Cisterna D, et al. Antigenic and molecular characterization of rabies virus in Argentina. Virus Research 2005; 109: 139–147. 12. Kuzmin I, Rupprech C. Bat rabies. In: Jackson A, Wunner B, eds. Rabies, 2nd edn, 2009, pp. 259–381. 13. Smith J S, et al. Epidemiologic and historical relationships among 87 rabies virus isolates as determine by limited sequence analysis. Journal of Infectious Disease 1992; 166: 296–307. 14. Hughes GJ, Orciari LA, Rupprecht CE. Evolutionary timescale of rabies virus adaptation to North American bats inferred from the substitution rate of the nucleoprotein gene. Journal of General Virology 2005; 86: 1467–1474. 15. Davis PL, Bourhy H, Holmes EC. The evolutionary history and dynamics of bat rabies virus. Infection, Genetics and Evolution 2006; 6: 464–473. 16. Favi M, et al. First case of human rabies in Chile due to an insectivorous bats virus variant. Emerging Infectious Disease 2002; 8: 79–81. 17. De Mattos C, et al. Bats rabies in urban centers in Chile. Journal of Wildlife Disease 2000; 36: 231–240. 6 V. Yung and others