Estudio biopatológico y respuesta inmune en la coccidiosis caprina por "eimeria ninakohlyakimovae": implicaciones en el control de la enfermedad

estudio biopatológico y respuesta inmune en la cocidiosis caprina producida por eimeria ninakohlyakimovae: ImplIcacIones e n e l control d e l a enfermedad Lorena Mat o s Guedes Instituto Universitario de Sanidad Animal y Seguridad Alimentaria. Universidad de Las Palmas de Gran Canaria TESIS DOCTORAL 2015 UNIVERSIDAD DE LAS PALMAS DE GRAN CANARIA FACULTAD DE VETERINARIA Instituto Universitario de Sanidad Animal y Seguridad Alimentaria Programa de Doctorado: “SANIDAD ANIMAL” Título de la Tesis ESTUDIO BIOPATOLÓGICO Y RESPUESTA INMUNE EN LA COCCIDIOSIS CAPRINA POR EIMERIA NINAKOHLYAKIMOVAE : IMPLICACIONES EN EL CONTROL DE LA ENFERMEDAD Tesis Doctoral presentada por Dña. Lorena Matos Guedes Dirigida por el Dr. Francisco Rodríguez Guisado, Dra. María del Carmen Muñoz Ojeda y Dr. Antonio Ruiz Reyes El Director, El Director, F. Rodríguez Guisado M.C. Muñoz Ojeda El Director, La Doctoranda, Antonio Ruiz Reyes Lorena Matos Guedes Arucas, noviembre 2015 Departamento de Patología Animal, Producción Animal y Tecnología de los Alimentos D. Francisco Rodríguez Guisado , Doctor en Veterinaria y morfología animal, Dña. María del Carmen Muñoz Ojeda , Dra. en Veterinaria y Profesora Titular del Área de Medicina y Cirugía Animal, y D. Antonio Ruiz Reyes , Doctor en Veterinaria y Profesor Titular del Área de Parasitología del Departamento de Patología Animal, Producción Animal, Bromatología y Tecnología de los Alimentos (Facultad de Veterinaria, Universidad de Las Palmas de Gran Canaria), INFORMAN: Que la tesis doctoral que lleva por título “ Estudio biopatológico y respuesta inmune en la coccidiosis caprina por Eimeria ninakohlyakimovae : implicaciones en el control de la enfermedad ” ha sido realizada por la licenciada en Veterinaria Dña. Lorena Matos Guedes en el Departamento de Patología Animal, Producción Animal, Bromatología y Tecnología de los Alimentos de la (Facultad de Veterinaria, Universidad de Las Palmas de Gran Canaria) bajo nuestra dirección y asesoramiento, y consideramos que cumple la normativa vigente para optar al Grado de Doctor en Veterinaria por la Universidad de Las Palmas de Gran Canaria. En Arucas (Las Palmas) a 17 noviembre de 2015 Fdo. Francisco Rodríguez Guisado Fdo. Mª Carmen Muñoz Ojeda Fdo. Antonio Ruiz Reyes 35413-ARUCAS-LAS PALMAS-ESPAÑA TELÉFONOS: (928) 451099/451137 – FAX: (928) 451142 – E-mail: [email protected] ÍNDICE 1. Agradecimientos .......................................................................................... 1 2. Introducción y Objetivos ............................................................................ 5 3. Revisión Bibiográfica ................................................................................... 9 3.1. Coccidiosis caprina...................................................................................... 9 3.1.1. Introducción .............................................................................................. 9 3.1.2. Etiología ....................................................................................................10 3.1.2.1. Encuadre Taxonómico ...................................................................11 3.1.2.2. Morfología......................................................................................12 3.1.2.3. Biología de los coccidios ................................................................14 3.1.3. Epidemiología ...........................................................................................16 3.1.4. Patología ...................................................................................................20 3.1.5. Diagnóstico ...............................................................................................24 3.1.6. Tratamiento...............................................................................................25 3.1.7. Control ......................................................................................................28 3.2. Respuesta inmune frente a la coccidiosis .................................................... 35 3.2.1. Introducción ..............................................................................................35 3.2.2. Respuesta inmune inespecífica ..................................................................36 3.2.3. Respuesta inmune Humoral ......................................................................42 3.2.4. Respuesta inmune Celular .........................................................................44 i ÍNDICE 4. Presentación de Artículos......................................................................... 51 4.1. Artículo 1 “ Isolation and infection of an E. ninakohlyakimovae strain“ ........ 51 4.2. Artículo 2 “ Acute coccidiosis of goats by E. ninakohlyakimovae” ................ 67 4.3. Artículo 3 “ Antibody response to E. ninakohlyakimovae ” ............................ 83 4.4. Artículo 4 “ Age-related immune responses to goat coccidiosis” .................... 97 4.5. Artículo 5 “ Immune response during prepatency in goat coccidiosis” ......... 117 5. Conclusiones .............................................................................................133 6. Resumen - Summary ................................................................................135 7. Referencias ................................................................................................139 ii 1. agradecimientos AGRADECIMIENTOS La realización de esta tesis ha sido financiada en base a los siguientes proyectos de investigación: Agencia Canaria de Investigación, Innovación y Sociedad de la Información (ACIISI) PROYECTO: SolSub 200801000244 PROTEÓMICA E INMUNOLOGÍA APLICADA A LA PROFILAXIS Y CONTROL DE LA COCCIDIOSIS CAPRINA PRODUCIDA POR EIMERIA NINAKHOLYAKIMOVAE Ministerio de Educación y Ciencia, Innovación Tecnológica 2008-2010 PROYECTO: AGL2007-63415 RESPUESTA INMUNE Y MECANISMOS DE PATOGENICIDAD EN LA COCCIDIOSIS CAPRINA: IMPLICACIONES EN LA PROFILAXIS Y CONTROL DE LA ENFERMEDAD 1 INTRODUCCIÓN Y OBJETIVOS parasitadas. Además de la activación de las células T, esta red atrae a células de la respuesta innata, como macrófagos y polimorfonucleares. Cada vez son más los estudios que destacan la importancia de la respuesta humoral en la inmunidad adquirida frente a infecciones por Eimeria . Este tipo de respuesta se desarrolla rápidamente y se caracteriza por la aparición de un título alto de anticuerpos en el suero del animal infectado, con un incremento inicial de IgM, seguido por IgG; también pueden aparecer otras inmunoglobulinas específicas como la IgA y, en general, la cantidad de anticuerpos va aumentando si los animales están continuamente expuestos a los ooquistes. Se ha demostrado que la respuesta inmune humoral desempeña un papel importante en las reinfección por coccidios en rumiantes, aunque, por sí sola, no provoca una inmuprotección absoluta. 7 INTRODUCCIÓN Y OBJETIVOS 2.2. OBJETIVOS 1.2.1. Objetivo general # Analizar la respuesta inmune frente al coccidio caprino Eimeria ninakohlyakimovae mediante infecciones experimentales y su relación con los mecanismos de patogenicidad del parásito y las posibles implicaciones en la profilaxis y control de la enfermedad. 1.2.2. Objetivos específicos # Aislar una cepa de Eimeria ninakohlyakimovae (Islas Canarias) y analizar su capacidad infectante, patogenicidad y aspectos generales de la respuesta inmune tras su inoculación a cabritos. # Valorar el grado de protección conferido por Eimeria ninakohlyakimovae tras reinfecciones homólogas con altas dosis infectantes (infección aguda) y su relación con la respuesta inmune desarrollada en el curso de las infecciones experimentales. # Estudiar y caracterizar la respuesta inmune humoral (sistémica y local) tras una infección experimental de cabritos con Eimeria ninakohlyakimovae. # Identificar péptidos reconocidos por anticuerpos del isotipo IgG en muestras séricas y su relación el desarrollo de repuestas inmunes protectoras. # Evaluar la influencia de la edad en el desarrollo de respuestas inmunes protectoras en la coccidiosis experimental caprina por Eimeria ninakohlyakimovae . # Analizar y caracterizar la respuesta inmune humoral y celular en la fase prepatente de la enfermedad producida por E. ninakohlyakimovae en cabritos y su relación con respuestas inmunoprotectoras frente a las formas inmaduras del parásito (esquizontes inmaduros) que se desarrollan durante esta fase del ciclo endógeno. 8 3. revisión bibliográfica REVISIÓN BIBLIOGRÁFICA 3.1. COCCIDIOSIS CAPRINA 3.1.1. INTRODUCCIÓN La coccidiosis es un proceso patológico causado por protozoos del género Eimeria (Schneider, 1875) , parásitos altamente específicos para el hospedador. Las infecciones que producen constituyen unas de las parasitosis más frecuentes y más ampliamente distribuidas en los sistemas de producción ganadera de grandes y pequeños rumiantes, representando, además, uno de los principales motivos de pérdidas económicas (Rashi y Tak, 2012; Hashemnia y cols., 2014) . Las pérdidas están ligadas al menor rendimiento zootécnico de los animales, incluso cuando las infecciones son moderadas y no aparecen signos clínicos. Las repercusiones económicas se reflejan, principalmente, en una disminución de las producciones y de la tasa de crecimiento, y en un aumento del índice de mortalidad que, en determinadas circunstancias, puede alcanzar tasas superiores al 20% ( Lima, 1981 ). Estas pérdidas aumentan y se agravan cuando la eimeriosis se asocia con infecciones producidas por otros protozoos y/o a infecciones por helmintos de patogenicidad diversa. Por todo ello, las coccidiosis se engloban en lo que han venido a denominarse “enfermedades económicas”. El problema puede aparecer en cualquier sistema de explotación, si bien, es en la explotación intensiva cuando la enfermedad se desarrolla de forma más manifiesta y, por consiguiente, el impacto económico es más alto, probablemente debido a la elevada densidad de animales. Los coccidios del género Eimeria son parásitos intracelulares de las células epiteliales del intestino delgado y grueso de los hospedadores, aunque no exclusivamente. Entre las localizaciones extra-intestinales más frecuentes se encuentran el hígado y el riñón aunque, en menor frecuencia, también se han observado en el bazo y en el pulmón (Collins y cols. 1988; Dai y cols., 1991; Morgan y cols., 2013) . Su acción patógena fundamental se debe a la destrucción celular que resulta de los ciclos de reproducción asexual y sexual de su ciclo endógeno ( Stockdale, 1980 ). Los animales adultos suelen hacerse resistentes después de sobrevivir al periodo crítico durante las primeras semanas de vida, transformándose entonces en reservorios y portadores inaparentes del parásito (Daugscheis y Najdrowski, 2005) . Por el contrario, los animales jóvenes son los más susceptibles de padecer la enfermedad, especialmente entre las 2 semanas y los 4 meses de vida (Ruiz y cols., 2006) . Los coccidios están presentes en todas las ganaderías de rumiantes, aunque esto no quiere decir que en todas ellas se desarrolle la enfermedad. No se conocen con exactitud los mecanismos que desarrollan una coccidiosis patente; lo que sí están claros son los factores de riesgo: destete, transporte, entrada en cebaderos, partos múltiples, madres mal alimentadas, ubres sucias, problemas de mastitis, etc., 9 REVISIÓN BIBLIOGRÁFICA situaciones todas ellas típicas del sistema de crianza y engorde de rumiantes, por ejemplo en corderos ( Sanz, 2000 ). En general, la coccidiosis en los rumiantes es el resultado de una compleja interacción entre huésped y parásito en la que intervienen, además, numerosos factores externos que pueden condicionar la severidad de la enfermedad, por lo que, a pesar de los avances logrados en los últimos años en el estudio de los ciclos de vida, la patogenia, la epidemiología y el control, la coccidiosis en los rumiantes presenta, aún en la actualidad, muchos aspectos sin aclarar. Además, muchos de los ciclos de vida de las especies que se consideran de menor importancia aún no se han dilucidado y existe controversia sobre la patogenicidad de algunas de ellas. En este contexto, se ha observado que los brotes de coccidiosis se producen con una frecuencia cada vez mayor, lo que contrasta con la escasa disponibilidad comercial de medicamentos para su tratamiento y prevención; asimismo, son limitadas las investigaciones que se han llevado a cabo sobre los métodos inmunológicos de control (Taylor y Catchpole, 1994 ). Han sido descritas al menos 18 especies de Eimeria parasitando a los caprinos con elevada prevalencia en diferentes localizaciones geográficas (Pellerdy, 1974; Soe y Pomroy, 1992; Smith y Sherman, 2009) . La especie E. ninakohlyakimovae se considera una de las más patógenas y más ampliamente distribuidas en todo el mundo (Balicka-Ramisz, 1999; Ruiz y cols., 2006; Taylor y cols., 2007 ), motivo por el cual ha sido objeto de infecciones experimentales en caprinos, fundamentalmente orientadas al estudio de los aspectos parasitológicos y biopatológicos de la enfermedad (Viera y cols., 1997; Dai y cols., 2006) , pero poco se conoce sobre la caracterización de la respuesta inmune frente a ésta y otras especies caprinas de Eimeria y, menos aún, sobre su posible repercusión en el desarrollo de respuestas inmunes protectoras. 3.1.2. ETIOLOGÍA Los coccidios son parásitos intracelulares altamente específicos y de ciclo directo (monoxenos) (Witcombe y Smith, 2014) , por lo tanto, no necesitan más de un hospedador para completar su ciclo. Se engloban dentro del phylum Apicomplexa, siendo su clasificación compleja (Lefevre y Blancou, 2010) por la gran cantidad de especies que se han descrito en todo el mundo. El phylum Apicomplexa está constituido actualmente por más de 300 géneros que, a su vez, incluyen cerca de 4600 especies de organismos. Los apicomplejos son parásitos intracelulares obligados durante su fase proliferativa y entre ellos se incluyen, probablemente, algunas de las especies responsables de las principales zoonosis del ser humano. Dentro de este grupo se encuentran los géneros Eimeria e Isospora , comúnmente conocidos como coccidios no formadores de quistes, así como otros parásitos de máxima importancia en medicina humana como Plasmodium spp. y Toxoplasma gondii (Champan, 2014) . 10 REVISIÓN BIBLIOGRÁFICA 3.1.2.1. Encuadre taxonónico La clasificación correcta de los Apicomplexa ha sido posible gracias a la utilización del microscopio electrónico, que ha permitido la observación detallada de la compleja estructura de estos organismos. Se han publicado diversas propuestas de clasificación para miembros de este phylum conocidos comúnmente como coccidios, siendo la siguiente la más aceptada para el género Eimeria (Tabla 1) (Schneider, 1975; Soulsby, 1982; Levine, 1988; Urquhart y cols., 1996) . Phylum Apicomplexa Clase Sporozoea Subclase Coccidia Orden Eucoccidia Suborden Eimeriina Familia Eimeridae Género Eimeria Tabla 1. Encuadre taxonómico de género Eimeria Aunque se creyó que ovinos y caprinos compartían los mismos coccidios, hoy se reconoce que cada hospedador tiene sus propias especies de Eimeria ( Amstutz y cols., 2000 ). De hecho, en base a la gran similitud morfológica entre las diferentes especies, se estableció una clave de determinación común para ovinos y caprinos ( Yvoré y Esnault, 1984; Yvoré y cols., 1985 ). Entre las nueve especies de Eimeria más frecuentes en la cabra, muchas de ellas son considerablemente parecidas a las propias del ganado ovino, pero los ensayos de infección cruzada han demostrado que se trata de agentes diferentes. A raíz de tales estudios, algunas especies no guardaban el mismo nombre para los dos huéspedes, mientras que otras fueron renombradas para cada una de las dos especies animales. Las especies reconocidas como más importantes en los caprinos son: E. arloingi (Sayin y cols., 1980 -Marotel, 1905-) , E. hirci (Lima, 1980) , E. christenseni (Lima, 1981-Levine, 1962-) , E. ninakohlyakimovae (Viera y cols., 1997 -yakimoff y Rastegaieff, 1930-) , E. caprovina (Lima, 1980 ), E. caprina (Lima, 1980) , E. alijevi (Kasim y cols., 1991 -Musaev, 1970-) , E. apsheronica (Musaev, 1970) , E. jolchijevi (Musaev, 1970) y E.kocharli (Musaev, 1970) . Como posiblemente compartibles entre oveja y cabra, con reservas, se han citado estas otras: E. marsica (Restani, 1971) , identificada en caprinos españoles por Romero (1984) y E. gilruthi (Soliman, 1970 - 11 REVISIÓN BIBLIOGRÁFICA Chatton, 1910) , hallada en la necropsia de una cabra y localizada en el cuajar. Sin embargo, se ha demostrado que estas dos últimas especies se desarrollan mejor en la oveja que en la cabra (Chatton, 1910; Restani, 1971; Maratea y Miller, 2007) . 3.1.2.2. Morfología Morfología de los Apicomplexa Los Apicomplexa se caracterizan por la presencia de un polo apical que se observa en las fases infectantes del ciclo del parásito, en concreto, en los esporozoítos, los merozoítos, los taquizoítos y los bradizoítos (Jolley y Barley, 2006) . Los caracteres de estas formas infectantes se emplean para la clasificación de las distintas especies. El polo apical o complejo apical proporciona al parásito la orientación y es el punto focal donde tiene lugar la exteriorización de los orgánulos secretores (Katris y cols., 2014 ). Además, existen elementos estructurales (como la actina y la miosina) dentro del complejo apical que son los que proporcionan la motilidad para la invasión de la célula hospedadora (Striepen y cols., 2007) . Por último, el complejo apical parece actuar directamente sobre la célula hospedadora a nivel de los microtúbulos, favoreciendo la replicación intracelular del parásito (Mehlhorn y Piekarski, 1993; Morrrissette y Sibley; 2002 ). Lo componen microtúbulos, micronemas, roptrias, gránulos densos, conoides y un complejo anillo-polar (Urquart y cols., 1996) . El conoide, de morfología tubular, se encuentra en el centro del complejo apical y es el encargado del proceso mecánico en la invasión intracelular (Mehlhorn y Piekarski, 1993; Morrrissette y Sibley; 2002) . Los micronemas, gránulos densos y roptrias secretan moléculas esenciales para la supervivencia del parásito en el interior de la célula parasitada (Morrrissette y Sibley; 2002) , y las roptrias, cuyo número puede variar de un género a otro dentro de los Apicomplexa (Blackman y Bannister, 2001) , se encuentran situadas a nivel del extremo apical y contienen proteínas que van a actuar en la adhesión a la membrana de la célula hospedadora (Alexander y cols., 2005 ). Por último, los microtúbulos son estructuras longitudinales que sirven de anclaje al anillo-polar, proporcionando estabilidad (Morrrissette y Sibley; 2002) . 12 REVISIÓN BIBLIOGRÁFICA Fig. 1. Zoíto de Apicomplexa Los ooquistes que salen en las heces de los animales infectados son los que tradicionalmente se han utilizado para diferenciar entre las distintas especies de Eimeria en base a sus características morfológicas. Sin embargo, hay que considerar que ésta no es más que una fase en el ciclo del parasito y que su morfología es mucho más compleja. La morfología y el tamaño de los ooquistes de las diferentes especies de Eimeria son muy variables, siendo las formas más comunes las esféricas, subesféricas, ovoides o elipsoides. Poseen una pared compuesta por dos o más capas (una a base de queratina y otra proteica) y, generalmente, son claras y transparentes, y presentan un contorno doble bien definido, aunque algunas especies pueden presentar una coloración que puede variar entre amarillo, marrón o, incluso, verde. El ooquiste puede o no tener una abertura en el extremo anterior que puede estar cubierto por un tapón llamado capuchón polar o cápsula micropilar. En el interior del ooquiste esporulado se desarrollan cuatro esporocistos que contienen en su interior dos esporozoítos cada uno. En algunas especies de Eimeria, en el extremo apical de los esporocistos puede observarse una pequeña protuberancia que recibe el nombre de cuerpo de “Stieda” (Long, 1990) . Los esporozoítos tienen forma de huso o de coma, y constituyen el elemento que emigra a través del hospedador e invade sus células. Eimeria ninakohlyakimovae En la actualidad se reconoce como especie exclusiva de los caprinos y presenta una distribución mundial. Se localiza en la porción posterior del intestino delgado, ciego y colon. Los ooquistes de E. ninakohlyakimovae son elipsoidales, a veces ovoides, con un tamaño medio de 23,1 µm de largo por 18,3 µm de ancho (con variaciones entre 20-28 µm) ( Christensen, 1938 ). Carecen de cápsula micropilar (Yakimoff y Rastegaieff, 1930) y, generalmente, el micrópilo es poco evidente o incluso puede no observarse. Los ooquistes son de pared fina, lisa y transparente, 13 REVISIÓN BIBLIOGRÁFICA ligeramente amarillo-marronácea, y, una vez esporulados, albergan en su interior cuerpos residuales y gránulos polares; los esporocistos presentan cuerpo de Stiedae (Ruiz y cols., 213) . La esporulación tiene lugar en uno o dos días (Opoku–Pare y Chineme, 1979; Deb y cols., 1981) y los esporozoítos que resultan de ella son alargados y con un tamaño aproximado de 11,6x 6,3 µm. A B Figura 2 . Representación esquemática de un ooquiste esporulado de Eimeria (A); ooquistes esporulados de las especies más frecuentes del género Eimeria en las cabras (Ruiz y cols., 2006 ): Eimeria arloingi (a) , Eimeria ninakohlyakimovae (b) , E. caprina (c) y E. alijevi (d) . 3.1.2.3. Biología de los coccidios El ciclo biológico endógeno de los coccidios del género Eimeria es continuo y más del 70% ocurre en el intestino delgado. Una vez ingeridos los ooquistes (día 1) se reproducen rápidamente en el yeyuno e íleon. A partir de los 10-12 días invaden y se desarrollan en el intestino grueso y, transcurridas aproximadamente dos semanas desde la infección, comienzan a liberar un gran número de ooquistes con las heces, los cuales, tras esporular en el medio y ser ingeridos por otros hospedadores, darían comienzo a un nuevo ciclo (Marshall y Williams, 1985) . En rumiantes, como en otras especies hospedadoras, el desarrollo tiene lugar en dos etapas (Sánchez y cols., 2005) : fase endógena, en la que se alternan ciclos de reproducción asexual (esquizogonia o merogonia) y sexual (gemetogonia), y fase exógena en medio, que consiste en un proceso de reproducción asexual mediante esporulación (esporogonia). Esporogonia El proceso de esporulación consiste en la segmentación del protoplasma en pequeños cuerpos infectantes llamados esporozoítos que se encuentran dentro de los esporocistos y éstos, a su vez, se encuentran dentro del ooquiste. Dependiendo de la especie de Eimeria , el tiempo de esporulación puede extenderse entre las 48 y las 104 a b c d 14 REVISIÓN BIBLIOGRÁFICA inmunidad específica. Por este motivo, los animales adultos son generalmente más resistentes y, a menos que la infección sea muy alta, no suelen presentar sintomatología, por lo que se considera una enfermedad autolimitante. En cambio, la introducción de animales susceptibles, generalmente jóvenes, en un grupo con portadores asintomáticos puede favorecer el desarrollo de infecciones serias y hasta fatales (Benavides y Romero, 2010) . En cualquier caso, habría que tener en cuenta que el desarrollo de la enfermedad está muy influenciado tanto por el ritmo de infección como por la cantidad de ooquistes ingeridos (Gregory y Catchpole, 1990 ). Los efectos de la eimeriosis pueden exacerbarse si están presentes varias especies en diferentes partes del intestino, así como cuando hay infecciones concurrentes, bien sea por helmintos, bacterias o virus (Taylor y Catchpole, 1994; Daugschies y Najdrowski, 2005; Witcombe y Smith, 2014) . En general, los coccidios del género Eimeria ejercen su acción patógena de la siguiente forma: • Lesionando las células epiteliales y, en ocasiones, endoteliales del intestino. Cada especie de Eimeria tiende a desarrollar su ciclo preferentemente sobre áreas concretas del tracto intestinal, pudiendo afectar a grandes tramos entéricos dejando al descubierto la lámina propia (Norton, 1986; Hermosilla y cols., 2008) . • Modificando la microflora intestinal: se pasa de un 16% de gérmenes gram-negativos a un 76%, (Mohamed, 2000) , lo cual supone un agravante en la aparición de diarreas. 3.1.4.2. Signos clínicos La exposición de los rumiantes a los ooquistes infectantes de Eimeria es constante pero, tal y como se comentó con anterioridad, dependiendo de determinados factores, la coccidiosis puede manifestarse de forma subclínica o, en el otro extremo, desencadenar una clínica severa. No obstante, en la mayoría de los casos, la infección por coccidios es bien tolerada por el animal (Daugschies y Najdrowski, 2005; Witcombe y Smith, 2014) . Así, en investigaciones sobre coccidiosis bovina desarrolladas por Rave y Zaraza (1985 ) en Colombia se encontró que tan sólo en el 40,6% de los casos positivos presentaban diarrea, en el 12,8% diarrea con sangre, y en el 50% no se apreció síntoma alguno que denunciara la presencia de la enfermedad. En este estudio, del total de animales analizados el 62% eran adultos (Lütcher, 2010) . La única forma de poder valorar el impacto de los coccidios en las infecciones subclínicas es realizar una comparación entre lotes tratados y lotes testigos sin tratar. Se observa claramente una disminución del apetito, pérdida de peso y un aumento del índice de conversión. Las coccidiosis clínicas son más esporádicas, con manifestaciones clínicas más evidentes tales como diarreas más o menos 21 REVISIÓN BIBLIOGRÁFICA hemorrágicas (según la especie de Eimeria ), retrasos evidentes del crecimiento y, eventualmente, muertes (Benavides y Romero 2010) . El primer síntoma de los animales es la presencia de diarrea de un color que puede oscilar entre amarillento y marrón oscuro, a veces con sangre, coágulos y mucus (Foreyt, 1990) . Suele coincidir con el inicio de la gametogonia y está asociada a la destrucción de las células de la mucosa del intestino grueso y, por consiguiente, a la disminución de su capacidad para absorber líquidos (Norton 1986 ). Esta fase puede durar entre 3-4 días hasta que se restablezca la mucosa (Koudela y Boková, 1998 ) pero, en los casos en los que la destrucción de la pared intestinal es masiva, la recuperación podría ser de varias semanas (Reeg y cols., 2005) . La diarrea en los rumiantes puede ir acompañada de signos clínicos generales como inapetencia, letargo, pérdida de peso, debilidad, anemia, hipoproteinemia y deshidratación; también suele apreciarse mal pelaje en los animales y, en ovinos, la lana suele volverse quebradiza ( Amstutz y cols., 2000 ). Aunque lo habitual es que se recuperen y que la mortalidad no supere el 10 % de los animales infectados, la coccidiosis puede estar asociada a una mortalidad mucho más elevada, a veces súbita y sin signos digestivos evidentes, sobretodo en animales entre los 2 y 4 meses de edad (Chartier y cols., 1994) . En el ganado vacuno, además de la coccidiosis intestinal descrita anteriormente, se han observado casos de coccidiosis nerviosa. Los signos clínicos asociados a este tipo de coccidiosis pueden variar en gravedad y frecuencia, con un rango que va desde una leve incoordinación muscular y temblores, hasta la pérdida de equilibrio con convulsiones intermitentes o continuas. Durante las convulsiones, los terneros afectados caen en decúbito lateral y exhiben una variedad de signos nerviosos (Isler y cols., 1987a) . Estudios de casos de coccidiosis entérica bovina, con y sin signos nerviosos, han concluido que la concentración de Na, K, Ca, P, Mg en sangre, la carencia de vitamina A, la deficiencia de tiamina, la anemia, la intoxicación por plomo, la uremia, las meningoencefalitis bacterianas, la gravedad de la infección por coccidios propiamente dicha y las alteraciones graves en la flora bacteriana intestinal no están involucrados en la patogenia de la coccidiosis nerviosa. Ese mismo año estos investigadores (Isler y cols., 1987b) llegaron a la conclusión que los signos nerviosos podrían asociarse a reacciones de autointoxicación producidas por toxinas de los coccidios que se absorben a través del epitelio dañado. 3.1.4.3. Lesiones En la necropsia, los animales muertos por coccidiosis muestran la zona perianal sucia por deyecciones diarreicas, pudiendo ser más o menos líquidas, con mucus o, incluso, contener restos entéricos (Mundt y cols., 2005; Ruiz y cols., 2014) . El intestino delgado aparece dilatado, congestivo y con la mucosa inflamada, frecuentemente con hemorragias y exceso de mucus. Sobre la mucosa, es común que aparezcan placas gruesas, blancas y opacas, visibles a simple vista, que suelen 22 REVISIÓN BIBLIOGRÁFICA corresponderse con macroesquizontes. Microscópicamente es común observar atrofia de las microvellosidades (Koudela y Boková, 1998; Daugschies y Najdrowski, 2005; Witcombe y Smith, 2014) . En general, la localización de las lesiones varía con el tipo de ciclo endógeno de la especie o especies de Eimeria que estén parasitando al animal. Por ejemplo, las especies que más frecuentemente parasitan a los caprinos, E.alijevi, E.caprina y E.ninakohlyakimovae se localizan tanto en intestino delgado como grueso, mientras que E. alijevi y E. christernseni lo hacen fundamentalmente en intestino delgado (Taylor y cols., 2007) . Como ya se ha comentado anteriormente, la especie E. ninkohlyakimovae se considera la más patógena entre las que parasitan a los caprinos. Las lesiones macroscópicas que produce incluyen engrosamiento de la pared cecal, petequias y leve congestión del colon, zonas de enteritis y aumento considerable del tamaño de los nódulos linfáticos mesentéricos (Dai y cols., 2006) . Histológicamente se observan áreas de erosión del epitelio y las criptas aparecen invadidas por esquizontes, gametocitos y ooquistes (Gregory y cols., 1989; Taylor y cols., 2003; Hashemia y cols., 2012) . Norton (1986) encontró que E. ninakohlyakimovae destruía las células madre en las criptas del intestino ciego y/o del colon, dejando la mucosa desprovista de epitelio, comparando su patología a la de E. ovinoidalis en ovejas. Las células que se encuentran parasitadas presentan un tamaño alrededor de cuatro veces el habitual y de hasta 15-20 veces si en su interior se desarrolla un macroesquizonte (Ruiz y cols., 2010; Razavi y cols., 2014) . Durante el estudio histológico también se han encontrado congestión y hemorragias capilares, así como una infiltración celular que corre a cargo de linfocitos, polinucleares neutrófilos y eosinófilos. Tras la muerte de los animales, después de padecer una enfermedad clínica, las poblaciones celulares que predominan son los macrófagos y los linfocitos (Long, 1990; Hashemia y cols., 2014) , mientras que en los casos de muerte súbita es más marcada la infiltración de neutrófilos y eosinófilos. En infecciones primarias, esta misma infiltración eosinofílica, además de hiperplasia de las células epiteliales, fueron descritas por Dai y cols. (2006 ) en infecciones experimentales con E. ninakohlyakimovae . Las infecciones por E. arloingi también pueden llegar a ser graves. Aparte de lo ya mencionado, lo más característico es la observación de placas amarillentas o blanquecinas (macroesquizontes) y formaciones papilomatoides en la mucosa (Catchpole y Gregory, 1985) . Por su parte, la coccidiosis por E. caprina se ha descrito que provoca hemorragias en la segunda mitad del colon y en la parte anterior del recto, el cual aparece vacío de heces, pero con mucus y restos de sangre coagulada. Los frotis de la mucosa muestran gran número de gametocitos y ooquistes en todo el ciego, colon y recto, mientras que el intestino delgado aparece relativamente normal. Como también ocurre con E. christenseni y en otras especies, las lesiones más importantes coinciden con el desarrollo de los estados gametogónicos y la producción de ooquistes (Gregory y cols., 1989) . 23 REVISIÓN BIBLIOGRÁFICA 3.1.5. DIAGNÓSTICO A la hora de abordar el diagnóstico ha de tenerse en cuenta la historia particular del brote de coccidiosis. Generalmente, existen evidencias de poca higiene en la explotación, tanto en los sistemas intensivos como en los extensivos, y diarreas en animales de entre 1-6 meses. No obstante, el diagnóstico ha de complementarse con la observación de las lesiones en el examen post mortem o con la realización de análisis coprológicos (Daugschies y Najdrowski, 2005; Silva y cols., 2013) . Por lo general, prácticamente todos los animales de la explotación presentan recuentos positivos, variando desde decenas de miles de ooquistes por gramo de heces (OPG) hasta varios millones en aquellos individuos que padecen coccidiosis clínica patente. Por tanto, la mera presencia de ooquistes en las heces no es un motivo suficiente para el diagnóstico de coccidiosis. Además, hay que tener en cuenta que en infecciones agudas pueden aparecer los signos clínicos antes de la eliminación fecal de ooquistes, como consecuencia del daño producido por la multiplicación asexual del parásito. En resumen, para hacer un diagnóstico correcto, es necesario apoyarse en la clínica y epidemiología, en las lesiones que aparecen en el animal muerto y en los datos que puedan aportar los análisis coprológicos. 3.1.5.1. Diagnóstico clínico y epidemiológico Se sospecha de coccidiosis cuando se encuentran problemas digestivos, con o sin diarrea hemorrágica, en animales jóvenes criados en malas condiciones de higiene o en sistemas intensivos. Por otro lado, una mortalidad súbita hacia el destete también podría hacer sospechar de una coccidiosis sobreaguda. La disminución del crecimiento y el empeoramiento de los índices de conversión, incluso sin la presencia de signos gastroentéricos aparentes, también pueden ser indicativos de la presencia de coccidiosis en la explotación ( Sanz, 2000) . 3.1.5.2. Diagnóstico anatomopatológico Una buena necropsia debe permitir detectar lesiones típicas de la enfermedad, así como evidenciar las diferentes formas parasitarias del ciclo de Eimeria (Gregory y Catchpole, 1990) . 3.1.5.3. Diagnóstico laboratorial En los análisis coprológicos se deben de incluir técnicas de concentración por flotación, por ejemplo con ClNa. También es recomendable realizar una cuantificación de los ooquistes liberados y la determinación de las especies de Eimeria implicadas. La cuantificación suele realizare por la técnica modificada de McMaster ( Thienpont y col., 1979; Bangoura y Daugschies, 2007 ), aunque 24 REVISIÓN BIBLIOGRÁFICA recientemente se ha demostrado la utilidad de nuevas técnicas como el FLOTAC ® o el Mini-FLOTAC® ( O’Grady y Slocombe, 1980 ). La interpretación de los resultados coprológicos no es fácil, porque existe una gran variabilidad en la eliminación de ooquistes según las especies de Eimeria que estén parasitando al ganado y según el animal que se esté analizando (Chartier y cols., 1994) . En general, la determinación del número de ooquistes por gramo de heces (OPG) permite predecir el grado de parasitación, a diferencia de lo que ocurre en algunas helmintosis, donde recuentos bajos pueden estar asociados a cargas parasitarias elevadas (Eysker y Ploeger, 2000 ). Se estima que los recuentos fecales indicativos de una coccidiosis clínica serían del orden de 50.000 a 100.000 OPG. No obstante, hay ocasiones en las que los síntomas de la enfermedad pueden aparecer antes de la excreción de ooquistes (Wright y Coop, 2007) . 3.1.6. TRATAMIENTO Con el fin de evitar contagios entre los animales infectados y los no infectados, se considera importante el aislamiento y tratamiento de los animales positivos, especialmente aquellos que están padeciendo enfermedad clínica y, por tanto, eliminando gran cantidad de ooquiestes al medio (Catchpole y col., 1993) . El tratamiento prematuro e individual con antibióticos de amplio espectro orales, como la penicilina, sulfamidas con trimetropin o quinolonas, brindan resultados satisfactorios, ya que reducen las posibles septicemias bacterianas debidas al debilitamiento defensivo de la barrera mucosa intestinal durante la infección (Smith y Sherman, 2009) . El uso combinado con antiinflamatorios no esteroideos reduciría los casos de inflamación local y dolor agudo. Del mismo modo, el empleo de analgésicos y antipiréticos podría ser considerado entre los procedimientos terapéuticos para disminuir casos de fiebre en los animales sintomáticos. En casos graves, cuando se detecten problemas de deshidratación, también se recomienda realizar un tratamiento de recomposición. El uso de suero, bien por vía oral (a través de biberones, o con sonda directamente en los casos de letargia), o incluso vía intravenosa o subcutánea (menos usado en ganadería), estaría indicado para restablecer el desequilibrio electrolítico consecuencia de la deshidratación por las diarreas, casos de anorexia o incluso infecciones crónicas secundarias a la alteración de la mucosa intestinal (Argüello y Cordero del Campillo, 1999) . En todos los casos, la administración de vitaminas, vía oral o intramuscular, siempre es aconsejable. Existen numerosos fármacos específicos susceptibles de ser utilizados frente a esta enfermedad parasitaria. Entre los que caben destacar, por un lado, aquellos fármacos que se utilizan como aditivos y que se incluyen dentro de la alimentación de los animales. Estos fármacos, conocidos como coccidiostáticos, se emplean actualmente como estrategias de control de la emeriosis, actuando en las fases iniciales del ciclo del parásito. No obstante, se ha detectado cierta resistencia frente a algunos de ellos debido a la plasticidad genómica del género Eimeria , la cual permite 25 REVISIÓN BIBLIOGRÁFICA al parásito seguir replicándose en presencia de un fármaco que, en general, debería suprimir su multiplicación (Witcombe y Smith, 2014) . Incluso, se han detectado efectos secundarios no deseados en ganado tratado con este tipo de coccidiostáticos debido a su baja acción farmacológica en ciertas etapas del ciclo evolutivo del parásito (Coppens, 2013) . Por otro lado, los fármacos anticocciodiósicos son aquellos compuestos químicos utilizados, no tanto para prevenir que los animales se infecten, sino para tratar aquellos que ya se encuentran infectados, por lo que paralelamente se estimula de forma positiva el desarrollo de respuesta inmunitaria (Wang, 1997) . Aun así, los mejores resultados se obtienen cuando se administran en fases iniciales de la enfermedad (Foreyt y cols., 1990; Mundt y cols., 2005) , ya que reducen el contacto con el parásito y, por tanto, se disminuye satisfactoriamente la clínica y las lesiones producidas a nivel del digestivo. Muchos de estos fármacos interaccionan de forma específica frente al proceso de gametogonia del parásito, por lo que se reducirá drásticamente la formación de nuevos ooquistes. Algunos de ellos son capaces de disminuir la multiplicación del parásito, pero no lo eliminan completamente, por lo que limitan el control de la enfermedad (Daugschies y cols., 2007; Smith y Sherman, 2009) . Siguiendo estas premisas, a continuación se detalla el uso de los quimioterápicos que con mayor frecuencia se emplean frente a la eimeriosis. Sulfonamidas Tradicionalmente, las sulfamidas han sido unos de los fármacos más utilizados en el control de la coccidiosis de rumiantes. Así, en el ganado vacuno, la sulfaguanidina usada a una dosis de 2 g/día durante seis días, se ha observado que suprime la producción de ooquistes en infecciones subclínicas y previene la adquisición de infecciones naturales subsiguientes (Foster y cols., 1941) . Por otro lado, el alimento suplementado con una mezcla de aureomicina-sulfametazina (a partes iguales), en dosis de 100 mg y 500 mg/cordero/día, se ha demostrado que inhibe el desarrollo de una población de diferentes especies de Eimeria en corderos (Ajayi y Todd 1977) . Así mismo, en el ganado caprino, el empleo de sulfonamidas (sulfametazina y sulfaquinoxalina) interfiere en la replicación asexual del parásito (Daugschies y Najdrowski, 2005) y, como consecuencia, reduce la producción de ooquistes y la gravedad de la enfermedad. No obstante, el uso reiterado de estos fármacos ha motivado la aparición de resistencias en varias zonas del mundo (Champan y cols., 1993; Peek y Landman, 2006; Smith y Sherman, 2009; Champan y cols., 2013) . 26 REVISIÓN BIBLIOGRÁFICA Nitrofurazona Su uso terapéutico a dosis de 10-20 mg/kg/día hasta 5 días consecutivos es utilizado en ganado vacuno, ovino y caprino (Sanchez R. y cols., 2005 ). Igualmente, se ha observado que puede ser administrada con éxito en el alimento o en el agua de bebida, previniendo la mortalidad y reduciendo la morbilidad producida por E. ninakohlyakimovae, E. ovina, E. intrincata, E. parva, E. faurei y E. pallida. Amprolio Estudios clásicos indican que este compuesto es eficaz en coccidiosis de ovinos y caprinos administrado a dosis de 50-62,5 mg/Kg de peso en el agua de bebida o en el alimento de ovejas, o de 100 mg/Kg en el caso de cabras, durante cuatro o más días (Kauffma, 1996). Más recientemente, se ha corroborado que, a altas dosis, este fármaco no produce toxicidad neurológica en cabras (Young y cols., 2011) y que actúa a nivel de los merontes de primera generación impidiendo su diferenciación (Daugschies y Najdrowski, 2005) , por lo que no tendrían lugar las subsiguientes fases sexuales del ciclo del parásito ni la esporulación de los ooquistes en el medio (Arabkhazaeli y cols., 2013) . Toltrazuril y diclazuril Se ha demostrado una alta eficacia del uso de estas drogas en la fase prepatente de la enfermedad (Daugschies y Najdrowski, 2005; Ruiz y cols., 2012) actuando a nivel de la gametonia y merogonia del ciclo endógeno del parásito. A una única dosis de 20 mg/kg de toltrazuril y 0,25-1,0 mg/kg de diclazuril vía oral se ha conseguido una reducción de la sintomatología de la enfermedad y un menor número de ooquistes excretados por las heces, así como una mayor tasa de crecimiento en los animales tratados (Daugschies y Najdrowski, 2005; Taylor y cols., 2011; Ruiz y cols., 2012) . Decoquinato El decoquinato es un derivado de la quinolona desarrollado inicialmente para aves de corral en 1967 (Williams, 2006) que presenta actividad frente a los esporozoitos y los trofozoítos de diferentes especies Eimeria. También parece inhibir el transporte de electrones y la fosforilación oxidativa, actuando entre los días 1 y 10 del ciclo de los coccidios (Sanchez y cols., 2005) . Este fármaco se ha utilizado en el control de la coccidiosis en los rumiantes domésticos por más de 20 años, tanto en el tratamiento como en la prevención de la coccidiosis en terneros (Miner y Jensen, 1976; Conlogue y cols., 1984; Foreyt y cols., 1986b; Fitzgerald y Mansfield, 1989; Mage y cols., 1996) y corderos (Spelman y cols., 1989; Mage y cols., 1995a) . El uso del decoquinato también se ha demostrado útil para prevenir la coccidiosis en cabritos (Foreyt y cols., 1986a; Mage y cols., 1995b) . 27 REVISIÓN BIBLIOGRÁFICA Monensina, lasalocid, salinomicina Por último, el uso de estos fármacos, que actúan en las primeras fases del ciclo biológico del parásito, también ha sido muy extendido pero, en a diferencia del amprolio, a altas dosis sí pueden ser tóxicos (Smith y Sherman, 2009) . Romero y Sánchez (2010) , describen las acciones de los fármacos descritos anteriormente de la siguiente forma: • Diclazuril, decoquinato, toltrazuril: Inhibición del transporte de electrones y la fosforilación oxidativa. • Sulfamidas: Inhibición sinérgica del metabolismo del ácido fólico. • Amprolio: Inhibición competitiva del transporte de tiamina a través de la membrana celular. • Salinomicina, lasalocid y monensina: destrucción de la integridad de membranas. Pese al gran número de fármacos con actividad anticoccidiósica y su extendido uso en diversas especies animales para el tratamiento de esta importante enfermedad parasitológica, aún hoy en día, en Europa no hay ninguno registrado legalmente para su uso en caprinos y tampoco, ni con fines terapéuticos ni profilácticos. Este hecho hace imprescindible el establecimiento de unas estrategias de control rigurosas frente a coccidiosis en esta especie hospedadora. 3.1.7. CONTROL El control de la coccidiosis caprina se basa actualmente en empleo combinado de medidas de manejo (de la Fuente y Alunda, 1992) y tratamientos profilácticos con fármacos que presenten actividad anticoccidiósica (Vihan, 2002) . Esto último presenta, no obstante, algunos inconvenientes, como la limitación cada vez más estricta del uso de aditivos en la Unión Europea, entre ellos los coccidiostáticos. A este respecto, el informe presentado en pleno del Parlamento Europeo por Wiecher (2005) propone, en relación al uso de coccidiostáticos e histomostáticos como aditivos para piensos, que se permita su uso, ya que estas sustancias son imprescindibles actualmente en la avicultura. Sin embargo, establece que se prohíban a partir de 2009, si no hay legislación adicional, con el fin de ejercer presión suficiente para que se desarrollen productos alternativos adecuados. Un problema adicional lo constituye el desarrollo cada vez más frecuente de fenómenos de resistencia, más extendidos y estudiados en la eimeriosis aviar (Stephen y cols., 1997) , pero igualmente extensibles a las infecciones por Eimeria de pequeños y grandes rumiantes. El problema de la resistencia frente a los coccidiostáticos, 28 REVISIÓN BIBLIOGRÁFICA discutido hace ya más de una década por Chapman (1993) y el resto de limitaciones descritas anteriormente proporcionan un estímulo para el desarrollo, no sólo de nuevos fármacos, sino también de nuevas alternativas de control de la coccidiosis. En general, el control de la coccidiosis requiere de la puesta en marcha de técnicas de gestión encaminadas a reducir la contaminación del medio con ooquistes, el hacinamiento y el estrés, en combinación con un uso apropiado de coccidiostáticos y coccidiósicos eficaces disponibles en el mercado para prevenir la enfermedad clínica. Como en cualquier enfermedad parasitaria, lo primero que debe hacerse es prevenir la aparición de los signos clínicos en los animales. El segundo objetivo sería reducir la infección por coccidios durante los periodos de riesgo para asegurar un crecimiento óptimo de corderos, cabritos y terneros (Gregory y Catchpole, 1990; Chartier y cols., 2011) . 3.1.7.1. Profilaxis terapéutica El control de la coccidiosis en poblaciones de rumiantes susceptibles es una propuesta bastante difícil, por lo que se han depositado grandes esperanzas en los productos administrados con fines profilácticos (Tabla 2). El objetivo de la profilaxis anticoccidiósica es proporcionar un nivel de protección suficiente al animal expuesto para permitir que desarrolle inmunidad propia sin padecer enfermedad. Los fármacos empleados en este sentido reducen la magnitud del contagio y, por tanto, previenen la coccidiosis clínica, pero no impiden la infección (Daugschies y Naddrowski, 2005) . Sin embargo, la profilaxis terapéutica no ha de ser la única medida de control a utilizar, ya que un exceso de contaminación del entorno con ooquistes y, lo que aún es más importante, un exceso de estrés sobre el hospedador, son circunstancias que no se pueden combatir ni siquiera con el mejor de los medicamentos (Young y cols., 2011) . Como se mencionó anteriormente, el uso sistemático de coccidiostáticos en animales de recría no es estrictamente necesario. Un tratamiento mínimo en los momentos de riesgo puede ser lo más recomendable. Así, salvo casos raros, no se recomienda tratar a los animales antes de que cumplan un mes de edad. Es más, algunos trabajos recientes demuestran que la administración de tratamiento entre las tres y cuatro semanas retardaría la aparición de la inmunidad, lo que expondría a los animales a infecciones posteriores graves. En cabritos, por ejemplo, el primer tratamiento sistemático debería hacerse durante el destete, principal periodo de estrés y, por tanto, de riesgo. Un segundo tratamiento podría realizarse un mes más tarde con el fin de asegurar un mejor crecimiento de los animales. Determinadas situaciones, como los transportes entre granjas, la separación en lotes, la salida a los pastos, son capaces de exacerbar la infección por coccidios y, en estos casos, también puede estar justificado un tratamiento especial ( Benavides y Romero, 2010; Young y cols., 2011) . 29 REVISIÓN BIBLIOGRÁFICA Tabla 2. Relación de fármacos de uso terapéutico y profiláctico más frecuentes y su estrategia de empleo (Kauffman, 1996) En el contexto veterinario, la administración de fármacos puede realizarse como un procedimiento metafiláctico. La metafilaxis, al igual que la profilaxis, se aplica en forma grupal, pero no para prevenir la enfermedad sino para tratar enfermedades incipientes en determinados animales y evitar que el brote se extienda al resto. La metafilaxis aplicada al control de la coccidiosis de rumiantes debería causar significativas mejoras en las ganancias de peso, lo que resultaría en beneficios económicos para la producción (Forey, 1990) . Algunos compuestos han sido usados para este propósito en las últimas décadas, tanto para corderos (Foreyt y cols., 1979; Horton y Stockdale, 1981; Gjerde y Helle, 1991) como para terneros (McMeniman y Elliott 1995; Hasbullah y cols., 1996; Mundt y cols., 2005) , pero pocos ensayos se han realizado en coccidiosis caprina. Uno de los fármacos más utilizados y estudiados es el diclazuril, un derivado triazinona cuyo uso como medicamento metafiláctico se ha demostrado en ovinos, asociado a una reducción de la eliminación de ooquistes, a la USO DOSIS Amprolio Profiláctico Terapéutico 5-10mg/kg/día 21 días 100mg/Kg/día 5 días Nitrofurazona Terapéutico 10-20mg/kg/día 5 días Decoquinato profiláctico 0.5mg/kg 28 días (en alimento) Lasalocid Profiláctico 0.5-1.0mg/kg/día 6 semanas Monensina Profiláctico 1mg/kg durante 30 días Salinomicina Profiláctico 100ppm en alimento 3 semanas Diclazuril Terapéutico 20mg/kg tratamiento único Sulfametazina Profiláctico Terapéutico 5g/tonelada de alimento 50-100g/kg/día 4 días Toltrazuril Profiláctico Terapéutico 20mg/kg (dosis) cada 34 semanas 20mg/kg /tratamiento único 30 REVISIÓN BIBLIOGRÁFICA Los monocitos y macrófagos también participan en las reacciones inmunes innatas que se desarrollan durante el primer contacto con el patógeno. Su papel concreto en las infecciones producidas por el género Eimeria es menos conocido, pero se ha demostrado que en infecciones por E. bovis son capaces de degenerar el macroesquizonte (Friend y Stockle, 1980) . En este sentido, Taubert y cols. (2009 ) describieron que las reacciones inmunológicas desarrolladas por los macrófagos pueden afectar el resultado de las infecciones primarias y son cruciales para la transición a la respuesta inmune adaptativa frente a la eimeriosis. En sus experimentos investigaron las reacciones inmunes mediadas por monocitos y macrófagos tanto in vivo , in vitro como ex vivo frente a Eimeria bovis , una de las especies de Eimeria más patógenas en el ganado bovino. Observaron que los macrófagos se infiltraban de manera significativa en la mucosa intestinal de los terneros infectados, sobre todo después de la infección primaria. Además, los monocitos periféricos de los animales infectados, células precursoras de los macrófagos, experimentaron ex vivo un incremento de su actividad fagocítica y oxidativa después de la infección y hacia el final de la merogonia. Por otro lado, en la exposición in vitro de los macrófagos a esporozoítos, se observó una intensa fagocitosis del patógeno, no ocurriendo así con los monocitos. La fagocitosis se produjo independientemente de la viabilidad de los esporozoítos, lo que indica que la invasión activa de los parásitos no era un factor clave en el proceso. También se observó actividad fagocitaria en ausencia de suero inmune, pero la administración de suero la incrementaba significativamente, lo que sugiere que la citotoxicidad derivada de macrófagos podría ser dependiente de anticuerpos. Estos mismos autores también demostraron que el co-cultivo de macrófagos con esporozoítos y merozoítos inducía niveles distintos de transcripción génica de determinadas citoquinas y quimioquinas. En su conjunto, todos estos resultados sugieren que las reacciones inmunes innatas mediadas por monocitos y macrófagos juegan un papel importante en la respuesta inmune temprana a las infecciones por E. bovis en terneros (Taubert et., al 2009) . Los eosinófilos se consideran leucocitos multifuncionales de gran importancia en diversos procesos inflamatorios, alérgicos y parasitarios, sobre todo en las infecciones producidas por nematodos. En condiciones homeostáticas son abundantes en la lámina propia del tracto gastrointestinal, donde se ha postulado su participación en diversos procesos bilógicos de este órgano (Jung y cols., 2014) . Actúan tanto en la respuesta innata como adquirida. En general, en animales primoinfectados por el género de Eimeria se observa un aumento en los recuentos hematológicos de eosinófilos, tal y como se ha demostrado en pollos primoinfectados con E. acervulina (McFarlane y cols., 1989) , en ratones con E. vermiformis (Linh y cols., 2009) y en pavos primo-infectados con E. adenoeides (Gadde y cols, 2009 ). Aunque menos documentado, también en rumiantes se ha demostrado la participación de los eosinófilos en la respuesta inmune frente a la 37 REVISIÓN BIBLIOGRÁFICA coccidiosis, aunque no claramente en las respuestas primarias. Así, en un estudio sobre la eficacia del toltrazuril al 5% en suspensión frente a especies de Eimeria de rumiantes ( E. bovis y E. zuernii ) el examen histológico demostró que los recuentos de eosinófilos eran más comunes en el íleon y colon de los animales clasificados como resistentes y tendían a estar asociados a concentraciones más elevadas de TNFα. Por otro lado, se ha observado que, aproximadamente, el 70% de los caprinos de raza pirenaica, en los que se encontró un porcentaje de parasitación por coccidios del género Eimeria del 84,32%, presentaba una marcada eosinofilia (Fernández del Palacio y cols., 1985) . Las células dendríticas son un tipo celular especializado del sistema inmunológico, cuyas funciones principales son capturar y procesar los antígenos y presentarlos a los linfocitos B y T para que éstos inicien repuestas inmunológicas específicas. Por este motivo, se consideran mensajeras entre el sistema inmune adaptativo e innato (Clark y cols., 2000) . Las células dendríticas están presentes en pequeñas cantidades en los tejidos que están en contacto con el ambiente externo, por lo que se encuentran principalmente en la piel, en el revestimiento interno de la nariz, los pulmones, el estómago y, en general, en todo el tracto gastrointestinal. También pueden encontrarse en estado inmaduro en la sangre. Las células del sistema inmune, incluyendo las células dendríticas, poseen los denominados receptores de reconocimiento de patrones moleculares ( PRR-Pattern recognition receptor ), que son capaces de reconocer distintos patrones moleculares asociados a patógenos ( PAMP-Pathongen-associated molecular pattern ) presentes en virus, bacterias, hongos y protozoos, como pueden ser su material genético, lipopolisacáridos (LPS), etc. Los PRR mejor estudiados son los receptores de tipo Toll ( TLRToll-like Receptors ), muy importantes en la biología de las células dendríticas (Reis y Sousa, 2004) . Las distintas vías de activación de estas células pueden ser dependientes o independientes de los PAMPs. Además de los TLRs, responsables de señalizaciones intracelulares, existen otros PRR incluidos en la familia de las lectinas de tipo C que están siendo objetivo de numerosos estudios (Figdor y cols., 2002; Kanazawa, 2007) . Las células dendríticas están frecuentemente implicadas en la iniciación de la respuesta inmune temprana del hospedador frente a patógenos microbianos, pero su actividad funcional se extiende más allá de este importante papel durante el inicio de la actividad (Denkers y cols., 2004) ya que, una vez activadas, estas células son capaces de segregar un importante número de citoquinas proinflamatorias (Moser y Murphy. 2000) . Este tipo celular es fuente importante de IL-12 en el sistema nervioso central durante la infección crónica de Toxoplasma , lo cual podría estar relacionado con la protección conferida frente al parásito (Fischer y cols., 2000) . Estos resultados coinciden con otros donde se muestra que la producción continua de IL-12 durante los procesos crónicos es necesaria para evitar la reactivación del parásito (Yap y cols., 2000) . También se ha demostrado que las células dendríticas de ratón pueden ser activadas mediante INF-γ y desarrollar una actividad microbiana frente a Toxoplasma dependiente de oxígeno (Aline y cols., 38 REVISIÓN BIBLIOGRÁFICA 2002) , y que antígenos proteicos de protozoos intestinales como Eimeria son estimuladores muy potentes in vivo de la liberación de citoquinas a partir de células dendríticas (Rosenberg y cols., 2005) , todo lo cual subraya el papel de este tipo celular en la infecciones por Apicomplexa. Los mastocitos son leucocitos derivados de precursores hematopoyéticos. Circulan en la sangre en su forma inmadura hasta que migran a tejidos vascularizados, donde se diferencian con ayuda del stemcell factor y de otras citoquinas segregadas por células endoteliales y fibroblastos. Se localizan en la mayoría de los tejidos, particularmente en aquellas localizaciones que están en contacto con el exterior, por ejemplo, piel, vías aéreas y tracto gastrointestinal (Urb y Sheppard, 2013) . Contienen mediadores primarios como proteasas, proteoglicanos o histamina, que pueden ser almacenados y liberados sin necesidad de sensibilización previa. Además, poseen mediadores secundarios como algunas prostaglandinas y citoquinas que se relacionan con el aumento de la permeabilidad vascular y la inducción de la respuesta inflamatoria. Los mastocitos se activan inicialmente al contactar con moléculas del parásito, o del tejido dañado, con moléculas de la vía alternativa del complemento o con bacterias (Balic y cols., 2000) . Debido a su localización, su plasticidad y la variedad de mediadores que producen, los mastocitos se consideran células inmunomoduladoras y efectoras importantes que pueden ejercer un puente entre las respuestas innatas y adaptativas (Salinas y cols., 2007; Metcalfe, 2008) . Confirmando su papel como células inflamatorias primarias, en infecciones por E. tenella y E. acervullina en pollo (Petrone y cols., 2002; Metcalfe, 2008) se ha observado que el número de mastocitos aumenta en la mucosa del ciego durante el proceso inflamatorio agudo. Los mastocitos intraepiteliales se consideran su última fase efectora, al haberse liberado sus gránulos con el fin de alterar el medio ambiente parasitario; en este estado, los mastocitos reciben el nombre de leucocitos globulares (LG) (Balic y cols., 2000) . Las células NK proceden de la familia de las células linfoides innatas (Spits y Di Santo, 2011) y presentan un progenitor común (Spits y Di Santo, 2011) . Se consideran esenciales como parte de la respuesta inmune innata, pero también se ha descrito que colaboran en la respuesta inmune adaptativa (Vivier y cols., 2008) . Además de sus acciones defensivas frente a agentes externos, las células NK tienen la propiedad de respetar las células propias de cada individuo, reconociendo así su identidad biológica. Esto se debe a la presencia en este tipo celular de moléculas HLA-I que actúan a modo de escudo protector. Por ello, cuando las propias células pierden estas moléculas, son destruidas por las células NK. Su participación en la respuesta frente a la coccidiosis se ha descrito en diversas especies de Eimeria aviares, entre ellas, E. acervulina, E. tenella o E. maxima (Lillhoj, 1989; Lillhoj y Bacon, 1991) . Concretamente, en infecciones por E. acervulina se ha observado que la actividad de las células NK esplénica e intestinal se incrementa durante la fase temprana de la infección. Entre las barreras físicas que participan en la respuesta inmune innata frente a 39 REVISIÓN BIBLIOGRÁFICA la coccidiosis cabe resaltar el papel del revestimiento endotelial de los vasos sanguíneos y linfáticos. El endotelio , además de participar en la modulación de la homeostasis metabólica y en la hemodinámica vascular e intercambio celular, debido a su ubicación, también presenta una importante función inmunorreguladora, por lo que las células endoteliales se encuentran entre las primeras células que interactúan con los patógenos externos (Mai y cols., 2013) . Las células endoteliales son las células hospedadoras específicas de muchos parásitos Apicomplexa in vivo , sobre todo, de distintas especies del género Eimeria que forman macroesquizontes (Taubert y cols., 2006; Alvarez y cols., 2014) . Durante la infección y desarrollo del parásito, las células endoteliales actúan modulando la transcripción génica de moléculas inmunorreguladoras de la respuesta inmune, como citoquinas, moléculas de adhesión y quimioquinas. Se ha demostrado, por ejemplo, que la infección in vitro de células endoteliales del cordón umbilical de bovinos (BUVEC) con esporozoítos de E. bovis y taquizoitos de T. gondii y de N. caninum (Taubert y cols., 2006) produce una activación de la célula hospedadora que resulta de un aumento de la transcripción de genes que codifican moléculas proinflamatorias y inmunomoduladoras (GRO-a, IL-8 e IP-10, CC2, GM-CSF, COX-2 e iNOS, entre otras), que son importantes para la respuesta inmune innata y la transcripción hacia una respuesta adquirida o específica. Como describieron Taubert y cols. (2006) , las diferencias que presenta E. bovis respecto a T. gondii y N. caninum indicarían una particular estrategia de evasión de los esporozoítos de E. bovis (Taubert y cols., 2006) , debido a la necesidad de persistir en el hospedador por un tiempo más largo, durante el cual podría evitar el que se desencadene un proceso inflamatorio. En este mismo sentido, se ha comprobado que las especies de Eimeria que desarrollan merogonias en el interior de las células endoteliales tienden a desencadenar una modulación compleja del transcriptoma y el proteoma para asegurar su desarrollo (Taubert y cols., 2010) . Además, el desarrollo de E. bovis parece que previene la apoptosis de la célula hospedadora endotelial mediante la regulación de la expresión génica de moléculas anti-apoptóticas, según describieron Lang y cols., (2009) . Por otro lado, se ha podido constatar que el crecimiento y desarrollo de los macroesquizontes de esta especie de Eimeria bovina demandan una gran cantidad de moléculas para la biogénesis de la membrana celular, como el colesterol y ácidos grasos (Hamid y cols., 2014) , de ahí que la transcripción génica de la biosíntesis y el metabolismo de los lípidos de la célula hospedadora endotelial infectada con E. bovis se encuentre incrementada (Taubert y cols., 2010) . Los coccidios están considerados autotróficos en la síntesis del colesterol, por lo que necesitan utilizar el colesterol de la célula hospedadora para la biogénesis de su membrana durante el desarrollo de la fase de replicación intracelular, como se ha demostrado en T. gondii y C. parvum (Coppens y cols., 2000; Ehrenman y cols., 2013) . En las especies patógenas de Eimeria en rumiantes, que producen un número mayor de merozoítos, los requisitos de colesterol son, incluso, más altos (Hamid y cols., 2014) . 40 REVISIÓN BIBLIOGRÁFICA Recientemente, se ha encontrado un nuevo mecanismo relacionado con la respuesta inmune innata que consiste en la formación de trampas extracelulares (ETs). Este mecanismo fue Inicialmente descrito en PMN, por lo que recibió el nombre de NETs (Brinkmann y cols., 2004; Brinkmann y Zychlinsky, 2007; Hellenbrand y cols., 2013; Jenne, 2013) , pero actualmente también se ha observado en otros tipos celulares, tales como eosinófilos (Yousefi y cols., 2008) , mastocitos (von köckritz-Bliclwede y cols., 2008) , monocitos (Muñoz-Caro y cols., 2014; Pérez y cols., 2015) y macrófagos (Aulik y cols., 2012; Bonne-Année y cols., 2014) frente a diferentes patógenos. La liberación de ETs puede promover la muerte extracelular y/o la inmovilización de agentes patógenos virales, bacterianos o fúngicos, levaduras de distintos géneros y determinados parásitos. La formación de ETs se ha observado como respuesta innata en numerosos vertebrados e invertebrados (Hermosilla y cols., 2014; Silva y cols., 2014) , ya no sólo asociadas a situaciones patológicas de origen infeccioso. Tales estructuras están formadas por redes de matrices de ADN mitocondrial y proteínas celulares que resultan de un complejo proceso en el que parece cobrar un importante protagonismo el sistema NADPH oxidasa como mecanismo iniciador; aparte, la mieloperoxidasa (MPO) y la elastasa de los neutrófilos (NE) también parecen contribuir a regular la liberación adecuada de ETs (Brinkmann y cols., 2004; Brinkmann y Zychlinsky, 2012) . Behrendt y cols. (2010) describieron por primera vez la formación de ETs como un mecanismo efector adicional de los PMN en respuesta a la coccidiosis producida por el género Eimeria , concretamente en E. bovis; posteriormente, se demostró que también E. arloingi era capaz de estimular el desarrollo de ETs en neutrófilos (Silva y cols., 2014a) . En ambos casos, las denominadas “trampas extracelulares” se demostraron mediante la visualización, a través de ensayos de fluorescencia y microscopia electrónica de barrido (SEM), de delicadas fibras procedentes de la red extracelular, originadas a partir de PMN, capaces de atrapar esporozoítos y otros estadios de ambas especies de Eimeria . Posteriormente, también se ha demostrado que los monocitos pueden sufrir ETosis en respuesta a la infección por esporozoítos de E. bovis y taquizoítos de B. besnoiti siguiendo un mecanismo similar (Muñoz-Caro y cols., 2014) . La formación de ETs se ha puesto de manifiesto, igualmente, en otros protozoos del phylum Apicomplexa, entre ellos Plasmodium falciparum y Toxoplasma gondii (Baker y cols., 2008; Abi Abdllah y cols., 2012) . 3.2.3. Respuesta inmune específica en rumiantes La respuesta inmune específica se caracteriza porque es efectiva ante aquellos antígenos frente a los cuales se ha iniciado y desarrollado. Este tipo de respuesta es mediada por los linfocitos, que pueden ser de dos tipos: linfocitos B y linfocitos T. Los linfocitos T, a su vez, se diferencian en linfocitos T colaboradores (Th), linfocitos T citotóxicos (Tc) y linfocitos T supresores/reguladores (Ts). 41 REVISIÓN BIBLIOGRÁFICA La respuesta inmune específica puede ser de dos tipos, humoral y celular, aunque esta separación es más de tipo didáctico que real. En general, se considera que la respuesta es de tipo humoral cuando el elemento efector final son las inmunoglobulinas formadas por los linfocitos B, mientras que cuando participan los linfocitos T, tanto colaboradores (Th) como citotóxicos (Tc), la respuesta inmune suele catalogarse como de tipo celular. Ambos tipos de respuesta se han descrito implicadas en la coccidiosis de rumiantes (Daugschies and Nadjdrowski 2005) . 3.2.3.1. Respuesta inmune humoral frente a la coccidiosis en rumiantes Aunque, tradicionalmente, se ha considerado que la respuesta inmune celular tiene más relevancia en la coccidiosis, cada vez hay más trabajos centrados en el estudio de la respuesta inmune humoral y su papel en la inmunidad protectora frente a infecciones por diferentes especies de Eimeria . En el estudio de la respuesta inmune innata se comentó que los mastocitos presentan en su membrana receptores de alta afinidad para la IgE, por lo que, en animales previamente sensibilizados al antígeno, la unión de la inmunoglobulina puede provocar su degranulación (Miller, 1996) . Por este motivo, se ha sugerido que los mastocitos podrían ejercer de células puente entre la respuesta inmune innata y adaptativa (Salinas y cols., 2007; Metcalfe, 2008) . Las células NK también disponen de receptores de inmunoglobulinas (FCR) capaces de reconocer objetivos recubiertos con anticuerpos; cuando esto ocurre, la célula inicia su actividad como mecanismo de citotoxicidad dependiente de anticuerpos. Este tipo celular también produce citoquinas inflamatorias que influyen de manera decisiva en el desarrollo de respuestas inmunes adaptativas (Vivier y cols., 2008) . La respuesta inmune humoral frente a la eimeriosis caprina no ha sido tratada en profundidad. En los escasos estudios realizados en este sentido destacan los trabajos de Kanyari (1994) , quien analizó mediante inmunohistoquímica la antigenicidad de diversas formas evolutivas de E. apsheronica en la cabra y el perfil de anticuerpos en dos razas de caprino sujetas a infecciones multiespecíficas de Eimeria que incluían principalmente E. christenseni (49%) , E. apsheronica (29%), y menores proporciones de E. arloingi, E. hirci, E. ninakohlyakimovae y E. alijevi (Kanyari, 1988) . Para el análisis de anticuerpos desarrollaron un ELISA indirecto utilizando como antígeno homogenizados de ooquistes esporulados de estas mismas especies de Eimeria. Relativamente más numerosos son los estudios inmunológicos realizados sobre eimeriosis bovina y ovina. Así, en bovinos, se ha observado que la respuesta inmune humoral en animales infectados por coccidiosis se desarrolla rápidamente, llegándose a observar un alto título de anticuerpos en el suero. Inicialmente, se produce un incremento de la IgM y, más tarde, de la IgG, pudiendo aparecer, además, otro tipo de anticuerpos específicos como la IgA (Hughes, 1985) . En la mayoría de los casos, la respuesta humoral se ve potenciada cuando los animales se 42 REVISIÓN BIBLIOGRÁFICA ven expuestos continuamente a los ooquistes. Del mismo modo, en infecciones experimentales con E. faurei y E. ovinoidalis en ovejas se demostró la presencia de anticuerpos, tanto en infecciones primarias como secundarias (Nolan y cols., 1987) . En dichos trabajos se demostró que existían considerables reacciones cruzadas entre las especies y que, en general, el incremento sérico de las inmunoglobulinas aparecía demasiado tarde como para ser de utilidad para un diagnóstico individual, aunque sí podría serlo para un diagnóstico a nivel de rebaño. El estudio de la respuesta humoral y su importancia para el control de la enfermedad se ha abordado más ampliamente en la coccidiosis aviar, donde se ha demostrado la capacidad de los anticuerpos para bloquear la invasión, el desarrollo y la trasmisión del parásito, así como la existencia de una inmunidad pasiva (Wallach, 2010) . En otras coccidiosis, como las producidas por Cryptosporidium y Toxoplasma gondii en cabras, también se ha señalado la importancia y el papel que juegan los anticuerpos, no sólo a nivel periférico, sino también en la mucosa intestinal de los animales infectados (Gomez Morales y cols., 2022; Conde y cols., 2001) . Por ejemplo, el análisis de la respuesta de IgG en cabras infectadas experimentalmente con Toxoplasma gondii utilizando un ELISA indirecto reveló que los anticuerpos podían detectarse a los 14 días después de la inoculación (p.i.), alcanzando un pico al día 35 p.i. y mostrando fluctuaciones ligeras hasta el final del experimento (91 p.i.) (Conde y cols., 2001) . En el mismo estudio se identificaron mediante Western-blot un panel de péptidos reconocidos específicamente por la IgG. En general, en las infecciones por coccidios se ha demostrado que los principales isotipos de inmunoglobulinas implicados son la IgG2, la IgM y la IgA (Faber y cols., 2002 ). Aunque la IgG2 se considera la fracción principal en la respuesta humoral en la eimeriosis, el tipo de la respuesta serológica puede variar dependiendo del nivel infección (Hughes, 1985) . Los tres isotipos se han correlacionado positivamente con la eliminación de ooquistes, siendo dependiente esta correlación de la dosis infectante que el ternero haya ingerido durante la primoinfección y de la severidad de la enfermedad. A pesar de ello, se sabe que la inmunidad humoral no es suficiente como para controlar una reinfección por Eimeria (Daugschies y Najdrowski, 2005 ) y que, aunque los anticuerpos reflejan la exposición al parásito, la protección que confieren no es absoluta (Fiege y cols., 1992) . Los anticuerpos pueden transferirse por el calostro, lo que podría constituir un mecanismo de inmunidad pasiva frente a la coccidiosis en rumiantes. Este hecho fue publicado por Gregory y cols. (1989) y Fiege y cols. (1992 ), quienes, tras administrar calostro con un alto contenido en IgG a corderos infectados por E. crandallis , observaron un mayor título de anticuerpos en los animales alimentados con calostro que en aquellos que no lo recibieron. En las infecciones experimentales se observó un aumento considerable en los niveles de anticuerpos IgG1 e IgG2, mientras que los valores de IgM aumentaron sólo ligeramente. Profundizando en el estudio de la influencia del calostro sobre la transmisión pasiva de anticuerpos en la 43 REVISIÓN BIBLIOGRÁFICA coccidiosis, Fayer y cols. (1992) concluyeron que la administración de calostro procedente de vacas inmunizadas con diferentes antígenos de Eimeria acervulina (esporozoítos, merozoítos y antígeno recombinante de merozoíto) reducía el desarrollo in vivo e in vitro del parásito. Como resultado, con excepción de los inmunizados con antígeno recombinante, todos los pollos inmunizados eliminaron menos ooquistes por las heces y presentaron menos etapas de desarrollo parasitario en las secciones histológicas. Posteriormente, Faber y cols. (2002) , tras una infección experimental con E. bovis en terneros, no lograron demostrar inmunoprotección mediante la administración de anticuerpos del calostro materno, por lo que ha sido motivo de controversia el si los anticuerpos maternos tienen efectos inmunoprotectores o no en la coccidiosis de rumiantes (Catchpole y Devonshire, 1989 y Gregory y Catchpole, 1989; Gregory y cols., 1989b) . Sí se considera más aceptado, como se ha descrito en ovinos ( Reeg y cols., 2005) , el que los niveles de anticuerpos maternos presentan un marcado descenso después del nacimiento y un posterior aumento debido a nuevos anticuerpos específicos sintetizados. Dado que la vida media de la IgG1, el isotipo predominante transmitido en las ovejas con el calostro (Reynolds y Griffin, 1990) , alcanza un pico máximo a los 11-13 días (Klobasa y Werhahn, 1989; Watson, 1992; Domínguez y cols., 2001 ) el período de tiempo en torno a 40 días después del nacimiento podría representar un período en el que los anticuerpos maternos se superponen a los producidos por los corderos. De hecho, a esta edad se han observado correlaciones directas entre anticuerpos y el patrón de excreción de ooquistes, lo que sugiere que los corderos habrían desarrollado ya una respuesta inmune frente a las infecciones por coccidios. Sin embargo, Reeg y cols., (2005) no pudieron confirmar hipótesis previas según las cuales los animales con bajos niveles de anticuerpos iniciales frente a los antígenos de Eimeria tienden a alcanzar finalmente títulos altos y viceversa Gregory y Catchpole (1989) , aunque parece lógico desde el punto de vista inmune (Morein y cols., 2002) . De cualquier forma, la respuesta de anticuerpos en corderos ha de considerarse el resultado de la interacción entre la inmunidad maternal pasiva y activa (resultado de la infección natural), el tipo de antígeno y otros factores, por lo que las características y la intensidad de la respuesta no siempre son predecibles (Watson y Gill, 1991; Morein y cols., 2002). 3.2.3.2. Respuesta inmune celular en la coccidiosis La respuesta inmune de tipo celular es compleja en sus efectos y acciones finales, así como en su iniciación y desarrollo. Además, ha de tenerse en cuenta que, durante todo el ciclo del parásito dentro del tracto gastrointestinal, los distintos estados parasitarios pueden generar una respuesta específica, lo cual complica aún más la respuesta inmune celular que se desarrolla frente a los coccidios. De hecho, cualquier factor que afecte negativamente la capacidad de respuesta inmunológica 44 REVISIÓN BIBLIOGRÁFICA del hospedador puede favorecer que el parásito exprese todo su potencial de multiplicación, llegando a la etapa de gametogonia y, por tanto, incrementando el número de células afectadas y la gravedad de la enfermedad. Sin embargo, lo habitual es que la exposición natural al coccidio asegure un contacto continuo que permita ir desarrollando una inmunidad específica frente a las distintas formas parasitarias del ciclo endógeno del parásito, y así evitar casos clínicos en contactos posteriores (Daugschies y Najdrowski, 2005) . En condiciones normales, tras el pico de eliminación de ooquistes, la enfermedad es autolimitante en los terneros y, a partir de ese momento, se reduce la prevalencia y los niveles de excreción. Existen claras diferencias en la biología de coccidios de rumiantes y la especies Eimeria en roedores, lo que arroja dudas sobre la posibilidad de establecer comparaciones directas entre unas especies y otras, por ejemplo, a la hora de abordar las diferentes estrategias de evasión de la respuesta inmune del huésped en el curso de una infección primaria. Así, los coccidios de roedores parecen evadir los ataques del sistema inmune repitiendo antigénicamente diferentes generaciones de merozoítos de forma rápida hasta que se desarrollan las etapas sexuales (Long, 1978) , mientras que, al menos algunos coccidios en rumiantes, incluyendo E. bovis en el ganado vacuno y E. ninakohlyakimovae en caprinos, producen los llamados macroesquizontes, capaces de liberar grandes cantidades de merozoítos. Además, el ciclo endógeno en rumiantes suele ser mucho más largo. Por ejemplo, en E. bovis , la maduración de los merozoítos necesita de 14-18 días (Hammond y cols., 1964) ; posteriormente, las etapas sexuales se desarrollan rápidamente y los ooquistes se liberan después de 18-21 días p.i. (Hammond y cols., 1964) . Estas diferencias en cuanto a estrategias de evasión y duración del desarrollo serían el resultado de reacciones particulares del sistema inmune del huésped, posiblemente relacionadas con la respuesta inmune celular, pero los estudios realizados hasta el momento en rumiantes son escasos (Speer y cols., 1985; Hughes y cols., 1989; Aleksandersen y cols., 1995) . La inmunidad celular es considerada la más importante frente a la eimeriosis, dado que las formas intracelulares del parásito (esquizontes, macrogamentos, microgametos), teóricamente, no pueden ser alcanzados por los anticuerpos. Las células parasitadas expresan en su superficie antígenos que son reconocidos por el sistema inmune, implicando a diferentes poblaciones celulares de linfocitos T, algunas de ellas capaces de desarrollar mecanismos de citotoxicidad (Rose, 1987) . Numerosos estudios han demostrado que, probablemente, se produzca una polarización hacia una respuesta Th1 frente a las primeras etapas del desarrollo parasitario, por ejemplo, los esporozoítos o los esquizontes inmaduros (de < 8 días), lo cual se considera de gran importancia para el control del parásito después de la infección (Rose y cols., 1992b; Shi y cols., 2001a) . Por otro lado, al analizar la participación de las subpoblaciones de células T en el curso de infecciones primarias y en reinfecciones en ratones se ha observado que la población de linfocitos T CD4+ se involucra, particularmente, en resolver las 45 REVISIÓN BIBLIOGRÁFICA infecciones primarias, regular su duración y el nivel de la eliminación de ooquistes, mientras que los T CD8+ estarían más implicados en las reinfecciones (Rose y cols., 1992a; Findley y cols., 1993; Ovington y cols., 1995; Smith y Hayday, 2000; Shi y cols., 2001a) . Del mismo modo, Hermosilla y cols. (1999) observaron que en terneros inoculados con ooquistes de E.bovis, las células T CD4+ circulantes aumentaban durante la prepatencia de la infección primaria decayendo tras el período patente (a los 25 días p.i.). Coincidiendo con esta observación, las necropsias realizadas el día 35 p.i. demostraron un aumento de tamaño en todos los nódulos linfático, incluso de los esplénicos y de áreas más lejanas. Durante la postpatencia, el recuento de células T CD4+ en órganos linfáticos fue mayor que el de células T CD8+, lo que confirmaría que las células CD4+ están implicadas en la terminación de una infección primaria, mientras que los CD8+ median la inmunidad en reinfecciones (Rose y cols., 1992) . Los linfocitos T CD8+ tienen propiedades citotóxicas y pueden actuar sobre células con Ag fijados al Complejo Mayor de Histocompatibilidad (MHC). Este tipo celular se ha observado que aumenta en la circulación periférica al comienzo de la infección con E. bovis pero luego decae, incluso durante el periodo de patencia, lo que sugiere que su consolidación en la respuesta efectiva surge tras la primoinfección. En reinfecciones (posteriores al día 35 p.i.) aumenta la presencia local de células CD8+ a nivel de la mucosa intestinal, además de en otros órganos como bazo y ganglios; tal incremento coincidiría en el tiempo con un descenso en la circulación periférica (Hermosilla y cols., 1999) . También en aves parece ser esta línea de linfocitos la asociada a la respuesta en reinfecciones, lo cual se ha comprobado al comparar diferentes líneas genéticas de pollos con mayor y menor capacidad de respuesta inmune frente a la coccidiosis (Lillehoj, H. y cols., 2004). Un aumento en la expresión de CD2+ en células T se ha relacionado con la activación y el aumento de la capacidad de reconocimiento antigénico de estas células (Davis y Van der Merwe, 1996) , un proceso en el que, tanto las células T CD8+ como las T CD4+ pueden estar implicadas (Baldwin y cols., 1988) . Se sabe poco sobre la participación de las células T CD2+ en las infecciones de Eimeria . Sin embargo, su incremento porcentual, de forma persistente en sangre periférica durante la infección experimental con E. bovis, sugiere un intenso estímulo antigénico, probablemente, debido al desarrollo de la primera esquizogonia (Hermosilla y cols., 1999) . En estudios recientes se ha investigado el posible papel de los receptores celulares αβ+TCR y γδ+TCR, y se ha observado que estas últimas células también pueden reconocer antígenos de manera independiente de MHC (De Libero, 1997) y que se encuentran predominantemente en tejidos epiteliales (Stingl y cols., 1987; Bonneville y cols., 1988; Goodman y Lefrançois, 1988) , el área principal donde las especies de Eimeria se están desarrollando. Son especialmente numerosos en el ganado bovino y ovino (Mackay y Hein, 1989; Hein y Mackay, 1991) , pero su papel 46 PRESENTACIÓN DE ARTÍCULOS Estos ooquistes se emplearon para infectar experimentalmente cabritos que fueron adquiridos con 1-4 días y mantenidos bajo condiciones libres de parásitos. Los cabritos fueron separados en tres grupos (n = 3). En dos ensayos diferentes se evaluó la inmunoprotección conferida por primoinfecciones (Grupo I: S4PI) y posterior reinfección con ooquistes de E. ninakohlyakimovae (Grupo II: S7RI). Como control de infección se utilizaron animales no infectados (Grupo III: C). El nivel de inmunoprotección se evaluó en base a parámetros productivos (peso corporal), clínicos, parasitológicos (recuentos de ooquistes en heces) e histopatológicos. El periodo prepatente, tanto en animales primocomo reinfectados, osciló entre 14-15 días, siendo mayor la proporción de cabritos que comenzaron a liberar ooquistes en heces a los 15 días post-infección. En su conjunto, el número de ooquistes por gramo de heces fue menor en los animales reinfectados (grupo II) que en el correspondiente control de reinfección (Grupo I). Con respecto a los parámetros de producción, el grupo I mostró tasas de reducción de crecimiento entre las 5 y 9 semanas en comparación con controles, mientras que el grupo II tales diferencias se encontraron entre las 8 y 9 semanas. La tasa de crecimiento global fue significativamente mayor en el grupo control III en comparación con cualquiera de los animales infectados. A pesar de la gravedad de los signos clínicos, las alteraciones hematológicas no fueron tan sorprendentes como sería de esperar, sino relativamente moderadas. Los recuentos de leucocitos sanguíneos demostraron indicios claros de eosinofilia, así como un mayor número de monocitos en cabritos durante la infección primaria, en concreto a las 7 semanas. Sin embargo, las alteraciones leucocitarias no fueron significativas. A nivel anatomopatológico, se encontraron lesiones inflamatorias con extensas de infiltración eosinofílica en la mucosa intestinal e infiltración difusa de otras poblaciones celulares. La reducción significativa de los recuentos de OPG en los animales reinfectados por E. ninakohlyakimovae en comparación con los controles de infección, así como de la gravedad clínica de la enfermedad proporcionan claras evidencias de que la cepa GC es apta para inducir respuestas inmunes protectoras, un fenómeno bien documentado para otras especies de Eimeria . Estos datos indicarían que la cepa específica de E. ninakohlyakimovae aislada en Gran Canaria (GC) podría ser empleada para futuros estudios terapéuticos o inmunológicos en la coccidiosis de caprina. 52 PRESENTACIÓN DE ARTÍCULOS Isolation of an Eimeria ninakohlyakimovae field strain (Canary Islands) and analysis of its infection characteristics in goat kids 1,*Ruiz, A., 1Matos, L., 1Muñoz, M. C., 2Hermosilla, C., 1Molina, J. M., 3Andrada, M., 3Rodríguez, F., 1Perez, D., 1Lopez, A., 1Guedes, A., 2Taubert, A. 1Department of Pathology, University of Las Palmas de Gran Canaria, Gran Canaria, Spain 2Institute of Parasitology, Faculty of Veterinary Medicine, Justus Liebig University Giessen, Giessen, Germany 3Department of Anatomy and Compared Anatomy Pathology, Faculty of Veterinary Medicine, University of Gran Canaria, Gran Canaria, Spain Abstract The current study was conducted to isolate a field strain of Eimeria ninakohlyakimovae , characterize its infectivity and the response to challenge under experimental conditions. The isolated strain (GC) induced a prepatent period of 14-15 days p. i., a patency of 7 ± 2 days and a noticeable pathogenicity in infected goat kids. Challenge trials resulting in a decrease of oocysts per gram counts as well as a milder intensity of clinical signs in re-infected animals indicated the capacity of this strain to induce protective immune response. Altogether, the data reported in the present study suggest that the strain E. ninakohlyakimovae GC is a useful tool for the investigation of mechanisms of pathogenicity as well as host protective immune response in caprine coccidiosis, representing a valuable prerequisite for the development of future strategies in prophylaxis and control of this important parasitic disease in goat. Key words: Eimeria ninakohlyakimovae - Apicomplexa - Coccidiosis - Caprine *Corresponding author: Antonio Ruiz Reyes Department of Animal Pathology Tel: +9284511133; Fax: +92845341 Email :[email protected] 53 PRESENTACIÓN DE ARTÍCULOS 1. Introduction Infections with different subspecies of the apicomplexan genus Eimeria represent one of the most common parasitoses in goat productive systems worldwide (Koudela and Boková, 1998). Depending of the mode of management, caprine coccidiosis may affect 100% of goat kids within the age range of 4-10 weeks, and cause severe economic losses by affecting animal health and profitability of the goat industry (Koudela and Boková, 1998; Ruiz et al., 2010). Reports for the Canary Islands (Spain), where the goat industry represents an important resource for farmers (Fresno et al., 1994), indicate the most frequent species of Eimeria in caprine flocks are Eimeria arloingi Marotel 1905, E. ninakohlyakimovae Yakimoff and Rastegaieff 1930 emend Levine 1961, E. alijevi Musaev 1970 and E. caprina Lima 1979 (Ruiz et al., 2006). Within this spectrum, E. ninakohlyakimovae showed the highest pathogenicity in parasitized animals, especially in goat kids (Ruiz et al., 2006). Analogous to the bovine system, where the most pathogenic Eimeria species infect endothelial cells and develop into macromeronts (Vieira et al., 1997), E. ninakohlyakimovae also resides in this peculiar location and shows similar developmental features (Ruiz et al., 2010) leading to severe intestinal disease characterized by catarrhal diarrhoea, weight loss, dehydration and stunted growth (Koudela and Bokova, 1998). In contrast to other well characterized eimerian infections in the murine (Shi et al., 2001; Al-Quraishy et al., 2011), avian (Rothwell et al., 2004) and bovine (Hermosilla et al., 1999, 2008; Taubert et al., 2008, Sühwold et al., 2010) system, caprine eimerian infections have been neglected in the past and only limited numbers of studies were conducted on this topic. These reports are mainly restricted on some investigations concerning the biology or the pathogenic effects of E. ninakohlyakimovae in countries such as Brasil (Vieira et al., 1997) or China (Dai et al., 2006). The high prevalence and the severe pathogenicity of this particular Eimeria species in the Canary Islands suggest E. ninakohlyakimovae as an ideal model for the study of the host immune response and the mechanisms of pathogenicity in the caprine system. In general, the termination of Eimeria spp. primary infections as well as the control of homologous challenge infections is based on host cellular adaptive immune reactions (Sühwold et al., 2010). Several investigations dealing with rodent model systems suggest that responses to primary infections are predominantly controlled by CD4+ T cells with Th1-associated T cell reactions being key to the control of primary infection, whilst cytotoxic CD8+ T cells seem to be the major effector cell type against challenge infections (Rose et al., 1992; Findly et al., 1993; Ovington et al., 1995; Smith and Hayday 2000; Shi et al., 2001). However, data generated in rodent models may only be of limited value for the ruminant system, as most of the pathogenic Eimeria species in ruminants (e. g. E. bovis Zublin 1908 , E. zuernii Rivolta 1878 , E. bakuensis Musaev 1970 , E. arloingi, E. ninakohlyakimovae ) develop differently from the rodent ones with respect to primary host cells, the formation of macromeronts and duration of replication, i. e., features that will potentially influence developing immune responses. In addition, cellular adaptive immunity against Eimeria spp. is reported as a species specific (Rose 1987) or even strain specific reaction (Shirley and Bellatti 1988; Fitz-Coy 1992; Martin et al., 1997; Smith et al., 2002). In consequence, protective cross immunity is rare giving reasons for immunological studies performed on defined individual Eimeria species in the respective host. Owing to the current lack of investigations on cellular immune responses against Eimeria spp. affecting goats, there is an urgent need for defined caprine eimerian strains for basic research and for the development of strategies on prophylaxis and control of the disease. 54 PRESENTACIÓN DE ARTÍCULOS The present study aims to isolate a defined strain of E. ninakohlyakimovae from the field and to evaluate its infectivity, pathogenicity as well as the development of protective immunity in the caprine host. 2. Material and methods 2. 1. Animals Majorera breed goat kids were purchased from a local farmer at the age of 1-4 days, treated with a single dosis of 1mg/kg b.w. diclazuril (Vecoxan®, Janssen-Cilag) and halofuginone (Halocur®, Intervet) during 5 consecutive days at dosis of, assessed for parasitic infections and, when deemed parasite free, maintained under parasite-free conditions in autoclaved stainless steel cages until exposed to experimental E. ninakohlyakimovae infections. Goat kids were fed with substitute (Bacilactol®, Capisa) and commercial concentrate pellets (Starting Concentrate®, Capisa). Water and sterilized hay were given ad libitum. A total of eight goat kids were employed for the isolation of the E. ninakohlyakimovae strain and additional 9 goat kids were used for the experimental studies on infectivity, pathogenicity and immune response of the new strain. 2. 2. Parasite For the isolation of a defined E. ninakohlyakimovae strain, oocysts from naturally infected animals were collected according to the previously described protocol by Silva and Lima (2000), with minor modifications. Given that faecal samples contained E. ninakohlyakimovae oocysts at > 90% purity, samples were subjected to a flotation process (using saturated sodiumchloride solution (Panreac), 1.19 g/l density, 40 min RT). Oocysts were collected by flotation and suspended in 2% potassium dichromate (w/v) solution (Merck) according to Hermosilla et al. (2002). Sporulation of oocysts was achieved by leaving this suspension at room temperature (25 °C) and stirring the oocyst suspension daily to infuse oxygen. Thereafter, individual sporulated oocysts, diagnosed microscopically as E. ninakohlyakimovae (Levine and Ivens, 1986, Alyousif et al., 1992, Soe and Pomroy, 1992), were collected using a micromanipulator (Olympus ITM-2). A total of 2 x 104 of sporulated E. ninakohlyakimovae oocysts were isolated by this technique and resuspended in 2% potassium dichromate (w/v). All oocysts were administrated orally to one goat kid at the age of four weeks. Throughout patency faecal samples were collected and respective oocysts were isolated as described below. In total, three consecutive goat kid infections were performed in order to achieve a 100% pure E. ninakohlyakimovae strain. For harbouring oocysts, goat kids were orally infected at the age of 4 weeks with 2 x 105 sporulated E. ninakohlyakimovae oocysts. Excreted oocysts from experimentally infected animals were obtained from faeces collected after two weeks post infection according to the method of Jackson (1964). Briefly, the faeces were washed with tap water using decreasing pore size sieves for elimination of debris. For flotation, the resulting oocyst suspension was mixed 1:1 with saturated sugar solution (1.5 g/l density), transferred to rectangular plastic bowls and incubated for 2h at RT. Oocysts were collected by applying glass plates (25 x 25 cm) to the surface of the sugar/oocyst suspension. Oocysts attached to the glass plates were washed from the plates by rinsing with Aqua dest, concentrated by centrifugation (1,100 x g , 10 min), and treated to induce sporulation as described above. The resulting sporulated E. ninakohlyakimovae oocysts were stored at 4 °C in 2% potassium dichromate (w/v) until further use. 2.3 Experimental design of the challenge infection trial For experimental infections, goat kids were separated into three groups (n=3) and kept under parasite-free conditions. In two independent experiments, the 55 PRESENTACIÓN DE ARTÍCULOS immunoprotection induced by a primary infection was evaluated by the challenge infection with the same dose of E. ninakohlyakimovae oocysts. The level of immunoprotection was evaluated by productive (body weight), clinical (presence of clinical signs of coccidiosis, e. g. diarrhoea, variations within blood parameters), parasitic (oocyst counts in faeces) and histological (macroand micro-lesions in the intestine at the necropsy) parameters. Four week-old goat kids were primary infected by the oral inoculation of 2 x 105 sporulated E. ninakohlyakimovae oocysts per animal (W4PI) and challenge infected three weeks later (W7RI) by applying the same dose of oocysts (group I = challenge infected animals). Goat kids primary-infected at 7 weeks of age (W7PI) served as challenge-infection controls (group II = primary infected animals), and noninfected animals were used as negative controls (group III). Measurement of the body weight was performed weekly. At 10 weeks of age, all animals were euthanized by using sodium pentobarbital and subjected to further pathological analyses. 2.4. Coprological, pathological and histopathological analyses For coprological analyses, faecal samples were obtained daily on 13-21 days p. i. (in both primaryand challenge-infections). The non-infected controls were also subjected to coprological analysis in order to verify the absence of infection. The counts of oocysts per gram (OPG) in the faeces were determined in all groups of primaryand challenge-infected animals by the modified McMaster technique (Maff, 1989). Faecal samples were obtained rectally and subjected immediately to OPG analyses. For haematological analyses, blood samples were taken weekly by puncture of the Vena jugularis . Some additional blood samples were taken on days 16-17 p. i., coinciding with the highest peak of oocysts being shed. Total leukocyte counts and analysis of haemoglobin concentration were determined by using the haematological Lasercyte® (IDEXX). The values of packed cell volume (PCV) were calculated according to standard procedures by centrifugation of capillary tubes in a microhaematocrit centrifuge. Differential blood counts were performed manually by analysing 200 leukocytes in blood smears stained by Diff-Quick (QCA Labs.). After euthanasia of the goat kids, all animals were subjected to necropsy. All macroscopic lesions were annotated and tissue samples were collected from the intestinal mucosa (ileum, colon) and from the mesenteric lymph nodes. The tissue samples were fixed in 10% formalin and embedded in paraffin. Cross sections of 4-5 µm were stained by haematoxylin and eosin (H&E) according to standard staining procedures and the samples were analysed microscopically. 2.5. Statistical analyses Faecal oocyst counts were logarithmically transformed and added by one (log [OPG + 1]). For the estimation of bodyweight improvements, the data were expressed as growth rate ( ln weight 2 – ln weight 1)/t*100 ), with “t” representing the number of days between the sampling time points 1 and 2. The data were analyzed using the MannWhitney rank sum test (SigmaStat 2.03). 3. Results 3.1. Morphological characteristics of Eimeria ninakohlyakimovae GC strain. The oocysts of E. ninakohlyakimovae were a medium size of 22.5 x 16.7 µm with values varying from 16.3 x 28.4 µm (longitudinal diameter) and 12 x 20.8 µm (transversal diameter). The morphology of the oocysts was round to slightly oval; the micropyle was manifested weakly and the micropyle capsule was absent (Fig. 1). The duration of the sporulation process at room temperature (2225 °C) varied from 48-96 h. The sporocysts showed an elongated morphology, with a mean size of 11.6 x 6.3 µm. Within the E. ninakohlyakimovaesporocyst a Stieda body was detected. In addition, residual bodies and 56 PRESENTACIÓN DE ARTÍCULOS polar granules were observed within sporocysts. The inner layer of the oocyst wall had a shiny red-orange colour and dispersed granulation was frequently observed in between the sporocysts (Fig. 1). Figure 1. Sporulated oocysts of Eimeria ninakohlyakimovae GC after incubation at room temperature for one week in 2% sodium dichromate (A, B, C). Details of the Stieda body and dispersed granulation in between the sporocysts are marked by arrows in images B and C, respectively. Primary infected animals (group II) scarcely had parasitic stages in the examined tissue samples of ileum and colon, probably because necropsy was performed at the postpatent period, when most of the parasites had Figure 2. Histological sections of goat kids orally infected with 2 x 105 sporulated oocysts Eimeria ninakohlyakimovae GC and sacrifized 3 weeks later showing sexual stages of the parasite (oocysts and macrogametes) in the colon (A, B) and a segmented macroschizont in ileon mucosa (C). Fig. 2D represents a profuse infiltration of eosinophils at the ileum interstitium in one of the challenge infected animals. already finished their endogenous development; only some sexual stages of the parasite could be found as depicted in Fig. 2A and 2B. The same was observed in challenged goat kids, although in this case several macroschizonts were encountered in the ileum mucosa, some of them forming A B C 57 PRESENTACIÓN DE ARTÍCULOS compartments in the interior of the macromeront (Fig. 2C). Most of them were mature macroschizonts, measured 186.2 (117.3-320.1) x 132.6 (61.6-176.9) µm and contained a large number of merozoites. 3.2. Characterization of E. ninakohlyakimovae (GC strain) in primary and challenge infections The prepatent period in both primaryand challenged-infected animals varied from 1416 days, with most of the animals (68.4%) beginning to shed oocysts at 14 days p. i. Irrespective of the age of goat kids, no significant differences were observed between primaryand challenge-infected animals referring to the time point of patency onset. Patency of E. ninakohlyakimovae infection lasted from 4 to 10 days in primary infected animals and from 1 to 8 days in challenged ones. The oocyst shedding of primary-infected animals at 4 weeks of age (group I, W4PI) started at 14 days p. i. and reached highest levels at 17-18 days p. i. Thereafter, oocyst shedding gradually decreased and ceased between days 19 and 22 p. i. In challenge infected animals (group I, W7RI), the faecal oocysts counts were significantly lower than in the challenge control group II (W7PI) between days 14 to 18 p. i. ( P < 0.05), with an overall reduction of the OPG counts of 99.9% ( P <0.05) during the whole experiment (Fig. 3). Approximately the same reduction was achieved when OPG from reinfected goat kids of group I (W7RI) were compared with those recorded during the corresponding primary infection (W4PI) (99.8%, P <0.05) (Fig. 3). The maximum faecal count in primary infected animals was 10.1 x 106 OPG, whilst in challenged goat kids a maximum of 1.9 x 104 OPG was detected. No oocyst shedding was observed in the negative control animals (group III). Days post-infection Log [OPG+1] 0 1 2 3 4 5 6 7 W4PI W7RI W7PI 14 15 16 17 18 19 20 21 ** ** * ** Figure 3. OPG (oocysts per gram of faeces) counts of goat kids orally infected at week 4 of life (W4PI) with 2 x 105 sporulated oocysts Eimeria ninakohlyakimovae GC and challenged three weeks later with the same dose (W7RI). Triangles represent the OPG counts of animals primary infected with 2 x 105 sporulated oocysts at week 7 of life (W7PI). OPG counts are depicted as the logarithm of the OPG plus one (log [OPG + 1]) and represent the mean ± SEM in all the experimental groups. (*) P<0.05 and (**) P<0.01 represent significant differences between primary (W7PI) and challenge infection (W7RI). With respect to production parameters, group I (primary plus challenge infected goat kids, W4PI+W7RI) showed reduced growth rates between weeks 5 and 9 when compared to negative controls, whilst group II (challenge control animals) showed decreased growth rates exclusively between weeks 8 and 9 of the experiment (Fig. 4A). The overall growth rate was significantly higher in control group III compared to either primary or challenged infected animals ( P <0.05) (Fig. 4B). The highest differences in growth rates were detected one week p. i. in both group I and II with respect to control group III, indicating a reduction of body weight of approximately 15% in both cases (Fig. 4A). 58 PRESENTACIÓN DE ARTÍCULOS Weeks Growth rate -10 0 10 20 30 40 50 C W4PI + W7RI W7PI 4-5 5-6 6-7 7-8 8-9 9-10 * ** * * * Global growth rate 0 5 10 15 20 25 Controls W4PI + W7RI W7PI * Figure 4. Growth rates ( ln weight 2 – ln weight 1)/t*100 ) in goat kids orally infected at week 4 of life (W4PI) with 2 x 105 sporulated oocysts Eimeria ninakohlyakimovae GC and challenged three weeks later with the same dose (W7RI). Goat kids primary infected with 2 x 105 sporulated oocysts at week 7 of life (W7PI) were used as challenge controls and group C as uninfected controls. The rate of growth rates amongst different sampling times and global growth are depicted on Fig. 2A and 2B, respectively. Data represent the mean ± SEM in all the experimental groups. (*) P<0.05 represents significant differences between infected vs uninfected control animals. Although individual variations of clinical signs were apparent, all 4-weeks old primary infected animals showed clinical signs compatible with coccidiosis such as diarrhoea, dehydration, deterioration of the general animal condition, anorexia and, occasionally, prostration. Different degrees of faecal texture were observed, ranging from paste-like to completely liquid and sometimes explosive diarrhoea containing blood and pieces of mucosa (Table 1). More severely affected animals during primary infection with E. ninakohlyakimovae needed rehydration, which was performed by oral or subcutaneous saline application (Ringer Lactate, BRAUN), and vitamin treatment (Amicen’s Plus, CENASA). Clinical signs were observed in parallel to the onset of oocyst shedding from 13-14 days p. i. which continued for 6-10 days. After challenge infection, the clinical signs were significantly less pronounced (Table 1), in some cases not even apparant. However, when comparing the 4-weeks-old primaryinfected animals (group I – W4PI) with 7weeks-old primary-infected animals (group II– W7PI), it appeared that older E. ninakohlyakimovaeinfected animals showed milder clinical signs of coccidiosis (Table 1). As to blood parameters, the total protein serum level were moderately reduced in primary-infected goat kids (groups I and II) within days 14-17 p. i. (Table 2). No significant changes were observed in the red blood cell fraction, although a moderate increase of PCV was detected 14-17 days p. i. in primary infected animals (group I and II), coinciding with the patency of primaryinfected animals. Blood haemoglobin content and total leukocyte counts did not reveal any significant change during E. ninakohlyakimovae infection (Table 2). However, in animals primary infected at 7 weeks of age (group II – W7PI) a sustained eosinophilia was detected from 7 days p. i. onwards (Table 2). Counts of monocytes and 59 PRESENTACIÓN DE ARTÍCULOS Fig. 1 Faecal scores of goat kids primary infected and challenged with Eimeria ninakohlyakimovae . PRIMARY INFECTION Days post-infection 13 14 15 16 17 18 19 20 21 22 W4PI K1 K2 K3 K4 W7PI K5 K6 K7 C K8 K9 K1 0 CHALLENGE INFECTION 13 14 15 16 17 18 19 20 21 Days post-infection W7R I K1  K2  K3  K4  W7PI K5  K6  K7  C K8  K9  K1 0  Individual faecal scores of goat kids (K) primary infected at week four of life with 2 x 105 sporulated oocysts of E. ninakohlyakimovae (W4PI) and challenged (W4RI) three weeks latter with the same dose are represented. Three animals primary infected at week 7 of life were used as challenge controls and three uninfected kids (C) served as controls of infection. The arrow () point to the day of sacrifice and autopsy of the animals. The clinical evaluation of the diarrhoea was determined using the following score: Clean normal Dried soiling normal Wet soiling diarrhoea Diarrhoea down legs Explosive diarrhoea Fluid, watery or bloody diarrhoea 60 PRESENTACIÓN DE ARTÍCULOS lymphocytes varied irregularly and PMN counts appeared unaffected, although neutrophil bands were occasionally observed in some of the infected animals (Table 2). Transient increase in eosinophil counts were also recorded for challenged animals, whilst almost no changes were recorded in goat kids primary infected at 4 weeks of age. In general, the reactions observed both in the red and white series were weak or moderate, and the differences were not statistically significant. At necropsy, the intestines of infected animals showed no relevant macroscopic alterations. Histology revealed a moderate hyperplasia of the intestinal epithelium and hypertrophy of the mesenteric lymph nodes and Peyer’s patches (PP). Futhermore, a clear eosinophilic enteritis (Fig. 2D) and a diffuse infiltration of mast cells, lymphocytes and PMN was detected. Parasitic stages found in the large intestine of affected animals corresponded mainly to the sexual stages of Eimeria , and were mainly represented by immature and mature oocysts. Control animals did not show significant morphological changes. 4. Discussion In this investigation we isolated a field strain of E. ninakohlyakimovae from the Canary Islands (Spain) that exhibited morphological and biological features previously described for this caprine Eimeria species (Alyousif et al., 1992; Levine and Ivens, 1986, Vieira et al., 1997). Furthermore, we demonstrated that the E. ninakohlyakimovae GC strain is capable to cause coccidiosis in goat kids and to induce protective immune responses against challenge infections. The pathogenicity of E. ninakohlyakimovae GC was clearly demonstrated by the intensity of clinical signs. One of the most evident clinical effects was the diminished body weight gain in kids. Overall, the growth rates over time in primary infected and challenged animals were 9.5 ± 1.4 and 11.9 ± 0.5, respectively, in relation to uninfected controls (18.3 ± 3.1), which means a reduction of about 10% and, in consequence, a substantial economical loss for the farmers. Reduced body weights and a retarded physical development of infected animals are common features observed in coccidiosis (Fox, 1985; Alyousif et al., 1992; Daugschies et al., 2007) and are of economic importance for farmers worldwide. This clinical consequence of caprine coccidiosis may affect goat production in arid/semiarid areas even more profoundly than elsewhere, as goats are the only and ultimate source for milk and meat available. It is assumed that even subclinical ruminant coccidiosis can alter the conversion indices and, because of the frequency of subclinical coccidiosis, the economic losses might be higher than the ones caused by clinical coccidiosis (Fox, 1985). It can be easily be assumed that the impact of E. ninakohlyakimovae coccidiosis on productive parameters could be even more important under field conditions, especially in caprine farms with low hygienic and management standards, as shown previously for other ruminant Eimeria spp. (Daugschies and Najdrowski, 2005, Daugschies et al., 2007). The clinical signs generally corresponded to those previously reported in naturally infected goat kids (Koudela and Bokova, 1998), as well as in experimental infections with different inoculum sizes (Dai et al., 2006). In the later study, infection doses higher than 1 x 106 sporulated oocysts per animal resulted in a more severe clinical signs of infected animals, nevertheless, no deaths were reported. This observation is somewhat contradictory to our findings, as – using only 1/5 of the dosage – some of the E. ninakohlyakimovaeinfected animals developed such a severe clinical disease that an immediate emergency treatment was necessary for survival. These findings may indicate differences in the pathogenicity of strains as the E. ninakohlyakimovae GC strain appears more pathogenic than the Chinese strain used by Dai et al. (2006). Similar features have previously been reported for diverse strains of avian Eimeria species (Li et al., 2004, Loo et al., 2010). 59 PRESENTACIÓN DE ARTÍCULOS La excreción de ooquistes se monitorizó desde el día 14 p.i. en adelante mediante un examen fecal diario. Las muestras de sangre para hematología se obtuvieron en los días 8 y 16 después de la infección primaria y en los días 8 y 16 después de reinfección. La necropsia se llevó a cabo tanto en los animales que murieron durante la experiencia como al finalizar el estudio: 17 después de la infección. Se tomaron muestras de diferentes áreas del tracto gastrointestinal y ganglios linfáticos regionales para el análisis histopatológico. Los recuentos de OPG fueron ligeramente más altos en los animales reinfectados con 2 x 105 ooquistes esporulados. Sin embargo, el cuadro clínico sufrido por los animales reinfectados con la dosis mayor fue mucho más severo, e incluso condujo a la muerte de algunos de los animales. En el análisis histopatológico se evidenció que los animales infectados presentaban una importante infiltración de eosinófilos y linfocitos en la mucosa del íleon con recuentos significativamente mayores en los animales reinfectados con 1 × 106 ooquistes esporulados. En el tejido intestinal de animales parasitados, además de infiltración celular inflamatoria, se observaron zonas de necrosis focal rodeando a las criptas y alteraciones histopatológicas severas en otros órganos (con zonas de infiltración por eosinófilos y necrosis). Se encontraron formas parasitarias en los animales infectados, desde ooquistes hasta macroesquizontes en un animal que murió durante el periodo prepatente. En conclusión, los datos del presente trabajo demuestran que la infección primaria con E. ninakohlyakimavae en cabritos reinfectados posteriormente con una dosis de infección alta pueden padecer una coccidiosis aguda fatal y, en consecuencia, mostrar graves alteraciones intestinales. La excesiva respuesta inmune desarrollada frente a las etapas intracelulares de E. ninakohlyakimovae (esporozoitos/meronts I), tal como demuestra la elevada infiltración leucocitaria a nivel del intestino, podría haber contribuido al desenlace fatal durante la reinfección con la dosis infectante más elevada. Estos resultados enfatizan la necesidad de establecer medidas de gestión que prevengan la contaminación excesiva del ambiente con ooquistes. Por otra parte, nuestros resultados indican la importancia del fondo genético individual asociado a la capacidad de superar re-infecciones por E. ninakohlyakimovae . En todo caso, queda por determinar si los mecanismos de patogenicidad responsables del cuadro agudo desarrollado son consecuencia directa de las lesiones mucosas inducidas por el parásito, de una exacerbación de la respuesta inflamatoria o ambas circunstancias. 68 PRESENTACIÓN DE ARTÍCULOS Primary infection of goats with Eimeria ninakohlyakimovae does not provide protective immunity against high challenge infections. A. Ruiz a*, M.C. Muñoz a, J.M. Molina a, C. Hermosilla b, F. Rodríguez c, M. Andrada c, S. Martín a, A.Guedes a, D. Pérez a, L. Matos a, A.M. López a, A.Taubert b aDepartment of Animal Pathology, Faculty of Veterinary Medicine, University of Las Palmas de Gran Canaria, Gran Canaria, Spain bInstitute of Parasitology, Faculty of Veterinary Medicine, Justus Liebig University Giessen, Giessen, Germany cDepartment of Anatomy and Comparative Pathologic Anatomy, Faculty of Veterinary Medicine, University of Las Palmas de Gran Canaria, Gran Canaria, Spain Abstract Coccidiosis caused by the apicomplexan protozoa Eimeria ninakohlyakimovae has a high impact on animal health and profitability of the goat industry worldwide. Primary E. ninakohlyakimovae infections induce clinical disease mainly in goat kids, as homologous re-infections are commonly under immunological control. Nevertheless, there is evidence of fatal acute E. ninakohlyakimovae challengeinfections to occur in goat kids. So far, the nature of the immune response promoting protection or failure in E. ninakohlyakimovae challenge-infected goat kids has not been investigated. Therefore, we here analyzed cellular immune responses and histopathological alterations in the gut mucosa of fatal E. ninakohlyakimovae challenge-infected goat kids. Overall, a severe eosinophilic enteritis was observed in affected animals. Hyperplasia of epithelial cells, hypertrophy of globet cells and marked hyperplasia of Peyer’s patches and mesenteric lymph nodes draining affected areas were also relevant findings. The majority of goat kids suffering from acute E. ninakohlyakimovae challenge-infections showed severe diarrhea and some of them even died within 11-16 days post challenge, indicating that the mechanisms of pathogenicity might be related to the first generation macromeronts. Interestingly, infiltration of eosinophils and, in less extent, of intraepithelial lymphocytes and neutrophils was observed even within degraded E. ninakohlyakimovae first generation macromeronts. The excessive immune response mounted against intracellular E. ninakohlyakimovae stages (sporozoites/meronts I) as measured by intestinal leukocyte infiltration might result in severe pro-inflammatory reactions and contribute to the fatal outcome of the challenge infection. Keywords: Eimeria ninakohlyakimovae ; Challenge infection; Fatal coccidiosis; Caprine *Corresponding author: Tel.: +34 928451113 Fax: +34 928454341 E-mail: [email protected] (A. Ruiz) 69 PRESENTACIÓN DE ARTÍCULOS 1. Introduction The global goat population, which was estimated 879 million heads in 2009, experienced a percentual increase of 17.5% during the last ten years (2000-2009, FAOSTAT). This feature was particularly relevant in less developed and in developing countries with the former showing an increase of even 55.4%. Easy handling and effective conversion of limited food resources into meat and milk are key factors favouring the goat as a stock animal for farmers in poor socio-economic areas worldwide (Harper and Penzdorn, 1999). Caprine coccidiosis severely affects the profitability of goat industry particularly in rural semi-arid geographic regions depending economically on goat rearing, such as the Mediterranean Basin (Ruiz et al. 2006), Africa (Harper and Penzhorn, 1999; Kimbita et al., 2009), Asia (Faizal and Rajapakse, 2001) and Latin America (Cavalcante et al., 2011). Up to date, coccidiosis is considered as a major cause of enteric disease in goats and potentially pathogenic for animals of all ages, although clinical manifestations are mostly seen in goat kids (Koudela and Bokova, 1998; Ruiz et al., 2006). Caprine coccidiosis is caused by monoxenic apicomplexan parasites of the genus Eimeria , which parasitize different parts of the small and large intestine of goats. E. ninakohlyakimovae is considered highly pathogenic especially in goat kids (Harper and Pentzhorn, 1999; Ruiz et al., 2006). To a lesser extent, E. caprina and E. christenseni have also been reported to induce clinical goat coccidiosis (Taylor and Catchpole, 1994). E. ninakohlyakimovae coccidiosis is characterized by a severe non-haemorrhagic typhlocolitis with up to 30% mortality rates in goat kids (Koudela and Bokova, 1998). In some geographic areas, more than 96% of goat kids might be affected (Ruiz et al., 2006). Goat kids at the age of 10-12 weeks are particularly susceptible to clinical E. ninakohlyakimovae coccidiosis, which rather reflects a lack of protective immunity than age resistance (Ruiz et al., 2006), as most of the kids had previously suffered from primary infections. The existence of different virulent E. ninakohlyakimovae strains in combination with a lack of cross immunity, as reported for avian Eimeria spp. (Abu-Akkada and Awad, 2010; Loo et al., 2010), might also give a plausible explanation for the lack of protective immunity in challenge infections. Additionally, fatal E. ninakohlyakimovae challenge infections might be influenced by other factors such as the intensification of production, the poor hygiene in some farms and even the climate conditions (Ruiz et al., 2006), i. e., circumstances that promote massive Eimeria infections (> 106 OPG/day) in the weaning period (personal field observations). Coccidiosis produced by Eimeria spp. in ruminants (Catchpole et al., 1993) and in other host species (Shi et al., 2000) generally induces strong protective immune responses which prevent clinical disease derived from homologous challenge infections. The immune reactions developed against caprine Eimeria spp. have poorly been investigated, but studies conducted in cattle Eimeria infection indicate that both humoral (B-cell) and cellular (T-cell) responses are involved (Daugschies and Najdrowsky, 2005). In the bovine system, serum IgM, IgA and IgG2 levels negatively correlate with excretion of oocysts of E. bovis (Faber et al., 2002). However, T cell reactions markedly induced in Eimeria -infected animals are generally considered more effective than humoral responses for the development of protective immunity. In the ruminant system, the expansion of both CD4+ and CD8+ T cell subsets was demonstrated during primary E. bovis infection (Hermosilla et al. 1999) and recent data showing enhanced antigenspecific IFN-γ production in E. bovis infections in calves suggest Th1-dominated immune responses in prepatency (Taubert et al. 2008). It has also been reported that antigen-specific T cells proliferate effectively during a restricted time span during prepatency of primary infection (Hermosilla et al., 1999) but fail to do so after challenge infection suggesting early abrogation of re70 PRESENTACIÓN DE ARTÍCULOS infection (Sühwold et al., 2010). Accordingly, peripheral CD4+ T cells expanded during primary E. bovis infection, whilst after challenge neither the proportions of CD4+ or CD8+ T cell subsets nor those of γδTCR+ T cells were influenced. Overall, analyses of T cell infiltration into parasitized gut mucosa suggest a major involvement of CD4+ T cells in the termination of primary infection and a role of both CD4+ and CD8+ T cells in the control of re-infections (Sühwold et al., 2010). Additionally, macrophage-, monocyteand PMN-mediated innate immune reactions play an important role in the early immune response of coccidiosis in calves (Behrendt et al., 2008; Taubert et al., 2009). In the present study we simulated high dose challenge infections in goat kids in order to investigate the development of cellular immune reactions in association with histopathological alterations in the parasitized gut mucosa. For this purpose, primary E. ninakohlyakimovae infected goat kids challenged with either a moderate or high infective dose of homologous oocysts were monitored for clinical signs, oocyst shedding and changes of leukocyte populations in the parasitized gut mucosa. The results indicate that primary-infected goat kids reared in environments with excessive infection pressure might indeed suffer from severe acute E. ninakohlyakimovae challenge infections. The mechanisms causing the failure of protective immune responses need further investigations but the data rather suggest an exacerbation of the cellular immune response than direct mucosal lesions caused by the parasite as cause of the fatal outcome. 2. Material and methods 2.1. Animals Goat kids were purchased from a local farmer at the age of 1-5 days, treated with Vecoxan® (Janssen-Cilag) and Halocur® (Intervet), assessed for parasitic infections and, when deemed parasite-free, maintained under parasite-free conditions in autoclaved stainless metabolic steel cages until experimental E. ninakohlyakimovae infections were performed. The goat kids were fed with milk substitute Bacilactol® (Capisa) and commercial pellet concentrates (Starting Concentrate®, Capisa). Water and sterilized hay were given ad libitum. 2.2. Parasite maintenance The E. ninakohlyakimovae strain GC used in the present study was initially isolated in 2006 from naturally infected goats in Gran Canary Island (Spain) and maintained by passages in goat kids for oocyst production according to Ruiz et al. (2010). Briefly, E. ninakohlyakimovae oocysts were isolated from the faeces by the use of a micromanipulator (Olympus IMT-2) and then suspended in 2% potassium dichromate (w/v) according to Yim et al. (2011). Sporulation was achieved by leaving the oocyst suspension at room temperature (RT). Sporulated oocysts were collected and stored at 4 ºC until further use. For oocyst production, goat kids were orally infected at the age of 4 weeks with 2 x 105 sporulated E. ninakohlyakimovae oocysts which were less than six-month old. Oocysts were isolated from faeces beginning 14 days p. i. according to the method proposed by Hermosilla et al. (2002). 2.3. Experimental design Three groups of 4 weeks-old goat kids of the Majoreta milk aptitude breed were used in the E. ninakohlyakimovae challenge infection trial. Group 1, composed of eight goat kids, was orally infected with 2 x 105 sporulated oocysts/animal and challenged on day 21 p. i. with 1 x 106 sporulated oocysts/animal. Group 2, composed of four goat kids, was orally infected on day 0 as described above and challenged on day 21 with 2 x 105 sporulated oocysts/animal. Group 3 consisted of three uninfected goat kids and served as negative control. Shedding of oocysts was monitored from day 14 p. i. onwards by daily faecal examination. Clinical examination of all experimental animals was performed on a daily base. Blood samples for haematology 71 PRESENTACIÓN DE ARTÍCULOS were obtained on days 0, 8, and 16 after primary infection and on days 8 and 16 after challenge infection. Complete necropsy was carried out both in animals which died during E. ninakohlyakimovae challenge-infection and all the other animals from groups 1, 2 and 3 were humanely euthanized on day 17 post challenge. Samples from different areas of the gastrointestinal tract and regional lymph nodes, in addition to specimens from the rest of the organs, were taken for histopathological analysis. 2.4. Parasitological, biochemical and haemathological determinations The shedding of oocysts was quantified by faecal examination using the modified McMaster technique (Bangoura and Daugschies, 2007) and expressed as oocysts per gram (OPG) of faeces. Blood samples were obtained by venipuncture from the jugular vein. For haemathological determinations, samples were collected in VetCollect® tubes (IDEXX) and immediately processed by using the haemathology analyzer LaserCyte® (IDEXX). Packed cell volume (PCV) was determined by centrifugation with a standard capillary microhaematocrit centrifuge. Additionally, blood smears were stained with panoptic staining (Diff-Quick) to perform differential leukocyte counts. 2.5. Histopathological analysis During necropsies, tissue specimens were collected and fixed in 4% formaldehyde (Merck) in phosphate-buffered saline (PBS) for 24 h, dehydrated and embedded in paraffin according to standard procedures. Cross sections of 4 µm were stained with haematoxylin-eosin (H&E; Merck) and examined by light microscopy (Laborlux X Wild® microscope; Leitz). Quantification of leukocyte populations (lymphocytes, eosinophils and neutrophils) in tissue sections was performed on ileum and colon samples of uninfected and E. ninakohlyakimovae challenge-infected goat kids. Cells were counted using a 10x eye piece containing a calibrated graticule and 40x objective lens viewing an area of 0.05265 mm2. The counts were randomly taken on 20 graticule fields within the mucosal surface. The counts were expressed as number of cells per mm2 of mucosa according to Amarante et al . (2005). 2.6. Statistical analysis For the statistical analyses OPG counts, cell counts at the intestinal mucosa and haemathological parameters (all dependent variables) were evaluated under different Eimeria infection regimes (independent variables: uninfection, primary infection with 2 x 105, challenge infection with 2 x 105 and challenge infection with 1 x 106). Faecal oocyst counts were logarithmically transformed and added by one (log [OPG + 1]) to obtain normal distributions (KolmogorovSmirnov’s Normality Test). Normalization of the data was not necessary either for cell counts at the intestinal mucosa or the different haemathological parameters. One way factorial analysis of variance ANOVA and Tukey Multiple Comparison Test were used to compared OPG counts. Additionally, Student’s t test was employed to analyze cell counts and haemathological paremeters. All the statistical analysis were carried out using the SigmaStat 2.03 software. For all the variable analyzed, comparisons were considered significantly different at P < 0.05. 3. Results 3.1. Parasitological, biochemical and haemathological data The oocyst shedding of primary infected animals started at 14 days p. i. and reached highest levels at 16 days p. i. Thereafter, oocyst shedding gradually decreased up to day 21 p. i. In challenge infected animals, either with 2 x 105 or 1 x 106 sporulated oocysts, faecal oocyst counts were significantly lower than during primary infection between days 16 to 17 p. i. ( P < 0.05 (Fig. 1). Animals challenged with 2 x 105 sporulated oocysts showed slightly higher OPG counts than 72 PRESENTACIÓN DE ARTÍCULOS those challenged with the higher dose, although differences were not significant. Figure 1. OPG (oocysts per gram of faeces) counts of goat kids orally infected with 2 x 105 sporulated oocysts Eimeria ninakohlyakimovae (closed circles) and challenged on day 21 p. i. with either 1 x 106 (closed triangles) or 2 x 105 sporulated oocysts (open circles). The OPG counts are depicted as the logarithm of the OPG plus one (log [OPG + 1]) and represent the mean ± SEM in all the experimental groups. (*): P < 0.01 between primary and challenge infections. All goat kids challenged with 2 x 105 E. ninakohlyakimovae oocysts hardly showed clinical signs, whilst individual kids challenged with 1 x 106 sporulated oocysts succumbed from an acute fatal coccidiosis. Within this challenge trial, 62.5% of goat kids developed severe clinical disease and 60% of them died before the end of the investigation period (Table 1). Furthermore, 12.5% of the animals showed moderate signs of disease whilst only 25% remained unaffected (Table 1). In addition, there were also differences in the intensity of clinical signs already during the primary infection (Table 1), but no individual correlations were observed between the primaryand re-infection clinical phases. The signs of diarrhoea were accompained with dehydration, anorexia and recumbency in more severely affected goat kids. Besides, haemathological analyses revealed a moderate non-significant decrease in PCV and haemoglobin values especially in re-infected animals (Table 2). Although without significant differences, leukocytosis with neutrophilia and eosinophilia were also observed in re-infected animals, particularly in those challenged with 1 x 106 sporulated oocysts (Table 2). 3.2. Histopathological analysis In this investigation both eosinophils and lymphocytes significantly infiltrated the mucosa of the ileum in challenged animals (2 x 105: P <0.05; 1 x 106: P < 0.01, Fig. 2), with significantly higher counts in animals challenged with 1 x 106 sporulated oocysts ( P < 0.05, Fig. 2B, C). Similar data were determined for the colon mucosa, although in this case differences between the challenge groups were not significant for lymphocyte counts (Fig. 2). For illustration, Fig. 3B and 3E show exemplary images of eosinophil and lymphocyte infiltration into the ileum mucosa of animals suffering clinical disease, respectively. Interestingly, infiltration of mainly eosinophils could even be observed within degraded macromeronts. Moderate increased counts of neutrophils ( P < 0.05) were recorded in animals challenged with 1 x 106 sporulated oocysts, whilst in those challenged with 2 x 105 sporulated oocysts counts were similar to values found in the control tissue samples. Besides these two gut sections, increased counts of eosinophils and in a lesser extent of lymphocytes could also be observed in the duodenum, the jejunum and the caecum of animals suffering from clinical disease. In addition to inflammatory cellular infiltration, other pathological findings such as hyperplasia of the globet cells, hyperplasia of the Peyer’s patches (PP) in the ileum mucosa, areas of focal necrosis surrounded by hypertrophic crypts were observed in parasitized gut tissue. Intestinal lesions were also accompanied by severe histopathological alterations in other organs. Thus, mesenteric lymph nodes and the spleen showed a marked infiltration of eosinophils and reactive hyperplasia. Lungs were congestive and presented variable degrees of alveolar and interstitial oedema and liver samples displayed focal degeneration and necrosis. Signs of Days p. i. Log [OPG+1] 0 1 2 3 4 5Primary infection 2 X 105 Challenge infection 2 X 105 Challenge infection 1 X 106 15 16 17 18 19 20 21 ** 73 PRESENTACIÓN DE ARTÍCULOS Table 1. Faecal scores of goat kids primary infected and challenged with Eimeria ninakohlyakimovae oocysts. PRIMARY INFECTION Days post-infection 12 13 14 15 16 17 18 19 20 21 2x10 5 K1 K2 K3 K4 K5 K6 K7 K8 2x10 5 K9 K10 K11 K12 Ø K13 K14 K15 CHALLENGE INFECTION 8 9 10 11 12 13 14 15 16 17 Days post-infection 1x10 6 K1  K2  K3  K4  K5  K6  K7  K8  2x10 5 K9  K10  K11  K12  Ø K13  K14  K15  74 PRESENTACIÓN DE ARTÍCULOS Individual faecal scores of goat kids (K) primary infected on day 0 with 2 x 105 sporulated oocysts and challenged at day 21 with either 1 x 106 or 2 x 105 sporulated oocysts of E. ninakohlyakimovae are represented. Three uninfected kids (Ø) served as controls of infection. The arrow () point to the day of death and/or sacrifice and necropsy of the animals. The clinical evaluation of the diarrhoea was determined using the following score: Clean normal Dried soiling normal Wet soiling diarrhoea Diarrhoea down legs Explosive diarrhoea Fluid, watery or bloody diarrhoea Table 2. Haematological parameters in Eimeria ninakohlyakimovae primaryand challenge-infected goat kids. PCV % HGB g/dl WBC cells/µl NEU cells/µl BAN cells/µl LYM cells/µl MONO cells/µl EOS cells/µl TP g/dl Primary infection (2 x 105 sporulated oocysts) W4 29.5 9.2 8732.5 3329.0 0.0 5131.5 86.3 185.8 5.5 (0 dpi) (1.4) (0.7) (490.0) (951.0) (0.0) (548.8) (37.2) (117.7) (0.3) W5 27.3 8.8 9915.0 4822.3 0.0 4380.8 254.5 429.8 5.4 8 dpi (2.1) (0.7) (806.6) (324.1) (0.0) (845.2) (102.4) (312.7) (0.2) W6 27.0 8.4 8682.5 2884.3 0.0 4064.0 373.8 528.8 5.0 (16 dpi) (3.7) (1.0) (1705.7) (350.1) (0.0) (634.7) (205.7) (299.6) (0.4) Challenge infection (2 x 105 sporulated oocysts) W8 24.3 7.4 10487.5 5436.3 76.0 4458.3 92.5 424.0 4.7 (8 dpri) (2.8) (0.9) (1400.3) (1423.2) (51.4) (938.9) (41.0) (141.2) (0.2) W9 27.0 8.7 9125.0 4079.2 0.0 4856.0 24.8 465.3 4.9 (16 dpri) (3.0) (0.9) (1104.1) (294.7) (0.0) (908.5) (28.6) (158.4) (0.2) Challenge infection (1 x 106 sporulated oocysts) W8 23.3 7.3 15287.5 6295.0 22.5 6410.8 389.0 2056.3 4.9 (8 dpri) (1.9) (0.6) (2883.5) (679.4) (26.0) (879.3) (186.3) (431.2) (0.2) W9 28.5 7.9 34662.0 17033.5 1141.5 11663.5 3827.5 1618.3 5.1 (16 dpri) (14.8) (2.6) (4098.4) (290.6) (1614.3) (9337.3) (5080.6) (369.6) (0.8) Uninfected controls W4 29.3 9.0 13582.7 6046.4 0.0 6434.7 0.0 258.3 4.9 (2.3) (0.1) (697.2) (229.9) (0.0) (552.3) (0.0) (82.7) (0.1) W5 27.7 9.2 14314.0 7057.3 0.0 6313.5 25.7 331.7 (4.9 (2.2) (0.4) (1024.0) (883.3) (0.0) (1484.9) (20.0) (75.6) (0.1) W6 30.7 9.3 10834.0) 6148.6 0.0 5626.9 230.0 248.3 5.0 (1.1) (0.3) (947.1) (1274.1) (0.0) (832.7) (160.5) (74.5) (0.1) W8 29.7 9.6 12846.7 4785.0 0.0 7988.8 0.0 290.3 5.1 (2.2) (0.5) (1900.7) (1017.8) (0.0) (379.1) (0.0) (84.4) (0.2) W9 30.3 9.8 13680.0 3938.3 0.0 9855.4 121.7 162.0 5.1 (1.1) (0.2) (785.9) (59.0) (0.0) (594.4) (75.4) (96.5) (0.1) Goat kids (K) were primary infected at week four of life with 2 x 105 sporulated oocysts of Eimeria ninakohlyakimovae and challenged three weeks latter with either 1 x 106 or 2 x 105 sporulated oocysts. Three uninfected kids (Ø) served as controls of infection. TP: total proteins; PCV: packed cell volume; HGB: haemoglobin; WBC: while blood cells; NEU: neutrophils; BAN; band neutrophils; LYM: lymphocytes; MONO: monocytes; EOS: eosinophils. All he results are expressed as the mean ± (SEM) of different weeks of life (W), days postinfection (dpi) or days postre-infection (dpri). 75 PRESENTACIÓN DE ARTÍCULOS congestion and degeneration of muscle fiber could be found in heart samples and kidney sections revealed mild glomerular and interstitial infiltration of mononuclear cells, calcium deposits and protein accumulation in tubules. As a common histopathological finding, leukocytosis and vascular stasis were also observed. In accordance to the sloughing of the mucosa and haemorrhagies observed in clinical cases, histopathology of the corresponding animal samples showed clear signs of denudation of the mucosal surface. Groups of merozoites I could be detected within the denudated mucosa in the lumen of some ileum samples (Fig. 3D). Remaining intact mucosa of ileum sections from the goat kid which died during the prepatent period (day 11 p. i.) presented a high number of macromeronts of E. ninakohlyakimovae (Fig. 3A, 3C) , whilst in animals necropsied during the patent period (from day 15 to 17 p. i.) exclusively microgamonts, macrogamonts and oocysts were observed in samples from the colon mucosa. Figure 2. Differencial leukocyte counts, expressed as number of cells per mm2, in the mucosa of the ileum and colon of goat kids primaryand challenged-infected with two infective doses of Eimeria ninakohlyakimovae oocysts (2 x 105 and 1 x 106). (a): P < 0.05 between control and challengeinfected animals; (b): P < 0.01 between control and challenge-infected animals; (*): P < 0.05 between goat kids challenged with 1 x 106 and 2 x 105 sporulated oocysts. 4. Discussion In general, protective immunity developed by ruminant hosts against Eimeria spp. does not prevent subsequent homologous challenge infections but will restrain the clinical signs of disease. Continuous exposure to homologous Eimeria oocysts will maintain host protective immunity with the exception of animals showing a compromised immune status based on 1) nutritional deficiences, 2) stress produced by transport, sudden changes in feeding, weaning etc., or 3) concomitant diseases (Lucas et al., 2006; Rausch et al., 2010). This appears particularly important in young animals, although a failure of immunity against Eimeria infections has also been reported in adult goats during the periparturient period (Faber et al., 2002). The results of the present study give additional evidence that E. ninakohlyakimovae primary infected goat kids subjected to homologous challenge infections may also develop clinical disease under high infection pressure. Goat kids challenged with 2 x 105 E. ninakohlyakimovae oocysts hardly showed clinical signs. In contrast, individual goat kids challenged with 1 x 106 sporulated oocysts Neutrophils/mm 2 0 2 4 6 8 10 12 14 16 Uninfected controls Challenged 2x105 Challenged 1x106 Ileum Colon A a a Eosinophils/mm 2 0 50 100 150 200 250 300 350 Ileum Colon B a, * b, * b, * a, * Lymphocytes/mm2 0 50 100 150 200 250 300 Ileum Colon C b,* b,* b a 76 PRESENTACIÓN DE ARTÍCULOS Figure 3. Histopathological finding in Eimeria ninakohlyakimovae primary infected goat kids and subsequently challenged with 1x106 Eimeria ninakohlyakimovae sporulated oocysts. (A) First generation macromeront at 11th day postinfection within the ileum mucosa (H&E, 100X). (B) Detail of a macromeront surrounded by an intense infiltration of eosinophils, some of them inside the meront (H&E, 400X). (C) Low magnification detail showing lesions in the mucosa of the ileum at day 11th postinfection; the arrow points a macromeront in the surface of the mucosa (H&E, 40X). (D) Bulk of merozoites I within partially degraded mucosa inside the lumen of the ileum (H&E, 400X). (E) Infiltration of eosinophils and lymphocytes (arrow) in the ileum mucosa. 77 PRESENTACIÓN DE ARTÍCULOS Characterization of antibody response to experimental infection with Eimeria ninakholyakimovae in goat kids 1Matos, L., 1Muñoz, M. C., 1Molina, J. M., 3Rodríguez, F., 1Perez, D., 1Lopez, A., 1Martín, S., 2Hermosilla, C., 2Taubert, A., 1,*Ruiz, A., 1Department of Animal Pathology, Faculty of Veterinary Medicine, University of Gran Canaria, Gran Canaria, Spain 2Institute of Parasitology, Faculty of Veterinary Medicine, Justus Liebig University Giessen, Giessen, Germany 3Department of Anatomy and Compared Anatomy Pathology, Faculty of Veterinary Medicine, University of Gran Canaria, Gran Canaria, Spain Abstract Infections produced by different Eimeria species , which are some of most important parasitic diseases affecting profitability of most production system worldwide in ruminants, are under immunological control. Although cellular immune response seems to be crucial for the development of protective immune response, there are also evidences showing an active involvement of the humoral counterpart. In the present study, we have analyzed the antibody response to an experimental infection with Eimeria ninakholyakimovae in goat kids. Therefore a total of 22 kids were divided into three groups: (i) primary infected animals at 5 weeks of age, and reinfected three weeks later (W5PI + W8RI), (ii) primary infected animals at 8 weeks or challenge controls (W8PI) and (iii) uninfected animals (C). For infection, sporulated oocysts of a local (GC) strain of Eimeria ninakohlyakimovae were used. The levels of specific IgG and IgM antibodies were determined in serum samples taken weekly throughout the experiments; additionally, IgA levels were estimated in the ileal mucus applying indirect ELISA tests based on homogenized oocyst antigen. Infected kids produced significant increase of IgG levels that was consistently observed from 3 weeks post infection onwards. Furthermore, an increase of specific IgM and secretory IgA levels were observed in infected animals. A wide range of peptides from sporulated oocyst antigen (SOA) were recognized by specific IgG using SDS-PAGE and subsequent immunoenzimatic botting, however, as for the indirect ELISA results, no correlations were found with OPG counts. Altogether, these results suggest that antibody analysis of experimental infection with E. ninakohlyakimovae in goat kids certainly reflect the exposure to the parasite but is not directly associated to the protection conferred after challenge. Further studies should be addressed to clarify if the lack of correlation might be associated to the type of antigen used for the immunoenzimatic assays, the age of the animals or other factors. Key words: Eimeria ninakohlyakimovae , goat kids, IgG, IgM, IgA *Corresponding author: Antonio Ruiz Reyes Department of Animal Pathology Tel: +9284511133; Fax: +92845341 Email: [email protected] 85 PRESENTACIÓN DE ARTÍCULOS 1. Introduction The caprine coccidiosis caused by Eimeria spp. is one of the most common parasitic disease affecting goats in production systems worldwide (Faizal and Rajapakse, 2001; Donkin and Boyazoglu, 2004; Cavalcante et al., 2012; Silva et al. 2014). Although most of the animals of a herd may be infected with different Eimeria species, the disease mainly affect kids between 4 to 20 weeks, sometimes with high mortality rates (Koudela and Boková 1998, Ruiz et al. 2006). Among most frequent Eimeria species in goats, E. ninakholyakimovae is considered as one of the most pathogenic, able to produce massive damage of the intestinal mucosa, which results in a malabsorption syndrome and severe diarrhea, often accompanied by blood and mucus. The clinic involves weight loss, dehydration and stunted growth rates, which results in important production losses (Koudela and Boková 1998). Control of ruminant coccidiosis is mainly based on the combination of management practices and the use of anticoccidials, such as sulfonamides (Svensson 1998), toltrazuril (Mund et al. 2003) and diclazuril (Ruiz et al. 2012). However, because of the indiscriminate use of these products, mainly in poultry, anticoccidial drug resistance has already been described (Peek and Landman 2005). This circumstance, together with the growing demand of consumers for organic livestock products free of residues, has increased the interest for new alterative control methods. One of these alternatives is the development of vaccines, for which a prerequisite is a better understanding of basic immunology and hostparasite interactions. Although both cellular and humoral immune responses are triggered against ruminant coccidiosis after the first contact with the antigen (Daugschies and Nadjdrowski 2005), many studies indicate that the cellular counterpart, mediated by T-cells, is the main responsible for the development of protective immune responses against Eimeria spp. infections and other Apicomplexa, such as Toxoplasma , Neospora or Cryptosporidium (Abrahansem, 1998; Yap and Sher, 1999; Hermosilla et al., 1999; Correia et al., 2013). Accordingly, T-cells response was associated with lower excretion of oocysts in feces before reinfection with E. bovis in cattle (Hermosilla et al., 1999; Taubert et al., 2008; Sühwold et al., 2010). Besides, it has been demonstrated that T-cells involved in the primary immune response in cows are mainly CD4+ (Findley et al., 1993; Hermosilla 1999; Taubert et al., 2010) whereas cytotoxic CD8+ T cells may represent the major effector cell type against the parasites in case of reinfections. In addition, E. bovismediated T-cells activation may induce a molecular regulation of a network associated with the movement and trafficking of immune cells, probably through the production of certain cytokines such as IL2, IL4 and INFγ (Taubert et al., 2010). In contrast, humoral immune reactions of the host are not sufficient for the termination of Eimeria spp. primary infections (Daugschies y Najdrowski, 2005) and, even when antibodies may reflect the exposure to the parasite, the protection which they confer is not (Fiege y cols., 1992). The humoral response develops rapidly and involves high titers of antibodies in the serum of infected animals. Usually, there is an initial increase of IgM, followed by IgG and other specific antibodies such as IgA (Hughes 1985). Experimental infections with E. ovinoidalis and E. faurei in sheep demonstrate that specific antibodies increased, not only in the primary infections, but also in secondary infections (Nolan et al. 1987). Like CD4+ T cells and probably other lymphoid cells, antibodies can be transferred in colostrum to the calf resulting as a mechanism of passive immunity against coccidiosis (Fiege et al., 1992). Actually, lambs and calves fed with colostrum with a high content of IgG had higher levels of specific antibodies against Eimeria than those which did not received it. Studies on the role of humoral immune response in avian coccidiosis have also demonstrated the ability of antibodies to block the invasion, development and transmission of the parasite and, like in ruminants, the existence of passive immunity (Wallach 2010). In other Apicomplexa parasites, such Toxoplasma gondii and 86 PRESENTACIÓN DE ARTÍCULOS Crytosporidium , the involvement of both, systemic and local, antibody responses in parasitized goats has also been reported (Conde et al. 2001, Gomez Morales et al. 2002). The antibody response during E. ninakholyakimovae infection has not been studied before and, in general, there are few data on the immune response in caprine coccidiosis. The main objective of this work was to evaluate the humoral immune response of this Eimeria species by ELISA sequential analysis of peripheral IgG and IgM and local IgA in primary and challenge infected animals. Additionally, a preliminary approach for antibody-mediated recognition (IgG) of specific peptides was conducted by SDS-PAGE and immunoblotting methodology. 2. Material and methods 2.1. Parasites and parasitic antigen The E. ninakholyakimovae strain GC used in the present study was initially isolated in 2006 from the field of naturally infected goats in Gran Canaria Island (Spain) and maintained by passages in goat kids for oocysts production (Ruiz et al. 2014). To grow oocysts, goat kids were orally infected at the age of 4 week with 2 x 105 sporulated E. ninakholyakimovae oocysts. Oocysts were isolated and purified from feces after 2 week post infection according to the methods of Jackson (1964) and then concentrated. Sporulation of oocysts was achieved in a week by leaving this suspension at room temperature and stirring the oocysts suspension daily to infuse oxygen into the suspension. Finally, sporulated oocysts were stored in a 2% potassium dichromate solution at 4°C until further use. For the preparation of the antigen used in ELISA and immunoblotting determinations, a solution containing approximately 106 sporulated E. ninakholyakimovae oocysts was employed. The oocysts were mixed and crushed with glass beads in a 15ml tube (Nunc) ten times for 60s on a vortex at maximum speed. When the oocysts were partially broken, the glass beads were withdrawn and the solution was subjected to ultrasounds (Vibra Cell TM) at 80Hz until they were completely crushed. The ultrasound treatment was carried out ten times for 60s each, maintaining the solution in water ice for 1 min between each ultrasound pulse. Thereafter, the final solution was transferred to Eppedorf tubes and centrifuged during 30 min at 11000 g and 4°C (MPW 65-R Centrifugue). The supernatant was then transferred to sterilized tubes to calculate the antigen concentration by using the BCA method. Briefly, 2ml of BCA solution (22ml Bicinchoninic acid solution + 0.44 ml Coopper sulfate) (Sigma-Aldrich) were added to a tube containing 100 µl of sporulated oocyst antigen (SOA), different dilutions of a reference stock protein (Bio-Rad) and negative controls (distilled water). All the samples were incubated 1h at 60°C before reading the absorbance values in a SmartSpecTMPlus spectrophotomemter (Bio-Rad). Different aliquots of the resulting SOA antigen having a 103.678µg/ml concentration were stored at - 20°C until the ELISA (Enzyme-linked immunosorbent assay) and Western immunoblotting tests were carried out. 2.2. Animals, experimental design and sample processing A total of (21) goat kids of the Majorera breed was purchased from a local farmer at the age of 1-5 days and maintained under parasite-free conditions in autoclaved stainless steel cages in a restricted stable (Faculty of Veterinary Medicine, University of Las Palmas de Gran Canaria, Spain). The kids were treated with Vecoxan® (Janssen-Cilag) and Halocur® (Intervet) just at their arrival. Goat kids were fed with the milk substitute Bacilactol® (Capisa) and commercial concentrate pellets for weaning goat kids (Starting Concentrate, Capisa). Water and sterilized hay were always given ad libitum . All animal procedures were conducted in strict accordance with national ethics and by institutional review board-approved protocols. For experimental purposes goat kids were divided into the following three groups: group 1 (n=9, W5PI+W8RI: primary infected at week 5 of life with E. ninakholyakimovae 87 PRESENTACIÓN DE ARTÍCULOS sporulated oocysts and reinfected at week 8); group 2 (n=6, W8PI: animals infected with E. ninakholyakimovae sporulated oocysts at week 8 of age; group 3 (n=7, CONTROL: non infected animals). All infections were performed orally with a gastro-ruminal tube using an infection dose of 2 x 105 sporulated E. ninakholyakimovae oocysts. The animals were weekly bled from the jugular vein and the corresponding serum individual samples were stored at -20°C until the serological studies were performed. In order to have a monitoring of the parasitological and clinical course of the experimental infection, individual fecal samples were collected from 14 to 21 days post-infection (p.i.), both during primary and challenge infections, and the presence of clinical signs were daily inspected. The estimation of the faecal release of oocysts was determined by a modified McMaster technique (Thienpont et al., 1979) and expressed as OPG (oocyst per gram of faeces) counts. The animals were humanely euthanized at the end of the experiment and mucus samples, for the analysis of specific IgA, were collected from the ileum. For their preservation, the samples were suspended in a pH 7.1 buffer containing proteinases inhibitors (all compounds from SigmaAldrich): 0.1 M Na2HPO4, 0.05 M NaCl, 3 mM NaN3, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 5 mM ethylenediaminetetraacetic acid (EDTA). Mix solutions containing mucus were centrifuged at 5000 x g (Eppendorf Centrifuge 5804R) for 1h at 4°C and the resulting supernatant was conserved at -20°C for further analyses. 2.3. Enzyme – linked immunosorbent assay (ELISA) A total of 100µl of a SOA solution in carbonated buffer at a 5µg/ml concentration was dispensed in 96 wells ELISA plates (Corning Glass Works) and incubated overnight at 4°C. After three washes with 200µl of PBS-0.05 % Tween 20, 200µl of PBS3% (w/v) bovine serum albumin (BSA, SigmaAldrich) was added and the plates were incubated for 45 min at 37°C. After each new step a similar washing procedure was addressed. Following treatment with BSA, individual serums, positive and negative controls were analyzed in duplicate using a 1/25 dilution in PBS-Tween 20 0.02% sodium acid. For each sample, a volume of 100µl was added to the plates and a new incubation was performed at 37°C for 1h. The plates were then incubated with 100µl per well of 1/2000 anti-Goat IgG (anti-goat IgG peroxidase conjugate, IgG fraction of antiserum, SigmaAldrich) diluted in 0.01M PBS and the incubation was performed in the same conditions. Thereafter, a total of 100µl of substrate was added to each well, a mix of compounds that includes citric-phosphate buffer, 0.04% (w/v) dihydrochloride orthophenyleneadiamine (OPD, Sigma-Aldrich) and 30% hydrogen peroxide (Panreac) at a final concentration of 0.1% (v/v). The incubation with the substrate was performed at room temperature in darkness for approximately 10 min. Finally, the reactions was stopped by adding 35 µl per well of a 2M solution of sulfuric acid (Panreac) and the optical density (O.D.) measured at a wavelength of 492 nm (Termo Labsystems, Multiskan Ascent). For specific IgM and IgA indirect ELISA tests, a similar protocol was employed but using 1/3000 anti-goat IgM peroxidase and 1/7000 anti-goat IgA peroxidase conjugates, respectively (Sigma-Aldrich). Similarly, dilutions 1/25 of the different serum or mucus samples were employed as primary antibody. 2.4. SDS-PAGE and Western immunoblotting A 86.65 µg/ml antigen solution (SOA) and low molecular weight standards (Pierce Blue Prestained Molecular Weight Marker, Thermo Scientific) ranging from 215 to 18 KDa were run on 12% SDS-polyacrylamide gels in a cuvette (Mini-Protean® Tetra Cell BioRad) at 100V constant voltage at 4°C. After electrophoresis, proteins were either revealed with silver stain (Sigma-Aldrich) or transferred to a nitrocellulose membrane (blotting Pure Nitrocellulose Membrane, Bio Trace® NT, Life Sciences) by applying an electric field perpendicular to the membrane 88 PRESENTACIÓN DE ARTÍCULOS and gel. The transfer was performed in the same bucket than electrophoresis at 100V constant voltage for 1h at 4°C. After the transference was finished, the membrane was sliced and the resulting strips were firstly washed with PBS-0.05 % Tween 20 and then incubated with PBS containing 3% (w/v) BSA for 1 h at 37°C. After a new washing step, the membranes were incubated overnight at 4°C with different pool sera from the different experimental groups, previously diluted 1/25 in PBS-0.05 % Tween 20 containing 0.02% NaN3 (w/v) (Sigma-Aldrich). Next to a new washing, the strips were incubated for 1 h at 37°C with anti-Goat IgG diluted 1/1000 in PBS. Finally, the membranes were washed again with PBS-0.05 % Tween 20 and revealed using an AEC Chromogen Kit (AEC Staining Kit, SigmaAldrich). The determination of the molecular weight of the different polypeptide subunits present, both in the acrylamide gels that were stained with silver after electrophoresis and in the nitrocellulose membrane strips from the Western-blot immunoenzymatic reaction, was carried out by a regression analysis (Milton, 1994; Gardiner, 1997) using the migration of proteins of molecular weight marker as a reference. 2.4. Statistical analysis of data Faecal OPG were logarithmically transformed and added by 1 [log (OPG+1)] in order to obtain normal distributions according to the Kolmogorov-Smirnov’s normality test. Levels of the different immunoglobulins analysed in the study were expressed as the relative percentage of the optical density (O.D.) of a positive control pool in order to avoid inter-assay differences (Relative O.D.). One factorial analysis of variance, Tukey’s multiple comparison test and Student’s t-test were used to analyse the data between different experimental groups and the Pearson correlation test for the evaluation of the association between OPG counts and the antibody levels. For that purpose, an statistical software (SigmaPlot 12.0) was employed and the differences were regarded as significant at a level of p ≤ 0.05. 3. Results 3.1. OPG counts and clinical signs After primary infection, goat kids primary infected at week 5 of age (W5PI) with 2 x 105 E. ninakohlyakimovae sporulated oocysts showed a prepatent period ranged from 14 to 19 days p.i., and had a maximum average value of 4.197 at day 16 p.i., which corresponds to 157,245 OPG counts (Table 1). A similar variation for the prepatent period (15 to 18 days) was found in the challenge control group (W8PI), although the maximum average in this case was much higher, reaching peak values of 5,393 (approximately 2,5 x 106 OPG) also at 16 days p.i.. Faecal counts were consistently lower in challenged kids (W5PI+W8RI) with differences being significant when comparing to results of both primary infections (W5PI and W8PI) ( p < 0.05 to p < 0,001) (Table 1). There were no differences between clinical signs when comparing animals primary infected either at week 5 (W5PI) or at week 8 of age (W8PI). With variable intensity depending on the goat kid, different degrees of diarrhea, dehydration and anorexia were observed. In some cases, the diarrhea was profuse, bloody and contained small debris of mucosa. By contrast, less obvious or even imperceptible clinical signs were recorded for challenged animals (W5PI+W8RI). During the whole experiment, uninfected control animals showed no oocysts in their feces nor clinical signs. 3.2. Enzyme-linked immunosorbent assay (ELISA) Mean values for specific IgG against SOA (sporulated oocyst antigen) for the different experimental groups are depicted in Figure 1. 89 PRESENTACIÓN DE ARTÍCULOS dpi W5PI W5PI+W8RI W8PI 14 0,701 ± 0,468 (a) 2,152 ± 0,535 (a) 2,813 ± 0,977 15 3,270 ± 0,991 2,525 ± 0,804 (*) 4,467 ± 0,684 (*) 16 4,197 ± 0,986 2,511 ± 0,744 (**) 5,393 ± 0,558 (**) 17 3,863 ± 0,904 2,204 ± 0,650 (**) 4,783 ± 0,458 (**) 18 3,455 ± 0,775 2,109 ± 0,697 (*) 4,005 ± 0,615 (*) 19 3,097 ± 0,588 (a) 1,337 ± 0,536 (a) 2,451 ± 0,994 20 2,689 ± 0,627 (a) 0,674 ± 0,460 (a) 1,276 ± 0,893 21 0,985 ± 0,493 0,000 ± 0,000 0,000 ± 0,000 Table 1. OPG (oocysts per gram of faeces) counts of goat kids orally infected at week 5 of life (W5PI) with 2 x 105 sporulated oocysts of the GC strain of Eimeria ninakohlyakimovae and challenged three weeks later with the same dose (W5PI+W8RI). Animals primary infected with 2 x 105 sporulated oocysts at week 8 of life (W7PI) were used as challenge controls. OPG counts are depicted as the logarithm of the OPG plus one (log [OPG + 1]) and represent the mean ± SEM in all the experimental groups. (*) P < 0.05 and (**) P <0.01 represent significant differences between re-infected animals (W5PI+W8RI) and challenge controls (W8PI), while (a) P <0.05 indicates differences between primary (W5PI) and challenge infection (W5PI+W8RI). The analysis of the results show that increased IgG levels from primary infected goat kids at week 5 of age progressively increased during the next three weeks p.i. with significant differences to control animals being observed at week 8 p.i. The same animals, but challenged at week 8 (W5PI+W8PI), had sustained and increasing levels of IgG during the following weeks with peak values at the last sampling point (week 11). Significant different IgG values ranging from p < 0.05 to p < 0.001 were observed in challenged goat kids with respect to control group after reinfection. A similar IgG profile was found in animals which were primary infected at week 8 (W8PI, challenge control group), although in this occasion the levels of this immunoglobulin reached significant increased values ( p < 0.05) at two weeks p.i. and slightly decreased afterwards. There were no differences between IgG levels between the re-infected animals and the corresponding challenge control group. Significant increased levels of specific IgM were first recorded at 7 weeks p.i. in goat kids primary infected at week 5 ( p < 0.05) and remained high up to the end of the experiment (Figure 2). However, one week after challenge infection, serum samples of this group of animals (W5PI+W8PI) slightly decreased before showing new peak values at week 10. IgM levels gradually increased also in goat kids primary infected at week 8 (W8PI), with significant differences ( p < 0.05) being observed at the last sampling point compared to control group. No differences were found between re-infected goat kids and the corresponding challenge controls. Finally, the analysis of the immunoglobulin isotype A (IgA) levels showed that mucus samples from both reinfected (W5PI+W8PI) and challenge control animals (W8PI) had significant higher levels of specific IgA than controls ( p < 0,05), but there were no differences between these two groups. The analysis of correlations between oocyst excretion and antibody levels showed an inverse relationship between cumulative OPG counts (14 to 21 days p.i.) and specific serum IgM values in primary infected animals 3 weeks after infection (W8) but differences could not be proven statistically ( p = 0.0213); no correlations were observed to either IgG or IgA levels during primary infections. Considering antibody levels of challenged animals (W5PI+W8RI), only slight nonsignificant correlations to OPG counts were recorded. In this occasion, negative correlation coefficients were also found for IgG ( p = 0.127), while for local IgA the correlation was positive ( p = 0.158). 90 PRESENTACIÓN DE ARTÍCULOS 3.2. SDS – PAGE and Western blotting SDS–PAGE analysis of different concentration of sporulated oocyst antigen (SOA) employed showed that a number of polypeptides were present in the homogenate with high, medium and low molecular weights (Fig. 4). Most of the bands were concentrated in the range between 54 and 108 kDa, but numerous peptides of smaller molecular Figure 1. Evolution of mean IgG anti-SOA levels (±standard error) expressed as relative optical densities (O.D.) in sera from all the experimental groups: W5PI+W8RI = primary infected with 2 x 105 Eimeria ninakholyakimovae sporulated oocysts at week 5 of age and reinfected at week 8; W8PI= primary infected at week 8 of age or challenge controls; C= uninfected animals. (*) P<0.05 and (**) P<0.01 represent significant differences between infected animals and control group. Figure 2. Evolution of mean IgM anti-SOA levels (±standard error) expressed as relative optical densities (O.D.) in sera from all the experimental groups: W5PI+W8RI = primary infected with 2 x 105 Eimeria ninakholyakimovae sporulated oocysts at week 5 of age and reinfected at week 8; W8PI= primary infected at week 8 of age or challenge controls; C= uninfected animals. (*) P<0.05 and (**) P<0.01 represent significant differences between infected animals and control group. Figure 3. Levels of IgA anti-SOA levels ± SD in ileal mucus expressed as relative optical densities (O.D.) in sera from all the experimental groups: W5PI+W8RI = primary infected with 2 x 105 Eimeria ninakholyakimovae sporulated oocysts at week 5 of age and reinfected at week 8; W8PI= primary infected at week 8 of age or challenge controls; C= uninfected animals. (*) P<0.05 and (**) P<0.01 represent significant differences between infected animals and control group. weights were also identified between 16 and 38 kDa. The strongest bands corresponded to peptides of 74, 54, 23 and 20 kDa. For the representation of the Western blotting results, a selection of the clearest and most exemplary weeks was made (Figure 5). The staining of the nitrocellulose membrane with Ponceau-S confirmed a good transference of proteins, as shown for the molecular weight marker (MWM). IgG from primary infected animals at week 5 firstly recognized specific bands after 1 weeks p.i., both in kids primary infected at 5 (W6/W5PI+W8PI) and 8 weeks of age (W9/W8PI); the intensity of the bands was bigger the following weeks though. In contrast to the pool of all week negative control sera (C), those of infected goat kids reacted with polypeptides of different molecular weights, ranging from 108 to 21 kDa. Peptides of 108 kDa and 28 kDa were the most strongly recognized. No differences either on the band profile or the intensity of the reactions were found between sera from re-infected (W5PI+W8RI) animals and the corresponding challenge controls (W8PI). 91 PRESENTACIÓN DE ARTÍCULOS Figure 4 . Protein bands separated by SDS–PAGE analysis using different concentration of sporulated oocyst antigen (SOA). MWM: molecular weight marker in kDa. 4. Discussion Based on the data obtained in the present study, the experimental infection of goat kids with 2 x 106 E. ninakohlyakimovae sporulated oocysts results in a patent infection with moderate so severe clinical signs and high rates of oocyst excretion in primary infected animals, while challenged goat kids only displayed mild signs of illness or no symptoms at all and the OPG counts were significantly lower. This is in accordance to previous studies performed with E. bovis in cattle (Hermosilla et al.,, 1999; Taubert et al., 2008; Sühwold et al., 2010) and also to those found by our group in goats (Ruiz et al., 2013; 2014), which reinforce the conviction that this Eimeria species is able to develop protective immune responses in goats. Probably related to this response, increased serum levels of specific IgG and IgM and local IgA were found to be increased in all the infected animals. Additionally, peripheral IgG could recognize a panel of different antibodies against the SOA (sporozoite oocyst antigen) used for immunoenzimatic reactions. More than 20 years ago, Kanyari (1988) demonstrated significantly increased antibody titres post-infection in two breeds of goats (Saanen and Anglonubians) after an experimental infection with 200,000 sporulated oocysts comprising mainly E. christenseni (49%), E. apsheronica (29%) and lesser proportions of E. arloingi , E. hirci , E. ninakohlyakimovae and E. alijevi . At our knowledge, no further studies on the humoral immune response in goats have been performed afterwards. Relatively more information concerning antibody response against coccidiosis in sheep and cattle is available in literature (Nolan et al., 1987; Hughes et al., 1989; Fiege et al., 1992; Dominguez et al., 2001; Faber et al., 2002). The results of the study published by Dominguez et al. (2001) showed that the halflife time of IgG1, by far the predominant isotype transmitted with colostrum in sheep (Reynolds and Griffin, 1990), comes up to 11– 13 days in the lamb (Klobasa and Werhahn, 1989; Watson, 1992; Dominguez et al., 2001), which suggests that this, and probably other maternal antibodies, would not be longer present in serum samples from goat kids used in the present study, taking into account that all the animals were purchased having 1-5 days of life. So, the levels of antibodies founds throughout the experiment should be considered as de novo synthesis. Significantly increased levels of serum IgG were already observed after 3 weeks p.i., slightly after the oocyst shedding reached peak values, which suggest that the immune system needs a boosting for the production of specific antibodies. Specific IgG values remained high and progressively increased during the next sampling times, probably related to further stimulation induced by re-infection made at week 8. In accordance, IgG levels recorded in challenge control group did not last increased but declined after the three week peak. Similarly, serum IgG against sporozoites reached a peak of activity between 10 and 20 days p.i., coinciding with oocyst shedding on 92 PRESENTACIÓN DE ARTÍCULOS Figure 5. Immunorecognition using Western blotting of Eimeria ninakholyakimovae sporulated oocysts antigens (SOA) by pool sera from goat kids infected with 2 x 105 oocysts at different weeks p.i. (Lane 1) molecular weight markers (MWM) in kDa.; (Lane 2) negative control; (lines 3-6) W8PI= primary infected at week 8 of age or challenge controls; (lines 7-10) W5PI+W8RI = primary infected at week 5 of age and re-infected at week 8. days 17 to 24, and the antibody titers dropped to base levels by 40 days p.i. (Hughes et al., 1989). Faber et al. (2002) also found IgG2 as the main isotype in the humoral response, in association to Th2 responses against E. bovis . Apart from IgG2, the IgG1 subfraction and specific IgM can be transferred to calves via colostrum (Fiege et al., 1992), and serum IgM, IgA and IgG2 have been correlate significantly with excretion of oocysts of E. bovis, whereas IgG1 is not directly related to the course of the disease (Faber et al., 2002). In our study, IgM levels were equally boosted by E. ninakholyakimovae infection but, compared to IgG profile, significant high values were already demonstrated after two weeks p.i. However, for this immunoglobulin, differences between re-infected animals and challenge controls could not be found. Similarly, local IgA from ileal mucus was significantly increased in infected animals compared to controls, but without differences between primary and re-infected animals. Altogether, the analysis of the antibody response suggests that, although the different immunoglobulins actually reflect exposure to E. ninakholyakimovae an unambiguous correlation to the level of protection could not be demonstrated, as previously referred (Fiege et al., 1992). Accordingly, although occasional relationships between OPG counts and the levels of either IgG, IgM or IgA were found, correlations could not be proven statistically. Specific peptide fractions recognized by serum IgG of animals using sporulated oocysts antigens (SOA), with molecular weights ranging between 134 to 16 kDa, support the value of electroimmunoblotting as a technique to detect immunoreactive proteins in goat coccidiosis. This approach, which had not been used before in caprines against Eimeria infections, has been extensively employed in poultry coccidiosis (Reduker et al., 1986; Jenking and Dame, 1987; Karkhanis et al., 1991; Xie et al., 1992; Krücken et al., 2008. Likewise), could demonstrated a panel of both common and unique protein bands in extracts of merozoites and sporozoites E. bovis , ranging in molecular weight from 15,000 to 215,000. The authors showed that of 3 types of immune sera used to probe immunoblots, serum taken from a calf that had been inoculated with oocysts of E. bovis and boosted 10 weeks later consistently identified and reacted more intensely with more antigens of merozoites and sporozoites than the other immune sera tested. This observation however could not be demonstrated in our study. By contrast, the pattern of peptide of SOA recognition did not differ between primary infected and challenged animals, indicating that there was no correlation between natural protection by previous exposure to the parasite and the specific antibody response, as also discussed for the ELISA results. This circumstance might be related to the type of antigen used in the ELISA and EIB determinations, as different Eimeria antigens have been demonstrated to exhibit specific immunogenicity (Fayer et al., 1992). It should be also considered that the age of the animals could strongly affect the development of protective immune response, both cellular and humoral (Smith et al., 1985; unpublished personal observations). Nevertheless, Lillehoj and Ruff (1987), found similar antibody 93 PRESENTACIÓN DE ARTÍCULOS response y chickens with different susceptibility to coccidiosis. Besides, sublines of chickens selected for high (HA) or low (LA) antibody response that differed at the major histocompatibility complex (MHC) had no differences in response to E. tenella infections (Dunnington et al., 1992). Fiege et al. (1992) also found no correlations between the levels of any specific antibody or the recognition patterns and the status of immunity to a severe challenge in experiments performed on colostral transfer of E. bovis antibodies in cattle. Classical research studies carried out worldwide to try to elucidate the mechanism of protective immunity against coccidiosis usually conclude that cellular immunity is the key to protection against Eimeria , whereas humoral immunity plays a very minor role in resistance against infection. By contrast, other studies have pointed towards the ability of antibody to block parasite invasion, development and transmission and to provide passive and maternal immunity against challenge infection (Wallach et al., 2010). Altogether, the results of the present study contribute to understand the complexity of the antibody response against caprine coccidiosis. Further investigations on the immunorecognition of other antigen preparations would be valuable for the immunoprophylactic approach for coccidiosis in livestock. 5. Acknowledgements This work has financially been supported by funding derived from the Spanish Ministry of Science and Innovation (MICIN) and the ACIISI (Agencia Canaria de Investigación, Innovación y Sociedad de la Información). All animal experiments included in the current study comply with the present laws of the Spanish government. 6. References Abrahamsen MS (1998) Bovine T cell responses to Cryptosporidium parvum infection. Int J Parasitol 28:1083-8. Review Cavalcante AC, Teixeira M, Monteiro JP, Lopes CW (2012) Eimeria species in dairy goats in Brazil. Vet Parasitol 183:356–8 Conde M., J.M. Molina J.M., Rodríguez-Ponce E., Ruiz A., González J (2001) Analysis of IgG response to experimental infection with RH Toxoplasma gondii in goats. Comparative Immunology, Microbiology and Infectious Diseases 24:197-206 Correia A, Ferreirinha P, Costa AA, Dias J, Melo J, Costa R, Ribeiro A, Faustino A, Teixeira L, Rocha A, Vilanova M (2013) Mucosal and systemic T cell response in mice intragastrically infected with Neospora caninum tachyzoites. Vet Res . 10;44:69. Daugschies A, Najdrowski M (2005) Eimeriosis in cattle: current understanding. J Vet Med B Infect Dis Vet Public Health 52:417–27 Dominguez, E., Perez, M.D., Puyol, P., Sanchez, L., Calvo, M (2001) Specific immunoglobulins in serum of newborn lambs fed with a single dose of colostrum containing anti-peroxidase IgG. Res. Vet. Sci . 70:275–279. Donkin, EF and Boyazoglu, PA (2004) Disease and mortality of goat kids in a South African milk goat herd. S Afr J Anim Sci 34:258–261. Dunnington EA, Gross WB, Martin A, Siegel PB (1992) Response to Eimeria tenella of chickens selected for high or low antibody response and differing in haplotypes at the major histocompatibility complex. Avian Dis 36:49-53. Faber JE, Kollmann D, Heise A, Bauer C, Failing K, Bürger HJ, Zahner H (2002) Eimeria infections in cows in the periparturient phase and their calves: oocyst excretion and levels of specific serum and colostrum antibodies. Vet Parasitol 104:1–17. 94 PRESENTACIÓN DE ARTÍCULOS serum of the infected animals, with an initial increase of IgM, followed by IgG; also other specific immunoglobulins such as IgA, may also appear. In general, the amount of antibodies increases if the animals are continually exposed to the oocysts (Reeg et al., 2005; Daugschies and Najdrowski, 2005). The antibodies can even transmitted through the colostrum, as shown by Gregory et al (1989) and Fiege et al. (1992), sometimes in correlation with the intensity of the infection of the offspring (Faber et al., 2002). The age of the animals influences the immune response to infection, not only in coccidiosis, but also in other common parasitic diseases of ruminants. For instance, studies conducted in sheep, from two weeks to 10 months of age, infected with Teladorsagia circumcincta showed that younger animals were more susceptible to reinfections and displayed a poor local immune response against gastrointestinal nematodes ( Smith et al., 1985). Also in vaccination approaches, age-related immunoprotection seems to be a perspective to be considered. Thus, experimental challenge infection by immunization with excretory/secretory products (ES) from Haemonchus contortus , containing predominantly proteins of 15 and 24 kDa, depends on the age of the sheep; i.e. vaccinated sheep 9 and 6 months of age had reduced final worm burdens of 82 and 77, respectively, whereas no reduction in worm burden was found in 3-month-old lambs. This circumstance should be particularly considered for the development of immunoprophylactic and therapeutic strategies against ruminant coccidiosis, as vaccine-induced protection has to elicited within the first weeks of age, before the a clinical impairment of the disease occurs. Taking all these considerations into account, the objective of the present study has been to investigate the influence of age in the development of protective immune responses in experimental goat coccidiosis by Eimeria ninakohlyakimovae . 2. Material al methods 2.1. Parasites and animals Experimental infections were performed by using the Eimeria ninakholyakimovae strain GC isolated from our laboratory from field samples and maintained by passages in goat kids for oocysts production (Ruiz et al. 2013). For harboring oocysts, goat kids were orally infected at the age of 4 week with 2 x 105 sporulated E. ninakholyakimovae oocysts, and the oocysts were isolated and purified from faeces after 2 week post infection according as described previously (Jackson 1964; Ruiz et al., 2013). The purified oocysts were maintained at 4 °C in a 2% potassium dichromate solution to further use. A total of 24 goat kids of the Majorera breed purchased from local farmers (Gran Canaria) were used. Newborn kids of 1-5 days were washed with a diluted solution of sodium hypochlorite in warm water in order to eliminate oocysts that may be attached to the hair, then dried and distributed in separated boxes into metabolic cages equipped with heat sources. The day of the arrival and a week later, all animals were treated with Vecoxan® (Janssen-Cilag) and, for a week with Halocur® (Intervet), following the manufacturer's recommendations in both cases. They were fed with milk substitute (Bacilacto®, Capisa) and starter (Capisa) and, in all cases, sterile hay, minerals and water were available ad libitum. The animals were treated according to the guidelines adopted in the European Communities Council of 24 November 1986 (86/609 / EEX) and the current Spanish legislation for the use and care of animals RD 1201/2005 (BOE 252 / 34367-91, 2005). 2.2. Experimental design For experimental purposes, the animals were divided into three experimental age groups, each consisting of three subgroups: (PI+RI) primary infected animals 101 PRESENTACIÓN DE ARTÍCULOS at 3, 4 and 5 weeks of age, and three weeks later; (PI) primary infected animals at 6, 7 and 8 weeks or controls of re-infection; and (C) uninfected animals. Age groups were arbitrarily named as A (W3PI+W6RI; W6PI), B (W4PI+W7RI; W7PI) and C (W5PI+W8RI; W8PI). Both primary and challenge infection were performed orally with 2 x106 Eimeria ninakholyakimovae sporulated oocysts by using a gastric feeding tube. The protective immunity after primary infection and subsequent reinfection in the different age groups was assessed clinical parameters (signs of eimeriosis, i.e. diarrhea), production parameters (body weight), hematological, parasitological parameters (faecal oocyst counts), immunological parameters (antibody and cellular immune responses) and pathological parameters (gross and microscopic lesions in colon and ileum). For this purpose, individual faecal and blood samples were. The body weight and clinical condition of all the animals were also examined and, at the end of the experiment, all the goat kids were euthanized and further subjected to necropsy. 2.3. Clinical, coprological and haematological determinations Throughout the whole the experience the clinical signs of the animals was evaluated by inspecting the hydration state, food intake and the presence of diarrhea, evaluated according to their consistence, color, presence of blood or mucus. In addition the weight of the animals was weekly monitored. For coprological analyses, faecal samples were daily taken from day 14 postinfection (p.i.); non-infected controls were also subjected to coprological analysis in order to verify the absence of infection. For the quantification of the oocysts excretion a modified McMaster technique was used (Thienpont et al., 1979) and the results were expressed as oocysts per gram of faeces (OPG). In cases of very high OPG values, dilutions of the faecal suspension by 10 or 100 times were performed to enable accurate counting according to Bangoura and Daugschies (2007). For hematological analysis, blood samples were taken weekly by jugular vein puncture. The total leukocyte count and the hemoglobin concentration were determined in IDEXX VetConnect® 1-mL tubes by using a LaserCyte® hematology analyzer (Idexx). The hematocrit value was calculated by centrifugation using standard centrifuge capillaries. Differential white blood cell (WBC) counting was performed manually; for this purpose, 200 leukocytes were counted in stained blood smears (Diff-Quick). 2.4. Pathological and histopathological analysi s At weeks 9, 10 and 11 of the experiment, animals of different age groups were sacrificed in order to perform histopathology. Goat kids primary infected at week 3 of age and challenged at week 6 (W3PI+W6RI) and the corresponding challenge control subgroup (W6PI) (age group A) were euthanized at week 9 of the experiment, while weeks 10 and 11 of the experience were reserved animals from age groups B and C, respectively. During the necropsy, all macroscopic lesions were annotated and tissue samples were collected from the intestinal mucosa (ileum, colon). Additionally, mucus samples were taken from the ileum of the animals for the analysis of specific IgA levels. For their preservation, the samples were suspended in a pH 7.1 buffer containing proteinases inhibitors (all compounds from Sigma-Aldrich): 0.1M Na2HPO4, 0.05 M NaCl, 3 mM NaN3, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 5 mM ethylenediaminetetraacetic acid (EDTA). Mix solutions containing mucus were centrifuged at 5000 x g (Eppendorf Centrifuge 5804R) for 1 h at 4°C and the resulting supernatant was conserved at -20ºC up to further analyses. The tissue samples were fixed in 10% buffered formalin and embedded in paraffin. Cross sections of 4–5µm were stained by haematoxylin and eosin (H&E) and Giemsa according to standard staining procedures. Quantification of leucocyte populations (neutrophils, lymphocytes, eosinophils, globular leukocyte and mast cells) in tissue sections was performed on the ileum and colon samples of uninfected (C), challenge 102 PRESENTACIÓN DE ARTÍCULOS controls (PI) and challenge infected goat kids (RI). Cells were counted using a 10× eyepiece containing a calibrated graticule and 40× objective lens viewing an area of 0.05265 mm2. The counts were randomly taken on 20 graticule fields within the mucosal surface. The counts were expressed as number of cells per mm2 of mucosa (Amarante et al., 2005). 2.5. Enzyme – linked immunosorbent assay (ELISA) To determine the levels of specific IgG and IgM antibodies, sera samples weekly collected were kept at -20°C until the appropriate tests performed. For the IgG indirect ELISA test, a total of 100 µl of a SOA solution in carbonated buffer at a concentration of 5µg/ml was dispensed in 96 wells ELISA plates (CORNING Dispensable Sterile ELISA Plates, Corning Glass Works) and incubated overnight at 4°C. After three washes with 200 µl of PBS-0.05 % Tween 20, 200 µl of PBS-3% (w/v) bovine serum albumin (BSA, Sigma-Aldrich) was added and the plates were incubated for 45 min at 37°C. After each new step a similar washing procedure was addressed. Following treatment with BSA, individual serums, positive and negative controls were analyzed in duplicate using a 1/25dilution in PBS-Tween 20 0.02% sodium acid. For each sample, a volume of 100 µl was added to the plates and a new incubation was performed at 37°C for 1h. The plates were then incubated with 100 µl per well of 1/2000 anti-Goat IgG (anti-goat IgG peroxidase conjugate, IgG fraction of antiserum, Sigma-Aldrich) diluted in 0.01 M PBS and the incubation was performed in the same conditions. Thereafter, a total of 100 µl of substrate was added to each well, a mix of compounds that includes citric-phosphate buffer, 0.04% (w/v) dihydrochloride orthophenyleneadiamine (OPD, Sigma-Aldrich) and 30% hydrogen peroxide (Panreac) at a final concentration of 0.1% (v/v). The incubation with the substrate was performed at room temperature in darkness for approximately10 minutes. Finally, the reactions was stopped by adding 35 µl per well of a 2M solution of sulfuric acid (Panreac) and the optical density (O.D.) measured at a wavelength of 492 nm (Termo Labsystems, Multiskan Ascent). For specific IgM and IgA indirect ELISA tests a similar protocol was employed but using 1/3000 anti-goat IgM peroxidase and 1/7000 anti-goat IgA peroxidase conjugates, respectively (both from Sigma-Aldrich). Similarly, dilutions 1/25 of the different serum or mucus samples were employed as primary antibody. 2.6. Statistical analysis of data Faecal OPG were logarithmically transformed and added by 1 [log (OPG+1)] in order to obtain normal distributions according to the Kolmogorov-Smirnov’s normality test. Levels of the different immunoglobulins analysed in the study were expressed as the relative percentage of the optical density (O.D.) of a positive control pool in order to avoid inter-assay differences (Relative O.D.). One factorial analysis of variance, Tukey’s multiple comparison test and Student’s t-test were used to analyse the data between different experimental groups and the Pearson correlation test for the evaluation of the association between different parameters assessed in this study. For that purpose, a statistical software (SigmaPlot 12.0) was employed and the differences were regarded as significant at a level of p ≤ 0.05. 3. Results 3.1. OPG counts, clinical signs and body weights In all age groups of kids re-infected with E. ninakohlyakimovae sporulated oocyst (A, B and C) oocyst counts per gram of feces (OPG) were consistently lower than in animals from the corresponding challenge controls (Fig. 1). Similarly, after primary infection, goat kids subsequently challenged 3 weeks later had significantly reduced OPG counts ( p < 0.05 to p < 0.001). In general, the prepatent period ranged from 14 to 17 days p.i., with slightly longer records found in goat kids primary infected at younger age, mainly W3PI group. 103 PRESENTACIÓN DE ARTÍCULOS On the contrary, some of the re-infected animals (i.e. from W5PI+W8PI) extended their prepatent period up to day 18 p.i. Peak values were not certainly related with duration of the prepatent periods of the different animal groups, except for kids from primary infected at week 3 (W3PI) and subsequently challenged, in which the highest OPG counts were recorded slightly latter (day 17 p.i.) in both groups. All inoculated animals developed a patent infection, but the severity of the disease was less apparent, and even imperceptible, in re-infected animals in all age groups (A, B and C) (Fig. 2). Clinical signs, which included anorexia, weakness, impairment of general condition and different degrees of diarrhoea, were particularly intense in some primary infected animals at week 5 and 4, moderate in those infected at week 6, 7 and 8 and moderate to mild in 3 week infected ones. In terms of production, both the challenge infected (PI+RI) and challenge controls groups of animals (PI) had lower mean body weights that the corresponding non-infected animals. Statistical significance ( p < 0.05 to p < 0.01) was particularly evident when comparing the weight progress of W4PI+W7PI, although in this case the differences between re-infected animals and the corresponding challenge control (W7PI) could not be proven statistically. By contrast, slight significant differences ( p < 0.05) were found when comparing body weights of reinfected animals at week 6 (W3PI+W6RI) to those primary infected at week 6 (W6PI), at least in certain weeks. Hematologic changes were very mild in all groups and only a moderate increase in the total number of leukocytes, neutrophilia with left shift and temporary monocytosis was observed (data not shown). Finally, in the last weeks of the experiment, a moderate eosinophilia was observed, which was particularly evident in the group primary infected at 5 weeks and then challenged (W5PI+W8RI). 3.2. Pathological and histopathological analysis At necropsy, all intestinal organs of the infected animals were apparently normal, except for some evidence of congestion and thickening of the intestinal mucosa, mainly affecting colon caecum, but also some portions of the ileum. Histological examination showed a moderate hyperplasia and hypertrophy of the mesenteric lymph nodes, and Peyer’s patches, eosinophilic enteritis and mastocytosis with diffuse infiltration of lymphocytes, neutrophils and globular leukocytes. Uninfected animals did not show any alteration on gross morphology and microscopical examination did not reveal any obvious change either. With some differences depending on the age group, both the ileum and colon mucosa of the infected goat kids were significantly infiltrated by different inflammatory cells, including neutrophils, mast cells, eosinophils, globular leukocytes and lymphocytes when compared to tissue samples from uninfected control animals. Eosinophils in ileum were higher than in colon for all animals (Fig. 3). For ileum samples, the higher counts corresponded to challenged groups (W3PI+W6RI, W4PI+W7RI and W5PI+W8RI), which, except for group W5PI+W8RI, had significantly increased values compared to their corresponding challenge controls (W6PI and W7PI) ( p < 0.05 to p < 0.001). By contrast, approximately the same counts were recorded for colonic samples both in challenge and the corresponding challenge control groups. Significant differences with respect to controls were found for both challenged and control challenged goat kids in all age groups (Fig. 3). A similar pattern was found for lymphocyte counts, although in this occasion differences between ileum and colon were not so evident (Fig. 4). Like for eosinophils, lymphocyte counts were significantly higher in all the infected animals when compared to controls ( p < 0.001 to p < 0.0001) and, in this case, 104 PRESENTACIÓN DE ARTÍCULOS Figure 1 . OPG (oocysts per gram of faeces) counts from different age group (A, B and C) after primary or challenge infections with Eimeria ninakohlyakimovae sporulated oocysts. (A) primary infected at week 3 and challenge at week 6 (W3PI+W6RI) and primary infected at week 6 or challenge control (W6PI); (B) primary infected at week 4 and challenge at week 7 (W4PI+W7RI) and primary infected at week 7 or challenge control (W7PI); (C) primary infected at week 5 and challenge at week 8 (W5PI+W8RI) and primary infected at week 8 or challenge control (W8PI). OPG counts are depicted as the logarithm of the OPG plus one (log [OPG + 1]) and represent the mean ± SEM in all the experimental groups. (*) p < 0.05, (**) p < 0.01 and (***) p < 0.001 represent significant differences between primary and challenge infections between a same age group; (a) p < 0.05, (b) p < 0.01 and (c) p < 0.001 001 represent significant differences between challenge infected and challenge control subgroups. statistical differences between challenge and control challenge groups could not be proven in group WPI3+W6RI. Most of neutrophil counts were found to be increased as well in comparison to uninfected controls, with significant levels ranging from p < 0.05 to p < 0.001 (Fig. 5). However, in this cell population, higher counts corresponded to primary infected challenge controls, with differences ranging from p < 0.05 to p < 0.001 in relation to challenged groups. As for neutrophil counts, the number of globular leukocytes at the intestinal mucosa of primary infected or challenge kids were, in general, higher than counts recorded for challenge groups (Fig. 6) ( p < 0.05 to p < 0.01) and no many differences were found between ileal and colonic samples; all the infected animals had increased globular leukocyte counts though when compared to control samples ( p < 0.05 to p < 0.001). Finally, in samples from ileum, mast cells counts were significantly increased in groups W3PI+W6RI and W6PI ( p < 0.001 and p < 0.05, respectively) and also in group W7PI ( p < 0.05), while in colonic samples the three challenge control groups had increased records for this cell population ( p < 0.01 to p < 0.001) (Fig. 7). In colon, counts from challenge animals were generally lower than in the corresponding challenge groups, with significant differences ranging from ( p < 0.05 to p < 0.001) The global cell counts for lymphocytes and neutrophils were approximately the same among the three age groups considered, whereas younger groups, particularly those primary infected at week 3 105 PRESENTACIÓN DE ARTÍCULOS Figure 2. Body weights of goat kids from different age group (A, B and C) after primary or challenge infections with Eimeria ninakohlyakimovae sporulated oocysts. (A) primary infected at week 3 and challenge at week 6 (W3PI+W6RI) and primary infected at week 6 or challenge control (W6PI); (B) primary infected at week 4 and challenge at week 7 (W4PI+W7RI) and primary infected at week 7 or challenge control (W7PI); (C) primary infected at week 5 and challenge at week 8 (W5PI+W8RI) and primary infected at week 8 or challenge control (W8PI). CONTROL represents uninfected animals. Data represent the mean ± SEM in all the experimental groups. (*) p < 0.05 and (**) p < 0.01 represent significant differences between infected animals and CONTROL; (a) p < 0.05 represents significant differences between challenge infected and challenge control subgroups. and challenged week latter (W3PI+W6RI) and the corresponding challenge control (W6PI), had higher records for the remaining cell populations analyzed in the study. Correlations between the different cell scores and their respective parasitological and clinical data were not proven statistically. 3.3. Enzyme-linked immunosorbent assay (ELISA) The specific levels of IgG did not differ from uninfected controls profile in age groups A and B, while in those primary infected at week 5 and subsequently challenged at week 8 (group C) a gradual increase was observed up to the end of the experiment; the differences were not statistically significant though (Fig. 8). Peack values for specific IgM were recorded three week after the primary infection in group C and then gradually decreased up to the end of the experiment. By contrast, IgM relative O.D. for groups A and B slightly increased week to week from the beginning to the end of the experiment without a clear peak being detected. With some fluctuations, challenge controls of groups A, B and C slightly increased after infection and had weak peak values around 2 week p.i. No significant differences could be detected in any of the age groups (Fig. 9). At last, local IgA levels were proven to be significantly higher in challenge control animals of all age groups (( p < 0.05 to p < 0.01), while only challenged kids from age group A had increased relative O.D. for this immunoglobulin (( p < 0.05) (Fig. 10). Probably due to fluctuations of optical densities among animals and the scarce number of animals per group, as for cell counts, no correlations could be proven to parasitological or clinical data. 106 PRESENTACIÓN DE ARTÍCULOS Figure 3 . Eosinophils counts, expressed as number of cells per mm2, in the mucosa of the ileum and colon of goat kids from different age group (A, B and C) after primary or challenge infections with Eimeria ninakohlyakimovae sporulated oocysts. (A) primary infected at week 3 and challenge at week 6 (W3PI+W6RI) and primary infected at week 6 or challenge control (W6PI); (B) primary infected at week 4 and challenge at week 7 (W4PI+W7RI) and primary infected at week 7 or challenge control (W7PI); (C) primary infected at week 5 and challenge at week 8 (W5PI+W8RI) and primary infected at week 8 or challenge control (W8PI). CONTROL represents uninfected animals. Data represent the mean ± SEM in all the experimental groups. (*) p < 0.05 and (**) p < 0.01 represent significant differences between infected animals and CONTROL; (b) p < 0.01 and (c) p < 0.001 represent significant differences between challenge infected and challenge control subgroups. 4. Discussion There is enough evidence showing that vaccination approaches at early age have numerous advantages for many diseases and may be the only alternative for others. For instance, in chicken salmonellosis , which can occur by both vertical and horizontal transmission (Cox et al., 2000), initial infections of broilers usually takes place early post-hatch and the infected young chicks led to high levels of environmental contamination and rapid transmission of pathogens as a result of litter contamination (van Immerseel et al., 2005). Although high titres of specific maternal antibodies are transferred to new born chicks (Methner et a., 2002), they lasts no more than a few weeks, so there is a need control strategies that confer resistance just after hatching and maintain long-term protective effects. Vaccination of young birds themselves has the disadvantage that the very young bird is immunologically immature (Friedman et al., 2003). Accordingly, a number of studies have investigated the response of chickens to immunisation against a variety of antigens, with results showing varying humoral and T cell responses in birds of different ages. T cell responsiveness to mitogens, including ConA, does not fully develop in birds until 1 week of age (Lowenthal et al., 1994), whilst variation in the humoral response to infection with M. gallisepticum and antigenic stimulation by BSA has been seen up until 2 weeks of age (Mast et al., 1999). However, a number of vaccines are able to provide protection when administered in ovo (Rick et al., 1999; Lillehoj et al., 2005). In general, age-related immunity 107 PRESENTACIÓN DE ARTÍCULOS 108 PRESENTACIÓN DE ARTÍCULOS 109 PRESENTACIÓN DE ARTÍCULOS Figure 4 . Lymphocyte counts, expressed as number of cells per mm2, in the mucosa of the ileum and colon of goat kids from different age group (A, B and C) after primary or challenge infections with Eimeria ninakohlyakimovae sporulated oocysts. (A) primary infected at week 3 and challenge at week 6 (W3PI+W6RI) and primary infected at week 6 or challenge control (W6PI); (B) primary infected at week 4 and challenge at week 7 (W4PI+W7RI) and primary infected at week 7 or challenge control (W7PI); (C) primary infected at week 5 and challenge at week 8 (W5PI+W8RI) and primary infected at week 8 or challenge control (W8PI). CONTROL represents uninfected animals. Data represent the mean ± SEM in all the experimental groups. (*) p < 0.05 represents significant differences between infected animals and CONTROL; (b) p < 0.01 and (c) p < 0.001 represent significant differences between challenge infected and challenge control subgroups. Figure 5. Neuthophils counts, expressed as number of cells per mm2, in the mucosa of the ileum and colon of goat kids from different age group (A, B and C) after primary or challenge infections with Eimeria ninakohlyakimovae sporulated oocysts. (A) primary infected at week 3 and challenge at week 6 (W3PI+W6RI) and primary infected at week 6 or challenge control (W6PI); (B) primary infected at week 4 and challenge at week 7 (W4PI+W7RI) and primary infected at week 7 or challenge control (W7PI); (C) primary infected at week 5 and challenge at week 8 (W5PI+W8RI) and primary infected at week 8 or challenge control (W8PI). CONTROL represents uninfected animals. Data represent the mean ± SEM in all the experimental groups. (*) p < 0.05, (**) p < 0.01 and (***) p < 0.01 represent significant differences between infected animals and CONTROL; (a) p < 0.05, (b) p < 0.01 and p < 0.001 represent significant differences between challenge infected and challenge control subgroups. Figure 6 . Globular leukocyte counts, expressed as number of cells per mm2, in the mucosa of the ileum and colon of goat kids from different age group (A, B and C) after primary or challenge infections with Eimeria ninakohlyakimovae sporulated oocysts. (A) primary infected at week 3 and challenge at week 6 (W3PI+W6RI) and primary infected at week 6 or challenge control (W6PI); (B) primary infected at week 4 and challenge at week 7 (W4PI+W7RI) and primary infected at week 7 or challenge control (W7PI); (C) primary infected at week 5 and challenge at week 8 (W5PI+W8RI) and primary infected at week 8 or challenge control (W8PI). CONTROL represents uninfected animals. Data represent the mean ± SEM in all the experimental groups. (*) p < 0.05 and (**) p < 0.01 represent significant differences between infected animals and CONTROL; (a) p < 0.05, (b) p < 0.01 and p < 0.001 represent significant differences between challenge infected and challenge control subgroups. Figure 7. Mast cell counts, expressed as number of cells per mm2, in the mucosa of the ileum and colon of goat kids from different age group (A, B and C) after primary or challenge infections with Eimeria ninakohlyakimovae sporulated oocysts. (A) primary infected at week 3 and challenge at week 6 (W3PI+W6RI) and primary infected at week 6 or challenge control (W6PI); (B) primary infected at week 4 and challenge at week 7 (W4PI+W7RI) and primary infected at week 7 or challenge control (W7PI); (C) primary infected at week 5 and challenge at week 8 (W5PI+W8RI) and primary infected at week 8 or challenge control (W8PI). CONTROL represents uninfected animals. Data represent the mean ± SEM in all the experimental groups. (*) p < 0.05 and (***) p < 0.01 represent significant differences between infected animals and CONTROL; (a) p < 0.05, (b) p < 0.01 and p < 0.001 represent significant differences between challenge infected and challenge control subgroups. 110 PRESENTACIÓN DE ARTÍCULOS ARTÍCULO Nº 5 Estudio de la respuesta inmunitaria protectora durante el periodo prepatente en cabritos experimentalmente infectados con Eimeria ninakholyakimovae In preparation to: Veterinary Parasitology ▪ RESUMEN ▪ Sin descartar la importancia de la inmunidad humoral en la eimeriosis en rumiantes, las respuestas inmunes celulares parecen jugar un papel crucial en la resistencia adquirida después de reinfecciones con el protozoo Apicomplexa Eimeria ninakohlyakimovae . En las cabras, las respuestas inmunes celulares frente a la coccidiosis han sido poco investigadas, especialmente en la fase temprana de la infección. En este estudio hemos evaluado los cambios en las poblaciones de células inmunes en la mucosa intestinal en el período prepatente tras infecciones y posteriores reinfecciones con esta especie de Eimeria . Para ello, un total de 15 cabritos se dividieron en tres grupos: (i) cabritos primo-infectados a las 4 semanas de edad y reinfectados tres semanas más tarde (Grupo RI); (ii) animales primoinfectados a las 8 semanas de edad (= controles de re-infección, Grupo PI); y (iii) cabritos no infectados (Grupo C). Todos los grupos fueron sacrificados una semana después de la reinfección, es decir, dentro del plazo de prepatencia. Los animales se inocularon con ooquistes esporulados de la cepa GC de Eimeria ninakholyakimovae . La inmunidad protectora en los diferentes grupos se evaluó en base a parámetros clínicos, productivos, hematológicos, parasitológicos, inmunológicos (anticuerpos y respuestas inmunes celulares) y parámetros patológicos (lesiones macroscópicas y microscópicas en el colon y el íleon). Con este propósito, se tomaron muestras fecales y de sangre individuales. También se examinaron el peso corporal y la condición clínica de todos los animales y, al final del experimento, todos los cabritos fueron sacrificados y se realizó la necropsia de todos ellos. Se tomaron muestras de las diferentes secciones de íleon, colon y nódulos linfáticos mesentéricos (MLN). Así mismo, se identificaron las lesiones histopatológicas y se realizaron recuentos celulares de linfocitos, eosinófilos, neutrófilos y leucocitos globulares (identificados por hematoxilina y eosina) y de mastocitos (mediante tinción con azul de Giemsa). Además, se llevó a cabo un análisis de expresión génica de IL-2, IL-4, IL-10 e INFγ en íleon, colon y MLN, así como la caracterización de células inmunes por inmunohistoquímica en secciones de íleon y colon. La infección por E. ninakohlyakimovae fue de moderada a severa, se observaron diversos grados de diarrea y se acompañó de altos recuentos de OPG durante la infección primaria. 117 PRESENTACIÓN DE ARTÍCULOS También se observó un aumento en el recuento de casi todos los tipos de células inmunes analizados en comparación con los animales no infectados. Por otra parte, los recuentos de eosinófilos, linfocitos, leucocitos globulares y mastocitos fueron significativamente más elevados en los animales reinfectados que en los primoinfectados, mientras que ocurrió lo contrario para los recuentos de neutrófilos. La primoinfección también se asoció a un aumento moderados/leve de los niveles séricos de IgG e IgM e IgA local. Sorprendentemente, el número de esquizontes inmaduros encontrados en la mucosa del íleon fue estadísticamente mayor en el grupo de primoinfectados. Además de la reducción del número de esquizontes inmaduros, en los animales reinfectados se observó un aumento de los linfocitos y otras poblaciones celulares; en concreto, los recuentos de T CD4 + y T CD8 + se encontraron aumentados, lo que indica que las células T podrían estar relacionada con el desarrollo de la respuesta protectora. La respuesta inmune desarrollada fue, sin embargo, muy compleja, ya que células presentadoras de antígeno y otras poblaciones de células efectoras del sistema inmune innato, así como ciertas citoquinas, también estuvieron involucradas. En su conjunto, los resultados de este estudio contribuyen a comprender la complejidad de la respuesta inmune celular y humoral caprina frente a la coccidiosis, en particular durante la fase de prepatencia, lo cual puede ser de utilidad para el desarrollo de estrategias para la modulación de la respuesta inmune y para la identificación de compuestos anticoccidiales o vacunas. 118 PRESENTACIÓN DE ARTÍCULOS Study of protective immune responses during prepatency in goat kids experimentally infected with Eimeria ninakholyakimovae 1Matos, L., 1Muñoz, M. C., 1Molina, J. M., 3Rodríguez, F., 1Perez, D., 1Lopez, A., 1Ferrer, O., 2Hermosilla, C., 2Taubert, A., 1,*Ruiz, A., 1Department of Animal Pathology, Faculty of Veterinary Medicine, University of Gran Canaria, Gran Canaria, Spain 2Institute of Parasitology, Faculty of Veterinary Medicine, Justus Liebig University Giessen, Giessen, Germany 3Department of Anatomy and Compared Anatomy Pathology, Faculty of Veterinary Medicine, University of Gran Canaria, Gran Canaria, Spain Abstract Without dismissing the importance of humoral immunity in ruminant eimeriosis, cellular immune responses seem to play a crucial role in acquired resistance after reinfections with the apicomplexan protozoa Eimeria ninakohlyakimovae . In goats, the cellular immune responses against coccidiosis have poorly been investigated, especially in the early phase of infection. In this study we have evaluated the changes of immune cell populations in intestinal mucosa in the prepatent period of infections as well as after challenge infection with this Eimeria species. Therefore, a total of 15 goat kids were divided into three groups: (i) goat kids primary infected at 4 weeks of age and re-infected three weeks later (Group RI); (ii) primary infected animals at 8 weeks of age (= controls of reinfection, Group PI); and (iii) uninfected goat kids (Group C). All groups were sacrificed one week after challenge infection, that is, within the period of prepatency. For infection, sporulated oocysts of the GC strain of Eimeria ninakholyakimovae were used. The protective immunity after primary infection and subsequent reinfection in the different groups was assessed by clinical, productive, hematological, parasitological, immunological (antibody and cellular immune responses) and pathological parameters (gross and microscopic lesions in colon and ileum). For this purpose, individual faecal and blood samples were taken. The body weight and clinical condition of all the animals were also examined and, at the end of the experiment, all the goat kids were euthanized and further subjected to necropsy. Samples were taken from different sections of the ileum, colon and mesenteric lymph nodes (MLN). Histopathological lesions were identified and cell counts of lymphocytes, eosinophils, neutrophils and globular leukocytes (identified by hematoxylin and eosin staining) and mast cells (identified by Giemsa blue staining) were recorded. Additionally, gene expression analysis of IL-2, IL-4, IL-10 and INFγ of ileal, colonic and MLN were performed, as well as the characterization of immune cells by immunohistochemistry in sections of ileum and colon. The E. ninakohlyakimovae infection resulted in moderate to severe enteritis and different degrees of diarrhoea and was accompanied by high OPG counts during the primary infection and an increase of almost all immune cell types analyzed compared to uninfected control animals. Furthermore, the counts of eosinophils, lymphocytes, globular leukocytes and mast cells were significantly higher in reinfected than in primary infected animals, whilst the opposite was true for neutrophils counts. Challenge infection was also associated to moderate/mild increased levels of serum IgG and IgM and local IgA. Interestingly, the number of immature schizonts found at the ileal mucosa was statistically higher in challenge group compared to challenge control animals. Apart from the association of 119 PRESENTACIÓN DE ARTÍCULOS reduced number of immmature schizonts in reinfected animals to mean values for lymphocyte and other cell population, in this group of animals a greater number of CD4+ and CD8+ cells were observed, all indicating that T cell response could be related to the development of the protective response. The immune response developed was, however, very complex, as antigen presenting cells and other effector cell populations of the innate immune system, as well as certain cytokines, are involved. As a whole, the results of this study contribute to understand the complexity of the cellular and humoral immune response of the goat hosts against coccidiosis, in particular during the prepatency, which can be used for the development of strategies for modulation of the immune response and for the identification of anticoccidial compounds or vaccines. Key words: Eimeria ninakholyakimovae; prepatency; immune response; goats *Corresponding author: Antonio Ruiz Reyes Department of Animal Pathology Tel: +9284511133; Fax: +92845341 Email: [email protected] 120 PRESENTACIÓN DE ARTÍCULOS 1. Introduction In arid and semi-arid areas (Ruiz et al., 2006; Khodakaram-Tafti et al., 2013), but also in other different geographical areas having diverse climatic conditions (Balicka-Ramisz et al., 2012; Zainalabidin et al., 2013), coccidian infections are considered one of the most important parasitic disease affecting goat production. The clinical signs and pathological changes associated to this parasitic disease (Dai et al., 2006; Kheirandish et al., 2014) mainly affect young kids, leading to high economic losses in terms of production, sometimes difficult to be determined (Koudela and Boková 1998). Sporozoites of Eimeria ninakholyakimovae, one of the most pathogenic species of goats (Dai et al., 2006), primary invades endothelial cells of lacteal ducts of the villi of the distal ileum where they develop first generation schizonts (up to 166 µm x 124 µm in size) within 10-12 days post-infection (p. i.), which finally release thousands of merozoites I (Vieira et al, 1997; Behrendt et al., 2010). The development of the schizonts, their rupture and the subsequent merozoite release lead to an extensive destruction of the intestinal mucosa of the infected animals, which may result in a malabsorption syndrome, even before the start of the faecal oocyst release and typical clinical signs such as the diarrhea appear. Actually, when high doses of oocysts are ingested, the magnitude of the tissue damaged may conduct to the death of the animal. This may explain why, under certain conditions, coccidiosis can be associated with sudden death without previous digestive signs, especially in young animals of between 2 and 4 months old (Chartier et al., 1994). The control of goat coccidiosis is principally based on the combination of management practices with the use of coccidiostats and anticoccidials, as sulfonamides (Svensson, 1998) or toltrazuril (Mund et al., 2003). Although new drug have been proven to be effective for goat coccidiosis, i.e. diclazuril (Ruiz et al., 2012) and ponazuril (Love et al., 2015), up to date there are no drugs registered for goats in the E.U. This, together with the increasing appearance of anticoccidial drug resistance in different geographic areas (Kawazoe and di Fabio, 1994; Williams, 2006) has stimulated the search of new alternatives for control, such as the use of vaccines. Recently, Ruiz et al. (2014) demonstrated that X-rad attenuated oocysts induce an immunoprotective response against a homologous challenge with E. ninakholyakimovae oocysts, and there is extensive literature in poultry showing that both sexual and asexual derived antigens may be useful for the design of recombinant vaccines (Vermeulen, 1998; Tewari and Maharana, 2011). Both for using live, attenuated or recombinant vaccines a deep knowledge of the immune response against Eimeria infections are a prerequisite. Coccidiosis produced by Eimeria spp. in ruminants (Catchpole et al., 1993) and in other host species (Shi et al., 2000) generally induces strong protective immune responses which prevent clinical disease derived from homologous challenge infections. The immune reactions developed against caprine Eimeria spp. have poorly been investigated, but recent experimental studies conducted in E. ninakholyakimovae demonstrate that previous exposure to the parasite induce strong protective immune responses which involves both innate and acquired components (Ruiz et al., 2013a). However, the same authors also found that protection can be truncated in case of high challenge infections (Ruiz et al., 2013b), suggesting that the immune response against the parasite is certainly complex. In the bovine system, different studies performed in Eimeria infections indicate that both humoral (B-cell) and cellular (T cell) responses are involved (Daugschies and Najdrowsky, 2005). Actually, levels of IgM, IgA and IgG2 transferred through the colostrum to calves have been found to negatively correlate with excretion of oocysts of E.bovis (Faber et al., 2002). Besides, several investigations dealing with cellular immune response demonstrate that both CD4+ and CD8+ T cell subsets are involved during primary E. bovis infection (Hermosilla et al.,1999) and that enhanced antigen-specific IFN-production in E.bovis infections indicate a Th1 immune response in prepatency in calves (Taubert et al.,2008). It 123 PRESENTACIÓN DE ARTÍCULOS has also been reported that antigen-specific T cells proliferated effectively during an strict time span during prepatency of primary infection (Hermosilla et al.,1999) but fail to do so after challenge infection, suggesting early abrogation of re-infection (Sühwold et al., 2010). Targets for the innate immune response against ruminant Eimeria species involve both sporozoites and merozoites, as shown by numerous in vitro studies performed in E. bovis (Hermosilla et al., 2006; Taubert et al., 2009; Behrendt et al., 2010; Muñoz-Caro et al., 2014), E. arloingi (Silva et al., 2014) and E. ninakholyakimovae (Pérez et al., 2015). The specific mechanisms involved in the development of acquired immune responses in vivo against ruminant coccidiosis are much less investigated. In this regards, apart from their pathological implications, first generation schizonts developed during prepatency are of great interest in various aspects. For one, the length of the maturation of schizonts suggests that E. ninakholyakimovae is influencing and / or modifying the host cell to allow the persistence of the parasite. Moreover, there is abundant evidence that first generation schizonts represent an important target for immune protective reactions (Taubert et al., 2006). In the present study we have investigated both innate and acquired immune reactions during the prepatent period of the disease in experimentally infected goat kids by E. ninakohlyakimovae . For this purpose, parasitological, haematological, clinical and pathological parameters were evaluated. Additionally, the antibody response, the characterization of immune cells by immunohistochemistry and the gene expression of different cytoquines were assessed. 2. Material and methods 2.1. Parasites and animals The Eimeria ninakholyakimovae strain GC used in the present study was initially isolated from the field of naturally infected goats in Gran Canaria Island (Spain) and maintained by passages in goat kids for oocysts production (Ruiz et al., 2013a). For harboring oocysts, goat kids were orally infected at the age of 4 week with 2 x 105 E. ninakholyakimovae sporulated oocysts. Newly produced and purified were concentrated and stored in a 2% potassium dichromate solution at 4°C until further use after they fulfill the sporulation proces. A total of 15 goat kids of the Majorera breed was purchased from a local farmer at the age of 1-5 days and maintained under parasite-free conditions in autoclaved stainless steel cages in a restricted stable (Faculty of Veterinary Medicine, University of Las Palmas de Gran Canaria, Spain). The kids were treated with Vecoxan® (Janssen-Cilag) and Halocur® (Intervet) just at their arrival to the Faculty of Veterinary Medicine. Goat kids were fed with the milk substitute Bacilactol® (Capisa) and commercial concentrate pellets for weaning goat kids (Starting Concentrate, Capisa). Water and sterilized hay were always given ad libitum. All animal procedures were conducted in strict accordance with national ethics and by institutional review boardapproved protocols. 2.2. Experimental design For experimental purposes goat kids were divided into the following three groups: group 1 (n=6; re-infected or challenged animals): animals infected with E. ninakholyakimovae sporulated oocysts at day 1 of the experiment and challenged at day 21; group 2 (n=5; animals primary infected or challenge controls): animals inoculated with E. ninakholyakimovae sporulated oocysts at day 21; group 3 (n=4; uninfected control): no infected animals. Day 1 of the experiment corresponds to 4th week of age in all groups. All infections were performed orally via gastro-ruminal tube using an infection dose of 2 x 105 E. ninakholyakimovae sporulated oocysts. The animals were weekly bled from the jugular vein and with the same interval their body weight was recorded. During the primary infection, faecal samples were individually taken for coprological analysis and the presence of clinical signs 124 PRESENTACIÓN DE ARTÍCULOS were daily monitored. The animals from all experimental groups were euthanized at day 28th of the study (one week after the challenge infection) and subsequently subjected to necropsy. Apart from a gross morphological examination, tissue samples from mesenteric lymph nodes, ileum and colon, as well as ileal mucus, were taken during the necropsy. 2.3. Parasitological, clinical and haemathological analysis The number of oocysts per gram of feces (OPG) for all the animals from group 1 during the primary infection was determined from day 14 to day 21 of the experiment by using a modified McMaster technique (MAFF, 1989). During the whole experiment, the weight of the animals was weekly taken in order to evaluate production parameters. The presence of clinical signs compatible with coccidiosis was also daily monitored, paying special attention to characteristic of the diarrhea. Individual blood samples were assessed by using a hematology analyzer (LaserCyte® hematology analyzer, Idexx) in order to determine total leukocyte counts and the hemoglobin concentration. The hematocrit value was calculated by centrifugation using standard centrifuge capillaries and differential white blood cell (WBC) counting was performed manually; for this purpose, 200 leukocytes were counted in stained blood smears (Diff-Quick). 2.4. Pathobiological and histolopathogical analysis All gross lesions were recorded during the necropsy and, afterwards, tissue samples from the ileal and colonic mucosa, as well as mesenteric lymph nodes (MLN), were taken and fixed in 4% buffered formalin. Next, tissue samples were included in paraffin blocks and 4-5 µm sections were cut and stained by hematoxylin and eosin (H&E) for the histopathological study and quantification of leucocyte populations (neutrophils, lymphocytes, eosinophils and globular leukocyte). A Giemsa staining was performed in order to determine mast cell counts. Finally, for the demonstration immature parasitic forms of E. ninakholyakimovae during the early prepatency, a PAS staining ( Periodic Acid Schiff ) was employed according to previously described methods (Barnhill et al., 2010). In all cases, cells or parasitic forms were counted using a 10× eyepiece containing a calibrated graticule and 40× objective lens by viewing an area of 0.05265mm2. The counts were randomly tak en on 20 graticule fields within the mucosal surface. The counts were expressed as number of cells per mm2 of mucosa (Amarante et al., 2005). Mucus samples were taken from the ileum of the animals for the analysis of specific IgA levels. For their preservation, the samples were suspended in a pH 7.1 buffer containing proteinases inhibitors (all compouds from Sigma-Aldrich): 0.1 M Na2HPO4, 0.05M NaCl, 3mM NaN3, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 5mM ethylenediaminetetraacetic acid (EDTA). Mix solutions containing mucus were centrifuged at 5000 x g (Eppendorf Centrifuge 5804R) for 1h at 4°C and the resulting supernatant was conserved at -20 ºC up to further analyses. 2.5. Enzyme – linked immunosorbent assay (ELISA) To determine the levels of specific IgG and IgM antibodies, sera samples collected weekly from the different groups were pooled (one pool per group and week), aliquoted and kept at -20°C until the appropriate tests was performed. For the IgG indirect ELISA test, a total of 100 µl of a SOA solution in carbonated buffer at a concentration of 5µg/ml was dispensed in 96 wells ELISA plates (CORNING Dispensable Sterile ELISA Plates, Corning Glass Works) and incubated overnight at 4°C. After three washes with 200 µl of PBS-0.05 % Tween 20, 200 µl of PBS-3% (w/v) bovine serum albumin (BSA, SigmaAldrich) were added and the plates were incubated for 45 min at 37°C. After each new step a similar washing procedure was done. Following treatment with BSA, individual 125 PRESENTACIÓN DE ARTÍCULOS serums, positive and negative controls were analyzed in duplicate using a 1/25dilution in PBS-Tween 20 0.02% sodium acid. For each sample, a volume of 100 µl was added to the plates and a new incubation was performed at 37°C for 1h. The plates were then incubated in the same conditions after adding 100 µl per well of 1/2000 anti-Goat IgG (anti-goat IgG peroxidase conjugate, IgG fraction of antiserum, Sigma-Aldrich) diluted in 0.01 M PBS. Thereafter, a total of 100 µl of substrate was incorporated to each well, a mix of compounds that includes citric-phosphate buffer, 0.04% (w/v) dihydrochloride orthophenyleneadiamine (OPD, Sigma-Aldrich) and 30% hydrogen peroxide (Panreac) at a final concentration of 0.1% (v/v). The incubation with the substrate was performed at room temperature in darkness for approximately10 minutes. Finally, the reactions was stopped by adding 35 µl per well of a 2M solution of sulfuric acid (Panreac) and the optical density (O.D.) measured at a wavelength of 492 nm (Termo Labsystems, Multiskan Ascent). For specific IgM and IgA indirect ELISA tests a similar protocol was employed but using 1/3000 anti-goat IgM peroxidase and 1/7000 anti-goat IgA peroxidase conjugates, respectively (both from Sigma-Aldrich). Similarly, dilutions 1/25 of the different serum or mucus samples were employed as primary antibody. 2.6. Immunohistochemistry For the characterization of the immune cell populations, additional samples of mesenteric lymph nodes (NLM) and intestinal mucosa from ileum and colon were taken at necropsy on each of the proposed experimental groups. Depending on the primary antibody used (Table 1), tissue samples of approximately 1 cm thick were preserved either in paraffin after fixation with 4% buffered formalin during 24 h or at - 80°C previous collection in liquid nitrogen. Before freezing, samples were included in OCT (Optimal Cutting Temperature, Tissue-Tek, Sakura Finetek Europe BV, Zoeterwoude, The Netherlands) and then immersed in a container with 2methylbutane (Merck, Darmstadt, Germany). For rapid freezing of tissue, the samples were subsequently submerged in liquid nitrogen and finally stored at -80 ° C until they were cut. Tissue samples 4 µm thick were cut by a cryostat (Reichert-Jung, 2800 Frigocut N, Germany) at -24°C, dried at room temperature for 40-60 min and fixed in acetone (Panreac) at 4°C for 5 min. Afterwards, tissue sections were dried again at room temperature for 30 min and routinely stained with H&E. Other cuts were stored wrapped in aluminium foil at -80°C up to IHC analysis were performed. The avidin-biotin-peroxidase (ABC) method (Navarro et al. , 1996), with some modifications, was used on sections of both formalin-fixed paraffin wax-embedded tissues and frozen tissues. The former were de-waxed and rehydrated, and endogenous peroxidase activity was blocked by incubation of the sections with 3% hydrogen peroxide in methanol for 30 min at room temperature. Sections were then treated with pronase (Sigma-Aldrich) 0.1% in Tris-buffered saline (TSB), pH 7.2 for 4 min at room temperature. When snap-froze tissue samples were used, endogenous peroxidase was blocked by incubation with phenyl-hydrazine (Sigma-Aldrich) 0.055 in PBS, without subsequent pronase treatment. After rehydration, all sections were incubated with 10% normal serum (Vector Laboratories) for 30 min at room temperature. The primary antibodies (Table 1) were then applied overnight at 4°C (formalin-fixed tissue) or for 2 h at 37°C (frozen tissue). Preliminary experiments were carried out to determine the optimal dilution for the different antibodies. All monoclonal (mAbs) and polyclonal antibodies (pAbs) were raised against immune cells of bovine origin but had been previously shown to cross-react with caprine antigens (Navarro et al., 1996; Rodríguez et al., 2000). Biotinated goat or rabbit anti-mouse IgG (for mAbs) and anti-rabbit IgG (for pAbs), both obtained from Vector Laboratories and diluted in 1 200, were used as the secondary reagents. An ABC complex (Vector) diluted 1 in 50 was applied as the third reagent. The sections were then incubated for 1 min with 126 PRESENTACIÓN DE ARTÍCULOS 3, 3’-diaminobenzidine tetrahydrochloride (Sigma) 0.035% in TBS containing hydrogen peroxide 0.1%. After rinsing in tap water, they were lightly counterstained with Harri’s haematoxilin, and mounted under DPX for microscopy. Sections in which the specific primary antibodies were replaces by TBS, normal goat or rabbit serum or inappropriate antibodies were included as negative controls. Sections from the lymph nodes of controls animals were used as positive control for all the primary antibodies. Positive labeled cells were counted in 20 selected fields (x 400 magnification) in each of the tissue sections (ileum or colon) from each goat kid. 2.7. Isolation of total mRNA and DNase treatment Total mRNA was isolated with the kit QuickPrep Total RNA (Amersham Pharmacia Biotech). The mRNA obtained was analyzed in a spectrophotometer Nanodrop 1000 (Thermo Scientific) in order to determine the concentration and purity of the sample. The purity of the mRNA was estimated by the A260/A280 relation and the alcoholic contamination agents by the A260/A230 relation (only samples with values between 1.8 – 2.1 and 2.02.1, respectively, were here employed). In addition, 1.2 % ( w/v ) MOPS (N-morpholine-propanesulfonic acid-3, Fluka Analytical) agar gels were used in order to prove the integrity and quality (Maniatis et al., 1982). Once checked, the mRNA sample was stored at -80 ° C until use. To further purify mRNA samples, genomic DNA was neutralized by using the kit RQ1 RNase-Free DNase (Promega) according to manufacturer's instructions. 2.8. Reverse transcription of total RNA and Real-time qPCR for the relative quantification of caprine IL-2, IL-4, INFγ and IL-10 gene transcripts The cDNA amplification process was performed in the iCycler (BioRad) thermocycler using a MyiQTM Single Color Real Time PCR Detection System (BioRad) and SYBR Green® I as fluorophore. To monitor the process, the iQTM 5 Optical System Software Version 2.0 for Windows 2000 and XP (BioRad) was used. For the amplifications, the GoTaq polymerase included in qPCR Master Mix® (Promega) at concentration 1X per sample was employed. The molecules analyzed in the present study were INF-γ, IL-2, IL-4 and IL-10. The β-actin gene was used as housekeeping, so the results were normalized according to the amplified copies of β-actin in each one of the samples assessed by using the software iQTM5 Optical System Software Version 2.0 for Windows 2000 and XP operating systems (BioRad). The primer pairs for each gene were designed using the corresponding goat or sheep sequences published in the Genbank (NCBI). The primer design was performed using the program Gene runner V. 3.0 and primer sequences were purchased from Invitrogen (Table 2). Based on the theoretical Tm of each primer and preliminary tests carried out at different temperatures, the optimal temperature (Tm) for all the primer pairs was set at 61°C. Thus, the amplification cycles and dissociation (melting-curve) for all genes were established as follows: denaturation 1 cycle at 95°C for 2 min, DNA replication 45 cycles at 94°C for 15 sec, 61°C for 20 sec and 72°C for 15 sec, and finally, one final elongation cycle at 72°C for 2 min. As referred before, the kit GoTaq® qPCR Master Mix (Promega) used for amplifications was provided as a simple-to-use stabilized 2X master mix which includes all components for quantitative PCR except sample DNA, primers and water. This formulation, which includes a proprietary dsDNA-binding dye, a low level of carboxy-Xrhodamine (CXR) reference dye, GoTaq® Hot Start Polymerase, MgCl2, dNTPs and a proprietary reaction buffer, produces optimal results in qPCR experiments. For β-actin, IL4, IL-2 and IL-10 primers, MgCl2 had to be increased up to a final concentration of 50 mM using a commercial MgCl2 solution (BioRad). For β-actin and IL-4, primer sets were used at a final concentration of 2 µM, while for IL-2, IL-10 and INF-γ a 4 µM final concentration of the corresponding primer sets were employed. For all the genes, 1:3 cDNA dilutions were added to each PCR 127 PRESENTACIÓN DE ARTÍCULOS reaction, 2.5 µl for β-actin and 5 µl for the remaining genes. Amplifications were performed in triplicates in a total volume of 25 µl using 10 µl of GoTaq® qPCR Master Mix. The reaction mixtures were always performed at 4 °C under sterile conditions using 96-well optical plates and each sample was analyzed in triplicates. To determine the linear range and amplification efficiencies and to confirm the specificity of the primer pairs of the β-actin and problem genes, DNA samples from lymphocytes isolated from caprine lymph nodes were here employed. In order to stimulate the gene transcription of IL-4, caprine lymphocytes were incubated with phorbol myristate acetate (PMA) (SigmaAldrich) at a 5 ng/ml final concentration (Gohin et al., 1997), whereas lymphocytes stimulated with ConA (Sigma-Aldrich) were utilized as positive controls for INF-γ, IL-2 and IL-10 gene amplifications. PCR reactions of these positive samples were run in duplicate in order to obtain standard curves. For the calibration of the curve, 6 serial dilutions of 1:4 from the cDNA were obtained in a pool out of all the samples used. The differences of the slopes between standard curves obtained for actin-β and the problem molecules (which should be <0.1 for reliable quantification) were plotted against the logarithm input of total RNA and a corresponding regression line was calculated. The data were visualized by the program iQTM5 Optical System Software Version 2.0 for Windows 2000 and XP operating systems. 2.10. Statistical analysis of data Faecal OPG were logarithmically transformed and added by 1 [log (OPG+1)] in order to obtain normal distributions according to the Kolmogorov-Smirnov’s normality test. Levels of the different immunoglobulins analysed in the study were expressed as the relative percentage of the optical density (O.D.) of a positive control pool in order to avoid inter-assay differences (Relative O.D.). One factorial analysis of variance, Tukey’s multiple comparison test and Student’s t-test were used to analyse the data between different experimental groups and the Pearson correlation test for the evaluation of the association between different parameters assessed in this study. For that purpose, the statistical software SigmaPlot 12.0 was employed and the differences were regarded as significant at a level of p ≤ 0.05. 3. Results 3.1. OPG counts, clinical signs and body weights Primary infected animals at 4 weeks of age already had high OPG counts at day 14 post infection (p.i.) with maximum values being observed at day 15 p.i., which corresponded to mean OPG counts of approximately 5.5 x 105 (Fig. 1). Thereafter, the oocyst excretion gradually decreased up to day 21st p.i. and, at day 22 p.i., negative counts were recorded in all animals. Challenge controls (group 2) and uninfected animals (group 3) had negative counts through the whole study. From day 14 p.i. up to day 22 p.i., faecal consistence was altered in all the five primary infected animals, ranging from slightly soft faeces to watery diarrhea with blood and small portions of gut mucosa. Clinical signs also included anorexia, weakness, dehydration and growth delay as shown by the weekly determination of body weights. In general, the severity of the disease was more evident between days 14-18 p.i., and, except for the presence of slightly soft faeces, clinical sings had already disappeared by day 22 p.i. In spite of treatment with fluidotherapy and vitamins, one of the goat kids died at day 20 p.i. Apart from some alterations due to dehydration, haematological determinations were not relevant. Any evidence of clinical signs compatible with coccidiosis was observed in the remaining animals during the whole experiment. 3.2. Pathologically and histological analysis Gross morphological examination of the animals from challenge group 1 did not revealed other changes than those observed in the intestinal mucosa, particularly of ileum, 128 PRESENTACIÓN DE ARTÍCULOS acquired immune responses, classical studies already demonstrated the immunogenicity of the first schizogony in poultry (McDonald et al., 1986). Many other publications also sustain the hypothesis that adaptive cellular immune responses occur in vivo already during the prepatency of E. bovis infections (Hughes et al. 1988, 1989; Fiege et al. 1992; Hermosilla et al. 1999; Taubert et al. 2007, 2008). Since sporozoites of this Eimeria species rapidly invade also endothelial host cells and then are situated intracellular, developing immune reactions should be directed against infected host cells as well. Given that endothelial cells generally have the capacity of antigen presentation (Wagner et al. 1984; Bosse et al. 1993; Knolle 2006; Behling-Kelly and Czuprynski 2007), it appears likely that T cells may act against early stages, such as intracellular sporozoites or meronts I, rather than meronts II and gamonts. Accordingly, mean values for lymphocyte counts recorded in the present study were increased in re-infected animals, like other cell populations such as eosinophils, mast cells and globular leukocytes. Likewise, in this group of animals a greater number of CD4+ and CD8+ cells were observed, all indicating that T cell response could be related to the development of the protective response. Contrarily to our data, Hermosilla et al. (1999) observed that calves inoculated with oocysts E. bovis had CD4+ T cells increased during prepatency in primary infections decaying after the patent period. The population of CD4+ T cells are actually admitted to be particularly involved in resolving primary infections by regulating the duration and level of oocyst shedding. On another hand, in agreement to our data, CD8 + T cells have been involved in Eimeria reinfections (Rose et al., 1992a; Findly et al., 1993; Ovington et al., 1995; Smith and Hayday, 2000; Shi et al, 2001a). The analysis of the T cells populations also showed that CD45+ T cells were increased in ileum of reinfected animals, which could be related to the mild increase of IgA in mucus samples. In contrast to T cell populations, except pan T CD3+, cell counts of other immune cell types did not changed or increased only in primary infected goat kids (challenge controls). For instance, MHII+ cells had significantly higher counts in ileal mucosa, as well as MAC387, which suggests an active recognition of antibodies and a dynamic participation of myeloid/histiocytic cells, probably involved in innate immune reactions. Finally, when the involvement of certain cytokines was evaluated we found some evidences that both Th1 and Th2 responses could be mounted against E. ninakohlyakimovae infections, as shown by increased relative expression of IL-2, IL-4, IL10 and INFγ. In general, during primary infection in ruminants, Eimeria species levels of INFγ and IL-2 gene expression are elevated in comparison to IL-4 transcription levels, which suggest an increased cytotoxic cellular response to parasite cells (Taubert et al., 2008). Furthermore, increased IFN and IL2 could stimulate lymphocyte activation and enhance the phagocytic activity of macrophages via an increase in nitrite production (Hermosilla et al., 1999). As a whole, the results of this study give evidence that protective immune responses may be addressed to early developmental stages of E. ninakohlyakimovae during the first schizogony, which probably results from a complex framework of mechanisms, effector cells and cytokines that involves both innate, humoral and cellular immune responses. The present data contribute to understand the complexity of the cellular and humoral immune response of the goat hosts against coccidiosis, which can be used for the development of strategies for modulation of the immune response and for the identification of anticoccidial compounds or vaccines. 5. Acknowledgements This work has financially been supported by funding derived from the Spanish Ministry of Science and Innovation (MICIN) and the ACIISI (Agencia Canaria de Investigación, Innovación y Sociedad de la Información). 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Trop Life Sci Res 26:1-8. 131 5. conclusiones CONCLUSIONES • PRIMERA.- La cepa GC de Eimeria ninakohlyakimovae aislada en nuestro laboratorio presenta no sólo una gran capacidad infectante sino un elevado poder patógeno, capaz de producir cuadros clínicos severos. • SEGUNDA.- Además, dicha cepa puede desarrollar respuestas inmunes protectoras, por lo cual constituiría un modelo idóneo para estudiar la respuesta inmune y los mecanismos de patogenicidad en las coccidiosis caprina. • TERCERA.- Aunque Eimeria ninakohlyakimovae induce una importante immunoprotección frente a reinfecciones homólogas con dosis infectantes moderadas similares a las empleadas en la primoinfección, la inmunidad puede ser sólo parcial cuando se utilizan dosis de infección masivas. En tales casos, el desenlace fatal durante la reinfección no está claro si se produce por la acción directa del parásito o por una exhacerbación de la respuesta inmune asociada. • CUARTA.- La infección y posterior reinfección con Eimeria ninakohlyakimovae estimula la producción de IgM e IgG séricas, así como la producción de IgA en el mucus del íleon. Estos niveles no se correlacionan con la producción de ooquistes y no se observan diferencias claras entre animales reinfectados y controles de reinfección, por lo que, aunque la respuesta de anticuerpos es reflejo de la exposición al parásito, parece no haber una asociación clara con el nivel de protección. QUINTA.- Utilizando como antígeno ooquistes esporulados de Eimeria ninakholyakimovae , la IgG sérica de los animales infectados logró reconocer un panel de péptidos específicos cuyos pesos moleculares oscilaron entre 134 y 16 kDa, apoyando el valor del electroimmunobotting como una técnica para detectar proteínas inmunorreactivas en coccidiosis caprina. Sin embargo, tampoco en esta ocasión se encontraron bandas de reconocimiento específico en los animales reinfectados. • SEXTA.- La infección de cabritos a las 3, 4 y 5 semanas de edad con Eimeria ninakohlyakimovae desencadena en todos los casos respuestas inmunes protectoras frente a reinfecciones homólogas realizadas 3 semanas más tarde. La inmunoprotección se tradujo en una reducción significativa de la producción de ooquistes y de la intensidad del cuadro clínico y estuvo asociada a componentes celulares, tanto innatos como adquiridos, de la respuesta inmune, no siendo tan destacada la implicación de la respuesta inmune humoral. 133 CONCLUSIONES • SÉPTIMA.- Sin embargo, cuando se realizó un análisis más detallado de los datos, se observaron algunas diferencias entre los tres grupos de edad, relacionado con el resultado de infección por Eimeria y la respuesta inmune resultante; tales resultados sugieren que los cabritos más jóvenes pueden no ser completamente inmunocompetentes, lo cual podría ser de interés para el diseño de estrategias inmunoprofilácticas para el control de la coccidiosis caprina. • OCTAVA.- La respuesta inmune adquirida frente a Eimeria ninakohlyakimovae puede estar dirigida hacia los estados parasitarios que se desarrollan durante la fase de prepatencia de la enfermedad, en concreto frente a los esquizontes inmaduros. Dicha respuesta resulta ser compleja, en tanto que intervienen diversas poblaciones celulares y mediadores químicos (por ejemplo citoquinas), además de células presentadoras de antígenos y células propias de respuestas inmunes innatas. 134 6. resumen