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EPIDEMIOLOGÍA DE LA SARNA SARCÓPTICA EN
FAUNA SILVESTRE DEL PRINCIPADO DE ASTURIAS
MEMORIA PRESENTADA POR
Álvaro Oleaga Ruiz de Escudero
PARA OPTAR AL GRADO DE DOCTOR
VºBº LOS DIRECTORES
DR. CHRISTIAN GORTÁZAR DRA. ROSA CASAIS DR. JOAQUÍN VICENTE
UNIVERSIDAD DE CASTILLA-LA MANCHA
INSTITUTO DE INVESTIGACIÓN EN RECURSOS CINEGÉTICOS
(CSIC – UCLM – JCCM)
DEPARTAMENTO DE CIENCIA Y TECNOLOGÍA AGROFORESTAL Y GENÉTICA
Para la realización de este trabajo se ha contado con la financiación de una Beca para la realización de la Tesis doctoral con cargo a Proyectos de Investigación en el
Consejo Superior de Investigaciones Científicas así como de los Convenios de Investigación establecidos entre el Principado de Asturias y CSIC.
A mis padres, hermanos y sobrinos, por estar siempre ahí a pesar de la distancia
A Patricia por su cariño, comprensión, paciencia y ayuda inagotables
A Aitor, por cambiarnos la vida de esta forma maravillosa: ¡suerte!
AGRADECIMIENTOS
Aunque generalmente prestamos poca atención a este apartado de
agradecimientos en las tesis ajenas (también yo, lo reconozco…), al dar los últimos
retoques a la mía y echar la vista atrás la perspectiva cambia la forma de ver las
cosas. A estas alturas comprendo que aparte de un objetivo profesional más o
menos importante o deseado, es de justicia entender la tesis como un “rendimiento
de cuentas” dedicado a todas esas personas que a lo largo de nuestra vida nos
orientaron y alentaron para dedicarnos a lo que nos gusta, y por otro lado como un
“resultado digno” del trabajo que muchas personas nos han ayudado a sacar
adelante en el desarrollo de nuestra tesis una vez planteada. Puesto que me
considero una persona parca en palabras, y soy consciente de que por desgracia no
doy las gracias todo lo que debería (y me gustaría), permitidme que aproveche este
comienzo de mi tesis para hacerlo.
El primero de mis agradecimientos ha de ser para mis padres, Marisol y Jesús,
que aparte de otros muchos valores que aprecio me educaron en el amor y respeto
a la naturaleza, y me dieron el privilegio de poder ser feliz con algo tan barato y
sencillo como su simple observación. Con el paso de los años se las ingeniaron
además para animarme y apoyarme a la hora de orientar mi futuro y elegir una
carrera, y gracias a ellos mi pasión pudo convertirse en mi trabajo. No puedo olvidar
a mis hermanos Marta, Rubén e Iñigo, cómplices durante nuestros años de correrías
por Vitoria y Retana, partícipes y responsables también de esa verdadera educación
que tanto valoro.
Con mis amigos de Vitoria no ha sido hasta ahora, pasados los años, cuando
he podido compartir esta pasión por la naturaleza, pero he de agradecerles los
estupendos años compartidos. Gracias Xabi, Íñigo, Gorka, Gorki, Javi, Eduardo, Jon,
Eneko, Iván, Gamboa… porque a pesar de la distancia y los años, sigue habiendo
siempre en la cuadrilla un rato para vernos, tomar unas cervezas u organizar una
cena cada vez que me escapo por Gasteiz. También de Vitoria tengo que dar las
gracias a Chema Fernández, veterinario de formación con alma de biólogo -como
yo- que me apoyó en mis pinitos como ornitólogo aficionado y continua, al igual que
Nerea, siendo un amigo y referente profesional para mí.
En la Facultad de Veterinaria de León conocí a un montón de gente magnífica
de entre los que me llevé muchos buenos amigos, con los que pasé años
inolvidables. Tanto los compañeros de piso de INEF mis primeros años (Pepe, Víctor,
Carlos) como los de veterinaria (Jorge, Roberto, David) y mi compañero de
aventuras durante el año de Erasmus en Lisboa (Jorge del Pozo) se convirtieron en
colegas incondicionales que además asumían el papel de familia, y son el mejor
ejemplo de los estupendos amigos que hice y que no me atrevo a enumerar porque
aquí no caben todos…
Mi aterrizaje en Asturias fue de la mano de Agüera, Jose y Alberto, que
depositaron su confianza en una persona sin experiencia como yo en el Centro
Veterinario de Tineo, algo que junto con el trato exquisito y la libertad que me
ofrecieron para organizar mi trabajo siempre les agradeceré. Allí pasé casi seis años,
y aparte de a trabajar, aprendí a disfrutar del trabajo en equipo y el verdadero
significado de la palabra “compañerismo”. Omar y Sergio además de compañeros de
piso fueron estupendos amigos y profesores en temas bovinos, con Chusa y María
como matriarcas en la gélida sierra tinetense.
De forma casual, durante un censo de aves invernantes (gracias David por
guiarme en el mundo de la naturaleza asturiana, y Emilio por el aviso y el trabajo
compartido posteriormente), me enteré de la posibilidad de trabajar con fauna
silvestre para la Administración y con ella, de convertir en profesión la pasión que
me impulsó a comenzar mis estudios de veterinaria. En este nuevo periplo fue Jaime
Marcos Beltrán el que confió en mí desde la primera entrevista, me dio la posibilidad
por parte del Principado de Asturias de plasmar en forma de trabajo el entusiasmo
manifestado, y al que debo agradecer su supervisión y apoyo en el mundo de la
sanidad y la fauna silvestre asturianas. Durante estos años he compartido con
muchos miembros de la Administración asturiana trabajo, preocupaciones y logros,
con la Guardería como mi vínculo más estrecho con el campo (donde además de
magníficos profesionales encontré estupendas personas y amigos que me han
enseñado mucho: Francisco, Jorge, Ramón, Obdulio, Angel…) y Paco Mier como mi
vínculo más directo con la Guardería y el “día a día”; muchísimas gracias a todos.
He de dar las gracias también dentro de la Administación a toda la gente que desde
Oviedo apoyó esta labor y su permanencia en el tiempo a pesar de las dificultades:
Jose Félix, Tito, Luis Miguel…
Mi trabajo para la Administración surgió en el marco de un Convenio
establecido con el IREC, donde Christian Gortázar me respaldó y apoyó desde un
primer momento y al que debo sin duda todo lo que a día de hoy he podido aprender,
hacer y disfrutar haciendo en mi trabajo con fauna silvestre. Como director principal
pero sobre todo como mentor y amigo, esta tesis es en gran medida suya.
Por si este aval no fuese suficiente, el grupo de auténticos cracks (como
profesionales de la ciencia y como personas) que lo rodean en el IREC me
acogieron, cuidaron y ayudaron desde un primer momento: Pelayo como conexión
asturiana, asesor permanente y protector en aquel nuevo ambiente para mí desde el
primer día que pisé el IREC (literalmente) allá por el 2006; Joaquín como claro
exponente de que la excelencia como investigador y como persona son compatibles,
mi “hospedador” en Ciudad Real e “instigador” de buena parte de mis trabajos, y que
no por ser Codirector de esta tesis podía faltar de esta lista de amigos con
mayúsculas que me llevo de Ciudad Real; Fran, gran profesor y estupendo amigo a
partes iguales; Isabel, la bondad hecha investigadora y trabajadora incansable;
Mónica, siempre con una sonrisa y el consejo acertado a todos los niveles; Encarni y
Paqui como mi “conexión permanente” con los despachos y la sala de necropsias
del IREC respectivamente, soluciones a cualquier problema que les pudiese plantear.
Pero he tenido el gusto de conocer (siempre menos de lo que me hubiese
gustado) a mucha gente magnífica en mis escapadas al IREC en Ciudad Real: de
Sanidad Animal, además de los ya mencionados, no puedo olvidarme de Vanesa,
Elisa, Mari Paz, Manolo, Raquel Sobrino, Ursula, Dolo, Vidal, María, Cristina, Elo,
Francis, Salva, Victoria, Pepe, Elena y La Raspa del Chaparrillo, Mariana, Raquel
Jaroso, Sandra, Rafita, Ricardo, Jesús, Mauricio, Bea, Valeria, José Ángel, Virginias,
Mario, Bianky, Lucca, Paolo, Tania, Caterina, Nelson, Tamara, Ernesto…; con
cualquiera de todos ellos (y la lista no es corta) era capaz de aprender más en 15
minutos de conversación en la cafetería en Ciudad real que en varios meses sólo en
Asturias. Del departamento de ecología pude disfrutar también de los sabios
consejos y la ayuda de Loren, Miguel, Rouco, Fabián, Javitxa, Paco, Viñuela, Dávila,
Jorge, Pablo, Rafa, Bea, Paqui, Marisa y María, Jesús, Julien, Catarina, Ainhoa,
Nacho… ; también conté siempre con la colaboración y buen hacer de Rafa, Pablo e
Inés en toxicología; muchas gracias a todos. No quiero dejar de agradecer a la gente
de servicios generales y “logística” del IREC que siempre se preocupasen de
facilitarme las cosas a pesar de la distancia: Arturo, Andrés, Lucía, Ana, Jorge,
Carolina, Angelines, Mercedes, y también a la gente de seguridad: Jorge, Terry y
Vicente. Cómo no dar las gracias a Julián Garde por su ayuda y asesoramiento
permanentes.
Ya centrándome en el trabajo en Asturias, mis primeros pasos fueron de la
mano de Gamarra, que junto con la hospitalidad y ayuda de toda la familia Álvarez
(Emilio, Pilar, María, Pepe…), hicieron que seguir la senda trazada por Diego
Villanúa en Asturias llegase a parecer posible en algún momento. La “sucursal”
asturiana del Irec tomó forma definitiva con la llegada de Oscar Rodríguez. Con sus
años de experiencia y estupendo hacer tanto en el campo como en los despachos,
junto con la incorporación posterior de María Suárez como apoyo logístico y experta
en el mundo aviar, consiguieron que la Red de Vigilancia Sanitaria se asentase y
todo comenzase a funcionar como una máquina bien engrasada, muchas gracias
por la ayuda durante estos años.
Además de aportar las infraestructuras y buena parte de los medios necesarios
para desarrollar mi trabajo en Asturias, el departamento de Sanidad Animal del
SERIDA ha sido desde mis inicios en este mundo y hasta la actualidad mi “otra casa”
tanto a nivel de investigación como de apoyo y ayuda incondicionales. Aparte de un
magnífico grupo de profesionales, tras estos años considero verdaderos amigos a
Ana Balseiro, Miguel, Alberto, Isabel, Ana del Cerro y como no a Rosa Casais, que
además es codirectora y corresponsable de que esta tesis exista. Muchas gracias a
todos.
Tras el paso por el IREC (pero sin dejar de contar en ningún momento con su
apoyo y asesoramiento científico) fue posible continuar con el trabajo desarrollado
en Asturias, y aunque en los ratos libres con la tesis, de la mano de diferentes
empresas: Fundación UCLM, TRAGSATEC, SERPA…gracias a toda la gente de
cada una de ellas que me ha ayudado durante este tiempo.
Los años de trabajo transcurridos me han dado tiempo para conocer a mucha
más gente estupenda que, aunque no pertenezcan a ninguno de los grupos
anteriores, tengo la suerte de considerar también amigos a estas alturas. Quiero
agradecer su colaboración desinteresada y permanente a Pablo Quirós, García,
Suso, Rafa, “Pablito” y María de Córdoba, Xeider, Natalia, Javi Millán, Jorge Jordi…
ha sido un placer haber podido contar con vuestros conocimientos y ayuda, pero
especialmente con vuestra compañía y amistad.
Finalmente, tengo que agradecer a Dolores Gavier-Widén y Paulo Célio Pereira
Martins Alves su amabilidad al acceder a evaluar esta tesis y la rapidez en su
respuesta.
He comenzado este apartado de agradecimientos hablando de mi familia en
Vitoria, y sólo puedo acabarlo dando las gracias a mi familia en Asturias, ya en el
sentido más literal de la palabra, que es la que en mayor medida ha “padecido” los
inconvenientes del desarrollo de esta tesis en mis ratos libres… Especialmente a
Patricia, que desde mis últimos años de carrera ha compartido, guiado y apoyado
incondicionalmente mis pasos con una paciencia, cariño y comprensión infinitos.
Esta tesis también es tuya. Gracias.
_____________________________________________________________________________Índice
ÍNDICE
INTRODUCCIÓN ......................................................................................... 1
I.1-Introducción general ............................................................................. 3
La enfermedad. Historia ........................................................................ 3
Taxonomía y morfología del ácaro Sarcoptes scabiei. .......................... 3
Ciclo de vida de S. scabiei ..................................................................... 5
Supervivencia y capacidad colonizadora del ácaro S. scabiei .............. 7
Manifestaciones clínicas y patogenia de la enfermedad ........................ 9
Métodos de diagnóstico ....................................................................... 10
Relevancia de la sarna sarcóptica ....................................................... 14
Sarna sarcóptica en fauna silvestre ..................................................... 15
Sarna sarcóptica en fauna silvestre en España ................................... 17
I.2-Hipótesis de trabajo ............................................................................ 21
I.3-Objetivos .............................................................................................. 22
I.4-Bibliografía .......................................................................................... 23
Capítulo 1. SARNA SARCÓPTICA EN CÉRVIDOS SILVESTRES DEL
PRINCIPADO DE ASTURIAS ................................................................... 31
1.1-Sarna sarcóptica en dos corzos (Capreolus capreolus) del Norte de
España
Sarcoptic mange in two roe deer (Capreolus capreolus) from Northern
Spain. European Journal of Wildlife Research 54, 134-137 ....................... 33
Resumen ............................................................................................. 35
Abstract ................................................................................................ 35
Introduction .......................................................................................... 36
Materials and methods ......................................................................... 37
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
Results ................................................................................................. 38
Discussion ........................................................................................... 40
Acknowledgement ............................................................................... 41
References .......................................................................................... 42
1.2-Sarna sarcóptica en ciervo ibérico: ¿enfermedad emergente o mejor
vigilancia sanitaria?
Sarcoptic mange in red deer from Spain: improved surveillance or disease
emergence?. Veterinary Parasitology 154, 103-113 .................................. 45
Resumen ............................................................................................. 47
Abstract ................................................................................................ 48
Introduction .......................................................................................... 48
Material and methods .......................................................................... 50
Results ................................................................................................. 55
Discussion ........................................................................................... 60
Conclusions ......................................................................................... 65
Acknowledgement ............................................................................... 65
References .......................................................................................... 66
Capítulo 2. PREVALENCIA DE ANTICUERPOS FRENTE A DIFERENTES
AGENTES PATÓGENOS COMPARTIDOS ENTRE REBECO
CANTÁBRICO Y CABRA DOMÉSTICA……………………………………….71
Prevalence of antibodies against selected agents shared between cantabrian
chamois (Rupicapra pyrenaica parva) and domestic goats. European Journal
of Wildlife Research 56, 319-325 ............................................................... 71
Resumen ............................................................................................. 73
Abstract ................................................................................................ 73
Introduction .......................................................................................... 74
Materials and methods ......................................................................... 76
_____________________________________________________________________________Índice
Results ................................................................................................. 78
Discussion ........................................................................................... 78
Acknowledgement ............................................................................... 83
References .......................................................................................... 84
Capítulo 3. SARNA SARCÓPTICA EN CÁNIDOS SILVESTRES DEL
PRINCIPADO DE ASTURIAS ................................................................... 91
3.1-Nuevas técnicas para estudiar una vieja enfermedad: sarna
sarcóptica en el lobo ibérico
New techniques for an old disease: sarcoptic mange in the Iberian wolf.
Veterinary Parasitology 181, 255-266 ........................................................ 93
Resumen ............................................................................................. 95
Abstract ................................................................................................ 96
Introduction .......................................................................................... 97
Materials and methods ......................................................................... 99
Results ............................................................................................... 108
Discussion ......................................................................................... 117
Acknowledgement ............................................................................. 122
References ........................................................................................ 122
3.2-Concomitancia e interacciones entre patógenos en el lobo ibérico
(Canis lupus)
Concomitance and interactions of pathogens in the Iberian wolf (Canis
lupus). Research in Veterinary Science (submitted) ................................ 131
Resumen ........................................................................................... 133
Abstract .............................................................................................. 133
Introduction ........................................................................................ 134
Material and methods ........................................................................ 135
Results ............................................................................................... 138
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
Discussion ......................................................................................... 140
Conclusions ....................................................................................... 146
Acknowledgement ............................................................................. 146
References ........................................................................................ 147
Capítulo 4. COMPARACIÓN DE LAS CARACTERÍSTICAS
PATOLÓGICAS E INMUNOHISTOQUÍMICAS DE LA SARNA
SARCÓPTICA EN CINCO ESPECIES SIMPÁTRICAS DE FAUNA
SILVESTRE DEL PRINCIPADO DE ASTURIAS……………………………151
Comparative pathological and immunohistochemical features of sarcoptic
mange in five sympatric wildlife species in Northern Spain. European Journal
of Wildlife Research 58, 997-1000 ........................................................... 151
Resumen ........................................................................................... 153
Abstract .............................................................................................. 153
Introduction ........................................................................................ 154
Materials and methods ....................................................................... 154
Results ............................................................................................... 155
Discussion ......................................................................................... 157
Acknowledgement ............................................................................. 159
References ........................................................................................ 160
Capítulo 5. CARACTERIZACIÓN Y COMPARACIÓN A NIVEL
MOLECULAR DE ÁCAROS Sarcoptes scabiei PRESENTES EN CINCO
ESPECIES SIMPÁTRICAS DE FAUNA SILVESTRE DEL PRINCIPADO DE
ASTURIAS ............................................................................................... 161
5.1-Estabilidad temporal en la estructura genética de Sarcoptes scabiei:
evidencias empíricas en fauna silvestre de Asturias
_____________________________________________________________________________Índice
Temporal stability in the genetic structure of Sarcoptes scabiei under the
host-taxon law: empirical evidences from wildlife-derived Sarcoptes mite in
Asturias, Apain. Parasites & Vectors 4, 151 ............................................. 163
Resumen ........................................................................................... 165
Abstract .............................................................................................. 165
Introduction ........................................................................................ 166
Methods ............................................................................................. 168
Results ............................................................................................... 170
Discussion ......................................................................................... 174
Conclusions ....................................................................................... 177
Acknowledgement .............................................................................. 177
References ........................................................................................ 177
5.2-Epidemiología genética de Sarcoptes scabiei en lobo ibérico en
Asturias
Genetic epidemiology of Sarcoptes scabiei in the Iberian wolf in Asturias,
Spain. Veterinary Parasitology 196, 453-459 ........................................... 183
Resumen ........................................................................................... 185
Abstract .............................................................................................. 186
Introduction ........................................................................................ 186
Material and methods ........................................................................ 189
Results ............................................................................................... 192
Discussion ......................................................................................... 196
Acknowledgement ............................................................................. 199
References ........................................................................................ 200
DISCUSIÓN GENERAL ........................................................................... 205
D.1-Síntesis de los hallazgos más relevantes ..................................... 207
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
D.2-Líneas de investigación y trabajos pendientes. ........................... 223
D.3-Conclusiones ................................................................................... 233
D.4-Bibliografía ....................................................................................... 235
INTRODUCCIÓN
I.1- INTRODUCCIÓN GENERAL
I.2- HIPÓTESIS DE TRABAJO
I.3- OBJETIVOS
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
2 _____________________________________________________________________________
________________________________________________________________________Introducción
______________________________________________________________________________ 3
I.1- INTRODUCCIÓN GENERAL
La enfermedad. Historia
La sarna sarcóptica es considerada
la primera enfermedad humana con un
agente etiológico conocido, al ser descrito
el ácaro implicado por los italianos
Bonomo y Cestoni en 1689 (Montesu &
Cottoni, 1991, Fig. I.1). Su identificación
supuso la primera mención a la “teoría
del parásito” en relación a las
enfermedades infecciosas, y el comienzo
de una nueva era en medicina.
La sarna sarcóptica es una enfermedad zoonósica que puede afectar a un gran
número de especies domésticas y silvestres, lo que le confiere gran importancia
económica, ecológica y en salud pública. Se trata de una parasitosis altamente
contagiosa que se transmite generalmente por contacto directo con individuos
enfermos (Arlian et al., 1988), si bien el contagio indirecto ha sido descrito y cobra
especial importancia entre animales, a través del uso de zonas comunes, como
refugios o rascaderos (Gerasimov, 1958; Arlian, 1989).
Taxonomía y morfología del ácaro Sarcoptes scabiei.
El agente etiológico de la sarna sarcóptica es el ácaro barrenador S. scabiei (L.
1758) que pertenece a la clase Arachnida, subclase Acari (=Acarina), orden
Astigmata y familia Sarcoptidae. Los sarcóptidos son parásitos obligados,
excavadores de la piel de mamíferos (Sarcoptidae), aves (Knemidokoptidae) o
murciélagos (Teinocoptidae). La familia Sarcoptidae incluye S. scabiei, Notoedres
cati (ácaro del gato), y Trixacarus caviae (ácaro del cerdo pigmeo) (Kettle, 1984).
Figura I.1- Dibujo de Bonomo correspondiente a la 1ª descripción registrada de Sarcoptes, realizada en 1689. Fotografía tomada de : (http://www.dermato.med.br/hds/bibliography/1998giovan-cosimo-bonomo.htm)
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
4 _____________________________________________________________________________
Todos los miembros del orden Astigmata son ácaros de reducida movilidad con
tegumento ligeramente esclerotizado y sin espiráculos o sistema traqueal
detectables. S. scabiei es de color blanco-crema con extremidades y aparato bucal
de coloración ligeramente más oscura (Figura I.2.A). La hembra adulta mide 300-500
µm de longitud por 230-340 µm de anchura, y el macho, más pequeño, 213-285 µm
de largo por 160-210 µm de ancho.
-
El tegumento tiene estrías transversales limitadas en la cara dorsal por un
parche central de escamas triangulares que presentan valor taxonómico. También a
nivel dorsal hay 6 ó 7 pares de espinas dispuestas de forma simétrica a ambos lados
de la línea media posterior así como 3 pares de espinas laterales a lo largo del
cuerpo (Figura I.2.B).
Tanto macho como hembra presentan “ventosas” pedunculadas en
extremidades I y II, que permiten al ácaro adherirse al sustrato. El tarso de las patas
III y IV termina en la hembra en largas “setas”, mientras que el macho tiene ventosas
en la extremidad IV. Adicionalmente los tarsos de ambos sexos soportan dos
espolones a modo de pinzas, si bien en el macho sólo hay un espolón en
extremidad IV. Las larvas difieren de adultos y ninfas en poseer únicamente 6 patas.
Las ninfas son similares a hembras adultas pero menores y sin oviporo (Fain, 1968;
Pence et al., 1975; Walton et al., 2004).
B A
Figura I.2- A: Imagen del aspecto de un ácaro S.scabiei identificado mediante el empleo de lupa durante el protocolo de búsqueda y aislamiento llevado a cabo en placas de incubación. B: Imagen microscópica de ácaro S. scabiei aislado de uno de los lobos objeto de estudio.
________________________________________________________________________Introducción
______________________________________________________________________________ 5
Las escasas diferencias morfológicas que presentan ácaros de S. scabiei
aislados de diferentes hospedadores no permitieron en su momento determinar si se
trataba de especies distintas o simplemente variedades diferentes de una misma
especie. A pesar de su similar morfología, se apreciaron de manera empírica
diferencias fisiológicas entre ácaros presentes en distintos hospedadores, que
parecían conferirles especificidad de hospedador (Fain, 1968; Pence et al., 1975) de
tal modo que algunos autores llegan a describir diferentes variedades (hominis,
bovis, ovis, caprae, cervi, canis) morfológicamente indistinguibles. Esta especificidad
puede atribuirse a numerosos factores del parásito (diferencias fisiológicas en los
requerimientos de las distintas variedades, antigenicidad del parásito, resistencia a
la respuesta inmune de su hospedador,…) y del hospedador (diferencias en las
propiedades dermatológicas, habilidad para desarrollar respuesta inmune,…).
Aunque se ha comprobado cómo las distintas variedades de S. scabiei sugeridas
están relacionadas antigénicamente, se han descrito también epítopos específicos
de cada variedad (Arlian et al., 1996). El reciente desarrollo de nuevas técnicas de
biología molecular ha permitido evaluar y confirmar algunas de las hipótesis
previamente formuladas acerca de la taxonomía de este ectoparásito. Los diferentes
trabajos efectuados han mostrado el estudio de microsatélites (secuencias
hipervariables de rápida evolución, capaces de poner de manifiesto las escasas y
sutiles diferencias genéticas presentes entre las distintas poblaciones del ácaro)
como una técnica adecuada de análisis a este nivel (Alasaad et al., 2008). Estos
estudios han permitido confirmar la idea mayoritariamente aceptada (Pence et al.,
1975; Andrews, 1983) de que el género Sarcoptes está constituido por una única
especie que presenta diferentes variedades adaptadas a sus distintos hospedadores
(Walton et al., 2004; Rasero et al., 2010; Gakuya et al., 2011).
Ciclo de vida de S. scabiei
El ciclo vital de S. scabiei incluye 4 momentos de desarrollo activo (figura I.3),
que generalmente acontecen sobre un mismo hospedador (Fain, 1968; Arlian &
Vyszenski-Moher, 1988). Tras contactar con su hospedador o tras alcanzar el
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
6 _____________________________________________________________________________
estadio de adulto sobre el que ya ocupaba, la hembra excava galerías en las que
deposita de 2 a 4 huevos al día. Las larvas emergen a partir de 50-53 horas tras la
puesta de los huevos, si bien este período puede verse prolongado en función de las
condiciones del hospedador, y migran a la superficie de la piel donde entre 3 y 4
días después, estas larvas mudan a protoninfas, que se desarrollan de 2 a 3 días.
Estas protoninfas mudan a (trito)ninfas, de las que emergerá un adulto tras un total
de 10 a 13 días de ciclo. Una vez transformados en adultos, el macho explora la
superficie en busca de hembras no fertilizadas, y es a este nivel donde se produce la
fertilización. Las hembras llenas de huevos excavan galerías donde los depositan y
en cuyo interior permanecen hasta 2 meses que dura su vida. Estas hembras
fertilizadas pueden reinfectar el hospedador del que proceden o, si las circunstancias
son las adecuadas, colonizar un nuevo hospedador en el que tratar de completar
nuevamente el ciclo (Bornstein et al., 2001).
Figura I.3- Representación del ciclo vital del ácaro S. scabiei (adaptado de Arlian & Vyszenski-Moher, 1988).
Es necesario tener en cuenta, no obstante, que muchas fases intermedias no
llegan a culminar el desarrollo, pudiendo verse alterada la duración aparente del
ciclo completo (Walton el al., 2004). Los períodos señalados para el ciclo del ácaro
________________________________________________________________________Introducción
______________________________________________________________________________ 7
pueden asimismo verse alterados cuando el hospedador sobre el que se desarrolla
no es el habitual.
Supervivencia y capacidad colonizadora del ácaro S. scabiei
Estudios llevados a cabo sobre ácaros presentes en humanos y perros (Arlian
et al., 1984) han demostrado que éstos son capaces de sobrevivir de 24 a 36 horas
a 21º C y 40-80% humedad relativa, manteniendo su capacidad colonizadora y de
penetración. Se comprobó asimismo que las hembras sobreviven durante más
tiempo que los machos bajo unas mismas condiciones ambientales. A este respecto,
durante nuestros trabajos pudo confirmarse tanto la supervivencia como la
persistencia de la capacidad colonizadora de ácaros S. scabiei aislados en conejo
silvestre tras dos días de traslado al laboratorio fuera de su hospedador a
temperatura ambiente.
Las bajas temperaturas (10-15º C) y humedad relativa alta favorecen la
supervivencia de S. scabiei; así por ejemplo, se ha comprobado cómo ácaros de la
var. canis son capaces de sobrevivir 19 días a 10º C y 97% de humedad relativa. Sin
embargo, el tiempo requerido por el ácaro para penetrar de nuevo a través de la
epidermis aumenta en función del tiempo que haya pasado fuera de su hospedador,
poniendo de manifiesto la debilidad adquirida durante su período de “ayuno”. A
temperaturas por debajo de 20º C S. scabiei permanece virtualmente inmóvil,
mientras que su actividad aumenta notablemente a 35ºC (Arlian, 1989).
Experimentos realizados una vez más con ácaros de origen canino han
demostrado que ácaros sacados de su hospedador responden al olor y estímulo
térmico del mismo, buscando de forma activa su fuente (Arlian et al., 1988). Si bien
el contacto directo entre el hospedador inicial y uno potencial parece ser la vía
habitual de transmisión de la enfermedad, es probable que fómites, objetos o el
propio medio ocupados por ácaros vivos y con capacidad infectiva jueguen un papel
relevante en la transmisión y mantenimiento de la sarna.
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
8 _____________________________________________________________________________
Figura I.4- La cabra doméstica (A) ha sido considerada como el origen del brote epizoótico de sarna sarcóptica que afecta al rebeco cantábrico (B) en Asturias desde 1993.
Todos estos factores, unidos a la capacidad de supervivencia y penetración de
S. scabiei, son cruciales en la transmisión y mantenimiento de esta parasitosis en
sus hospedadores. Un mayor conocimiento de todas estas variables y del
comportamiento y capacidad de supervivencia del ácaro en el medio natural se
antoja necesario para comprender la epidemiología de la enfermedad y tratar de
identificar las vías de transmisión inter e intraespecíficas del ácaro en poblaciones
de fauna silvestre.
La transmisión interespecífica ha sido demostrada experimentalmente entre
diferentes especies susceptibles (McCarthy, 1960a; Stone et al., 1972; Samuel,
1981). Estas “infecciones cruzadas” entre hospedadores de especies distintas
resultan normalmente autolimitantes, si bien pueden llegar a prolongarse hasta
varios meses durante los cuales el ácaro puede llegar a reproducirse. Esta habilidad
de ciertas variedades para desencadenar parasitaciones temporales en
hospedadores “extraños” parece haber permitido que animales temporalmente
infectados actúen como reservorios para la transmisión del parásito a su hospedador
“natural” (Rossi et al., 2007). La transmisión interespecífica de la sarna sarcóptica
fue documentada de rebeco a corzo, ciervo e íbice por Kutzer (1996), y en la
Península Ibérica de cabra hispánica a ciervo, gamo y muflón por León Vizcaíno et
al. (1992). En Asturias la transmisión de sarna sarcóptica entre cabra doméstica y
rebeco (confirmada experimentalmente por Lavín et al., 2000, Figura I-4) fue
A
B A
________________________________________________________________________Introducción
______________________________________________________________________________ 9
señalada como el origen más probable del brote epizoótico que viene afectando a la
población Oriental de rebeco cantábrico desde 1993 (Fernández-Morán et al., 1997).
Manifestaciones clínicas y patogenia de la enfermedad
Las manifestaciones clínicas de esta enfermedad cutánea varían en función de
la especie afectada. En general, las formas agudas cursan con prurito, eritema,
pápulas, seborrea y alopecia. En los casos crónicos se observan costras,
hiperqueratosis y aumento del grosor de la piel. Los efectos que la enfermedad
produce en cada animal están modulados por factores individuales (especie
hospedadora, sexo, edad, condición física y estado inmunitario) y ambientales
(generalmente afectando a la dosis infectiva o condición corporal: densidad de
hospedadores, uso de zonas comunes de rascado y reposo, presencia de individuos
u otras especies hospedadoras que actúen como reservorio, disponibilidad de
alimento…).
Aparte del efecto mecánico e irritante que los ácaros provocan durante la
excavación de los túneles y alimentación en un hospedador, la gran cantidad de
material antigénico depositado durante la afección por S. scabiei (excreciones y
secreciones de ácaros vivos, ácaros muertos, restos de mudas entre diferentes
fases, restos de huevos…) puede explicar buena parte de la patogénesis de la sarna
sarcóptica como una manifestación/reacción de hipersensibilidad del hospedador al
ácaro y sus restos (Pence & Ueckermann, 2002). En especies como el perro o el
cerdo se han descrito reacciones de hipersensibilidad tanto inmediata (Tipo I) como
retardada (Tipo IV, Bornstein et al., 2001; Davis & Moon, 1990), mientras en otras
especies como el zorro (Little et al., 1998a) y el coyote (Pence et al., 1983) sólo
pudo confirmarse la presencia de una respuesta de hipersensibilidad inmediata (Tipo
I). La respuesta de hipersensibilidad Tipo I puede ser considerada una forma de
“reacción alérgica”, mediada básicamente por inmunoglobulinas IgE. En el caso de
las reacciones de hipersensibilidad Tipo IV, son células pertenecientes al sistema
inmunitario las que intervienen como mediadoras (Gell & Coombs, 1963). En
diferentes especies de bovinos silvestres afectados por fuertes brotes epizoóticos de
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
10 _____________________________________________________________________________
sarna sarcóptica en distintas partes del mundo, como los pertenecientes a los
géneros Ibex y Rupicapra, la manifestación clínica del proceso parece poner de
manifiesto un cierto grado de hipersensibilidad tipo I (a tenor del tipo de lesiones
observadas) que sin embargo no llega a controlar el ácaro. La proliferación de S.
scabiei en estas circunstancias puede responder a la incapacidad del sistema
inmune de estas especies para un correcto control del ácaro o bien a un estado de
anergia o malnutrición de los individuos afectados que les impide desplegar una
respuesta inmunitaria satisfactoria (Pence & Ueckermann, 2002).
Métodos de diagnóstico
El punto de partida para el estudio de cualquier enfermedad es la realización de
un diagnóstico correcto. En este apartado se describen diferentes técnicas
empleadas en el diagnóstico de la sarna sarcóptica en fauna silvestre, así como
propiedades y limitaciones de las mismas:
A-) Signos clínicos: En el caso concreto de la sarna, la observación en
individuos susceptibles de lesiones y síntomas (inquietud, rascados, sacudidas, mala
condición corporal en casos avanzados…) compatibles resultan indicios de
inestimable ayuda cuando es efectuada por personal familiarizado con la
enfermedad o formado en su identificación. La utilidad de estos indicios resulta
evidente en el caso de la fauna silvestre (en la que habitualmente sólo seremos
capaces de observar al animal a distancia y durante períodos cortos de tiempo) y su
vigilancia epidemiológica, para la cual resulta imprescindible una adecuada
caracterización clínica del proceso e identificación de las lesiones y alteraciones más
representativas en cada una de las especies susceptibles.
No es posible sin embargo hablar de sarna (ni asegurar que sea sarcóptica) sin
un diagnóstico definitivo. Todos los indicios previamente señalados han de ser
tomados únicamente como alteraciones o lesiones “compatibles con sarna”,
debiendo ser especialmente cautos a la hora de equiparar este tipo de “diagnósticos
presuntivos” con casos clínicos o brotes de sarna confirmados. Esta confirmación de
________________________________________________________________________Introducción
______________________________________________________________________________ 11
sarna sarcóptica es sólo posible mediante la detección del ácaro S. scabiei y su
relación con las lesiones clínicas presentes en animales afectados.
Figura I.5- El protocolo de búsqueda y aislamiento de ácaros vivos incluyó la recogida en fresco de muestras de piel de 5 x 5 cm (A), su incubación durante 24 horas a 37 ºC (B) y finalmente su revisión y recogida mediante el uso de lupa (C).
B-) Aislamiento-identificación del ácaro: el aislamiento e identificación de S.
scabiei, sus huevos o sus restos son la base para un diagnóstico definitivo. El
aislamiento de ácaros vivos ha sido la técnica de elección en los trabajos
desarrollados en la presente tesis: el protocolo establecido consistió en la
introducción en placas de Petri, protegidas y selladas con Parafilm®, de muestras de
piel fresca, su incubación durante 24 horas a 37ºC para estimular la movilidad y
migración de los ácaros, y su posterior identificación y recogida con la ayuda de una
lupa y el empleo de rascadores en el caso de grandes cantidades del parásito
(Figura I.5). La identificación y recogida de S. scabiei a partir de raspados o
muestras de piel puede no obstante resultar muy complicada o incluso infructuosa a
pesar de la evidencia del cuadro clínico en determinadas circunstancias o especies;
así por ejemplo, en perros, solamente llegan a encontrarse parásitos en el 20-30%
de los animales afectados mediante la técnica habitual de raspado y estudio
microscópico (Arlian et al., 1995; Hill & Steinberg, 1993). Técnicas de digestión de
raspados o incluso trozos de piel (con KOH como el reactivo más empleado en
diferentes protocolos) para la posterior búsqueda de exoesqueletos o restos de
huevos de S. scabiei ha sido habitualmente empleada en distintas especies
animales (Alasaad et al., 2009). Sin embargo, y al igual que con el caso del
aislamiento de ácaros vivos, esta técnica presenta una sensibilidad muy baja en
aquellos casos en que el número de ácaros o restos presentes en piel es muy bajo,
A B C
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
12 _____________________________________________________________________________
como ocurre en fases iniciales de parasitación o en individuos inmunocompetentes
con capacidad de hacer frente a S. scabiei.
El desarrollo y empleo de otros métodos de diagnóstico pueden por ello resultar
necesarios para monitorizar la sarna sarcóptica en determinadas circunstancias o
especies.
C-) Estudio histológico: Las lesiones microscópicas provocadas por la sarna
sarcóptica permiten identificar dos formas patológicas principales: una forma
“paraqueratótica”, propia de una respuesta de hipersensibilidad Tipo I o inmediata, y
otra forma “alopécica”, que se corresponde con una respuesta de hipersensibilidad
Tipo IV o retardada (Bates, 2003; Skerrat, 2003).
Si bien ambos tipos de respuesta presentan unas características histológicas
definidas, la presencia de ácaros o sus túneles en cortes histológicos resulta
necesaria para confirmar a S. scabiei como desencadenante de la respuesta inmune
identificada mediante esta técnica. Dicha presencia resulta frecuentemente
confirmada en cortes histológicos en el caso de sarna sarcóptica de ciertas especies
o determinados cuadros clínicos, donde el abundante número de ácaros presentes
facilita su observación en alguna de las muestras histológicas revisadas al
microscopio; sin embargo, la misma escasez de ácaros que generalmente impide su
aislamiento in vivo en otras especies, cuadros clínicos o fases iniciales de la
enfermedad supone también la incapacidad de su detección mediante histología, y
por tanto la utilidad de esta técnica en el diagnóstico etiológico del proceso, al verse
extremadamente reducidas las posibilidades de “cortar” e identificar un ácaro ( o sus
túneles, huevos…) en las muestras de tejido fijadas para su estudio.
D-) Detección de material genético del ácaro (PCR): La detección de material
genético de agentes patógenos mediante la Reacción en Cadena de la Polimerasa
(PCR) ha supuesto en las últimas décadas un enorme avance sanitario en el
diagnóstico de multitud de procesos, especialmente víricos y bacteriológicos, gracias
a la gran especificidad que esta técnica proporciona. Sin embargo, al igual que
ocurría con otras técnicas como el aislamiento in vivo o la histología y su uso con
fines diagnósticos, en aquellos casos en que el número de ácaros es reducido la
________________________________________________________________________Introducción
______________________________________________________________________________ 13
sensibilidad de esta técnica resulta muy limitada debido a la escasa probabilidad de
que la pequeña porción de tejido cuyo material genético va a ser amplificado
contenga un ácaro.
E-) Detección de anticuerpos: Frente al diagnóstico clásico de la sarna basado
en la detección del agente etiológico in situ, la detección de anticuerpos contra S.
scabiei en suero aporta información sobre la exposición o contacto previos del
animal objeto de estudio con el ácaro. Los ácaros de S. scabiei inducen una
respuesta inmunológica específica en los animales que parasita, la cual puede ser
detectada mediante un enzimoinmunoensayo tipo ELISA (Allen & Nelson, 1982;
Bornstein et al., 1996). A pesar de que la presencia de anticuerpos frente a S.
scabiei indica exclusivamente la existencia de contacto en un momento dado entre el
animal muestreado y el ácaro, no pudiendo por tanto ser considerado como una
prueba diagnóstica, los test ELISA presentan una indudable utilidad en estudios
epidemiológicos de sarna en diversas especies, tanto domésticas como salvajes
(Bornstein et al., 2001).
Estos estudios han llevado al desarrollo de varios ELISAs indirectos, basados
generalmente en el uso de homogeneizados totales de antígeno procedentes de
distintas variedades de S. scabiei, para la detección de anticuerpos específicos
frente al parásito S. scabiei en distintas especies (Hollanders et al., 1997; Bornstein
et al., 1995; Bornstein et al., 1996; Bornstein & Wallgren, 1997; Van der Heijden et
al., 2000; Lower et al., 2001; Rambozzi et al., 2004). Los resultados obtenidos en los
estudios realizados con diferentes tests indican que la detección de anticuerpos
frente a Sarcoptes mediante ELISA depende en gran medida del test serólogico
elegido, puesto que la sensibilidad varía enormemente entre los distintos tests e
incluso entre distintos lotes del mismo test (Kessler et al., 2003). Por otra parte, el
uso de los preparados antigénicos procedentes de homogenizados totales conlleva
varios problemas:
1. Problemas de inespecificidad provocados por la complejidad del antígeno y
en ocasiones por la contaminación de la muestra con inmunoglobulinas de la
especie a partir de la cual se prepara el antígeno.
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
14 _____________________________________________________________________________
2. Escasez y dificultad de su obtención. No existe un sistema de cultivo de los
ácaros, por lo que tienen que ser obtenidos a partir de biopsias de piel de animales
infectados de forma natural lo que hace que las cantidades de antígeno que se
obtienen con este sistema sean muy pequeñas. Además, la obtención de animales
infectados no siempre es fácil.
3. Cada preparado antigénico es distinto, lo cual compromete la repetibilidad,
reproductibilidad y en definitiva la fiabilidad del ensayo.
Para evitar los problemas derivados del uso de homogeneizados totales de
ácaros en el presente trabajo se ha utilizado un ensayo inmunoenzimático (ELISA
indirecto), basado en un antígeno recombinante (Casais et al., 2007). En general, el
uso de antígenos recombinantes posee varias ventajas:
a- Mejora la especificidad del ensayo.
b- La obtención del antígeno no requiere el uso animales infectados de forma
natural o experimentalmente, consiguiendo abaratar los costes de producción del
test diagnóstico.
c- Los métodos de producción y purificación de proteínas recombinantes son
eficaces y están estandarizados.
Relevancia de la sarna sarcóptica
La relevancia de la sarna sarcóptica en la actualidad debe ser considerada
desde tres puntos de vista diferenciados:
1.- Importancia en salud humana: se trata de una parasitosis de escasa
relevancia hoy en día en países desarrollados (donde puede aparecer asociada a
problemas de respuesta inmunológica) debido al reducido número de casos y la
efectividad de los tratamientos; en países en vías de desarrollo, en cambio, la sarna
sarcóptica continúa presentando prevalencias elevadas, especialmente ante
situaciones de hacinamiento y pobreza (Heukelbach & Feldmeier, 2006; Walton &
Currie, 2007). Como apunte de interés a nivel regional, cabe señalar a este respecto
que en Asturias fue descrita una “epidemia de sarna” que afectó a personas de la
región en el año 1971 (Barthe & Martín, 1976).
________________________________________________________________________Introducción
______________________________________________________________________________ 15
2.- Importancia en ganado: un amplio listado de especies domésticas, tanto
de compañía como de abasto, son susceptibles de hospedar a este ácaro y padecer
sarna. Si bien el uso de tratamientos eficaces la convierte en una enfermedad
raramente grave, las pérdidas económicas derivadas de menores rendimientos
asociados a infecciones no diagnosticadas (en porcino principalmente), así como las
asociadas a su profilaxis y tratamiento en distintas especies de ganado (bovino,
ovino, caprino, y especialmente porcino) hacen necesario tenerla en consideración
(Alonso de Vega et al., 1998; Rehbein et al., 2003; Menzano et al., 2007). La
reciente aparición de fenómenos de resistencia del ácaro frente a determinados
antiparasitarios (Currie et al., 2004) y la alta toxicidad de algunos de ellos confirma
la necesidad de continuar con correctos protocolos de seguimiento y control de S.
scabiei tanto a nivel de ganado doméstico como en sanidad humana.
3.- Importancia en fauna silvestre: el gran número de especies susceptibles y
la dificultad o imposibilidad de aplicar tratamientos efectivos en poblaciones libres
hacen que sea en la fauna silvestre donde se manifiestan con más crudeza los
efectos de S. scabiei. Este último aspecto se desarrolla con mayor detalle en los dos
siguientes apartados.
Sarna sarcóptica en fauna Silvestre
La sarna sarcóptica ha sido descrita en 10 órdenes, incluyendo 27 familias y un
total de 104 especies de mamíferos (silvestres y domésticos) en todo el mundo
(listado completo en Bornstein et al., 2001). De las diferentes especies silvestres
afectadas por esta enfermedad a nivel mundial (Tabla I.1; Pence & Ueckermann,
2002), el proceso cobra especial relevancia en aquellos taxones o poblaciones
susceptibles que nunca habían entrado en contacto con la enfermedad en las que
llega a adquirir carácter epizoótico, ya que en dichas circunstancias acaba
generalmente afectando a su dinámica poblacional (Mörner, 1992; Balestrieri et al.,
2006; Rossi et al., 2007). Si bien estos efectos se han mostrado generalmente
reversibles en poblaciones autosuficientes (con paulatina recuperación de los
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
16 _____________________________________________________________________________
Europa Norteamérica África Asia Australia
Coyote (Canis latrans)
Pence et al., 1983; Pence & Windberg, 1994; Samuel, 1981; Todd et al.,1981; Trainer & Hale, 1969
Zorro (Vulpes vulpes) Gortázar et al., 1998; Holt & Berg, 1990; Mörner, 1981; Mörner, 1992
Little et al., 1998b; Samuel, 1981; Stone et al., 1972; Trainer & Hale, 1969
Gray, 1937; McCarthy, 1960b
Lobo (Canis lupus) Mörner, 1992 Samuel, 1981; Todd et al., 1981 Zorro ártico (Alopex lagopus) Mörner, 1992 Dingo (Canis familiaris dingo) McCarthy, 1960a
Lince boreal (Lynx lynx) Holt & Berg, 1990; Mörner, 1992; Ryser-Degiorgis et al., 2002
León (Panthera leo) Young, 1975; Gakuya et al.,2011
Guepardo (Acinonyx jubatus) Mwanzia et al., 1995; Gakuya et al., 2011
Rebeco (Rupicapra rupicapra) Fernández-Morán et al., 1997; Onderscheka et al., 1968; Rossi et al., 1995
Íbice (Capra ibex) Rossi et al., 2007 Cabra montés (Capra pyrenaica) León-Vizcaíno et al.,1999
Íbice siberiano (Capra siberica) Vyrypaev, 1985 Vyrypaev, 1985
Jabalí (Sus scrofa) Ippen et al., 1995
Wombat (Vombatus ursinus) Gray, 1937; Martin et al., 1998; Skerratt et al., 1998
Koala (Phascolarctos cinereus) Brown et al., 1981 Gorila de montaña (Gorilla gorilla berengei) Kalema et al., 1998 Impala (Aepyceros melampus)
Sachs & Sachs, 1968; Young, 1975; Zumpt & Ledger, 1973
Alcelafo (Alcelaphus buselaphus) Springbok (Antidorcas marsupialis) Gacela de Grant (Gazella gazella) Gacela de Thompson (G. Thompsoni) Ñu (Connochaetes taurinus) Búfalo (Syncerus caffer) Eland (Taurotragus oryx) Gran kudú (Tragelaphus strepsiceros) Antílope sable (Hippotragus niger)
Tabla I.1- Poblaciones de especies de fauna silvestre descritas en la literatura científica como afectadas por brotes epizoóticos de sarna sarcóptica (Adaptado de Pence & Ueckermann 2002).
________________________________________________________________________Introducción
_____________________________________________________________________________ 17
valores poblacionales iniciales, o al menos “saludables” -Lindström et al., 1994;
Pence & Windberg, 1994-), es necesario considerar el riesgo a nivel de conservación
que puede suponer en caso de afectar a especies amenazadas, con escasos
efectivos o poblaciones fragmentadas (Martin et al., 1988; Henriksen et al., 1993;
Pence & Ueckermann, 2002). Otra manifestación de la importancia de esta
parasitosis en conservación son sus posibles consecuencias en el ecosistema al
reducir de forma notable una especie “presa”, importante como fuente de alimento
para otras (como podría ser el caso del conejo europeo, Millán, 2010). No debemos
tampoco olvidar sus posibles implicaciones en el ámbito de la gestión y
aprovechamiento cinegético en caso de afectar a especies objeto de caza.
Sarna sarcóptica en fauna silvestre en España
Si bien su reflejo en bibliografía es escaso, la sarna sarcóptica es una
enfermedad reconocida en España desde antaño especialmente por su presencia
relativamente habitual y su relevancia en diferentes especies de ganado doméstico
(Gil Collado, 1953).
Por lo que a la fauna silvestre se refiere, la enfermedad es también conocida
desde antiguo en la Península Ibérica en el caso del zorro, especie en la que esta
parasitosis presenta desde hace décadas una situación enzoótica en buena parte de
España (Gortázar et al., 1998). En esta situación endémica de la enfermedad, las
poblaciones de zorro se ven periódicamente afectadas por rebrotes locales de la
misma que no llegan a desencadenar las dramáticas consecuencias descritas en la
irrupción del parásito a poblaciones de zorro de otros países donde no existía
contacto previo con este ectoparásito (Mörner, 1981; Soulsbury et al., 2007). El
momento en que este primer contacto con la población vulpina se produjo en la
Península se desconoce, y en la actualidad los periódicos rebrotes registrados a
nivel local tienen un efecto limitado a largo plazo o mayor escala sobre las
poblaciones de este pequeño carnívoro.
Fueron sin embargo los brotes epidémicos de sarna sarcóptica experimentados
desde finales de los 80 por tres especies diferentes de Bóvidos (Cabra hispánica -
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
18 _____________________________________________________________________________
Capra pyrenaica hispanica -, rebeco cantábrico - Rupicapra pyrenaica parva - y arruí
- Ammotragus lervia -) los que hicieron tristemente conocida a esta parasitosis,
convirtiéndola en la principal amenaza para las poblaciones de las 3 especies en
buena parte de su área de distribución en la Península, y constituyendo uno de los
procesos patológicos más relevantes a nivel de gestión y conservación de fauna
silvestre durante las últimas décadas en España. Si bien en los tres casos han sido
descritas unas tasas de morbilidad y mortalidad relativamente elevadas en un primer
contacto de sus poblaciones con la enfermedad, se ha confirmado asimismo un
posterior descenso paulatino de las mismas siguiendo una onda epidémica, tal y
como ha sido descrito en otras especies de la familia Bovidae a nivel europeo como
el íbice y el rebeco de los Alpes (Rossi et al., 1995; Rossi et al., 2007).
La primera de las especies de ungulados afectadas por un brote epizóotico de
sarna sarcóptica en la Península de la que existe registro fue la cabra montés. En
1987 se registró por primera vez la afección de este bóvido por sarna sarcóptica en
el Parque Natural de Cazorla (León-Vizcaíno et al., 1989), cuya población se vio
severamente afectada por dicha parasitosis (aún presente en la misma a día de
hoy), y en cuya área de distribución se describieron otras especies de ungulados –
cérvidos- afectadas de forma esporádica a raíz del citado brote (León-Vizcaíno et al.,
1992). La sarna sarcóptica ha sido posteriormente detectada afectando a otras
poblaciones de cabra hispánica del Sur y Este de España (Pérez et al., 1992;
Palomares & Ruiz-Martínez, 1993; Pérez et al., 1997; León Vizcaíno et al., 1999).
El arruí, una especie introducida de origen norteafricano, se vio también
afectado por S. scabiei en el Parque Regional de Sierra Espuña (Murcia) por un
brote de sarna sarcóptica detectado en 1991 y que redujo drásticamente su
población durante los 3 primeros años de afección, mostrando sin embargo una
rápida recuperación de los parámetros demográficos y poblacionales y la ausencia
de animales enfermos detectados ya en 1999 (González-Candela et al., 2004).
La bibliografía científica hace escasa referencia a la confirmación de sarna
sarcóptica en otras especies de fauna silvestre de la Península Ibérica. Cabe señalar
la descripción en 2008 por primera vez en España de sarna sarcóptica en lobo en
Burgos (Domínguez et al., 2008) entre las especies de carnívoros presentes,
________________________________________________________________________Introducción
_____________________________________________________________________________ 19
mientras que en 2010 fue descrita por primera vez afectando a conejos silvestres en
España (Millán, 2010; Navarro-González et al., 2010).
En Asturias, aparte de su situación endémica en zorro previamente señalada
para buena parte de la Península, la sarna sarcóptica fue detectada por primera vez
en el rebeco cantábrico en Mayo de 1993 en las proximidades del Pico Torres
(Concejo de Aller, en su límite con León). Desde entonces el área afectada por la
enfermedad se ha ido extendiendo de forma continua en “balsa de aceite”, habiendo
sido registrados hasta la fecha un número superior a los 1500 animales afectados
por esta parasitosis en las Reservas Regionales de Caza del Principado de Asturias
(Figura I-6, González-Quirós et al., 2002; González-Quirós et al., 2007), lo que
convierte a la sarna sarcóptica en el proceso patológico más importante de cuantos
afectan a día de hoy al rebeco cantábrico. En la actualidad la sarna sarcóptica puede
ser considerada enzoótica en la población Oriental de rebeco cantábrico de Asturias,
donde continúa extendiéndose y provocando un goteo continuo de animales
afectados por esta parasitosis, mientras que la población occidental de este bóvido
continúa aún libre de la enfermedad.
Tras el citado brote epizoótico de sarna sarcóptica registrado en rebecos del
Principado de Asturias, la aparición de casos esporádicos y mortales de esta
parasitosis en cérvidos simpátricos (ciervo -Cervus elaphus- y corzo –Capreolus
capreolus-) así como la escasa información recogida en la bibliografía científica
acerca de los efectos de S. scabiei en estas dos especies, plantearon la necesidad
de estudiar dichos casos y evaluar su origen y relevancia indagando en los efectos
que S. scabiei pudiese tener sobre las poblaciones de ciervo y corzo locales.
Del mismo modo, la detección por primera vez en Asturias durante 2008 de al
menos 6 lobos con sarna sarcóptica abatidos por Guardería en los controles de
población efectuados por el Gobierno del Principado planteó la necesidad de
estudiar dicha parasitosis y sus posibles efectos sobre este cánido y sus
poblaciones.
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
20 _____________________________________________________________________________
Figura I.6- Representación gráfica del número de rebecos detectados con sarna (línea discontinua) y la superficie afectada por esta parasitosis en dicho bóvido silvestre (línea continua) tras la aparición del brote epidémico registrado en Asturias en 1993.
Con el ánimo de profundizar en el conocimiento y comprensión de la sarna
sarcóptica y su epidemiología en la fauna silvestre del Principado de Asturias, la
presente tesis trata de aportar información sobre el efecto de esta parasitosis a nivel
individual (aspectos clínicos del proceso) así como a nivel de población dentro de
cada una de las especies simpátricas de fauna silvestre estudiadas. Una vez
caracterizados estos efectos, se intentaron relacionar los resultados obtenidos para
las 5 especies objeto de estudio y se trató de indagar en el tipo de relaciones inter-
específicas en que el ácaro se ve implicado en una región de dimensiones discretas
como el Principado de Asturias.
0
200
400
600
800
1000
1200
1400
1600
1800
2000
0
50
100
150
200
250
300
350
1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007
Rebecos con sarna detectados Área de sarna en rebeco
Km² Número de animales
detectados con sarna
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_____________________________________________________________________________ 21
I.2- HIPÓTESIS DE TRABAJO
La sarna sarcóptica, a la luz de los datos recogidos durante los últimos años,
puede ser considerada en Asturias como una enfermedad “emergente” en el caso de
especies como el ciervo, el corzo y el lobo.
Existen diferencias, tanto clínicas como epidemiológicas, en los efectos de la
sarna sarcóptica a nivel individual y poblacional entre las diferentes especies de
fauna silvestre afectadas por S. scabiei en Asturias.
Los casos de sarna sarcóptica registrados en ciervo y corzo en Asturias
durante los últimos años parecen guardar relación con el brote epizoótico de esta
parasitosis registrado en rebeco cantábrico en Asturias desde 1993, mientras que
los casos registrados en lobo durante 2008 podrían relacionarse con los brotes
periódicos registrados en zorros de la zona, en los que el proceso se encuentra
presente de forma endémica.
Tanto la aparición como los efectos provocados por la sarna sarcóptica en
poblaciones silvestres vienen determinadas por el tipo y eficacia de la respuesta
inmune desplegada por las diferentes especies afectadas, y pueden verse
influenciadas por la presencia de agentes patógenos concomitantes (especialmente
por aquellos con capacidad inmunosupresora).
El grado de especificidad o adecuación del ácaro S. scabiei a su hospedador
permite identificar diferentes “variedades” o ”clusters” adaptados a los diferentes
taxones parasitados, manifestándose en forma de una menor transmisibilidad y
morbilidad entre aquellas especies más alejadas filogenéticamente. La adaptación
de una misma variedad del ácaro a varios hospedadores próximos taxonómicamente
puede permitir el intercambio de ácaros entre distintos taxones, con la relevancia
que este hecho puede tener en cuanto a epidemiología y gestión de las especies
afectadas.
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
22 _____________________________________________________________________________
I.3- OBJETIVOS
1. Caracterización clínica de la sarna sarcóptica en las diferentes
especies de fauna silvestre afectadas por este parásito en Asturias (rebeco, ciervo,
corzo, zorro y lobo).
2. Determinación de las diferencias existentes en el tipo de respuesta
inmune desplegada y su eficacia, así como en la evolución clínica de la sarna
sarcóptica entre las diferentes especies objeto de estudio.
3. Profundización en la epidemiología de la sarna sarcóptica en las
distintas especies de fauna silvestre afectadas en Asturias durante las dos últimas
décadas así como en sus posibles efectos a largo plazo a nivel de población.
4. Determinación de las posibles relaciones interespecíficas existentes en
la transmisión y evolución de esta parasitosis en la fauna silvestre asturiana.
5. Evaluación del grado de amenaza que a día de hoy la sarna sarcóptica
puede suponer para las poblaciones de los diferentes taxones estudiados y su
conservación en Asturias.
6. Evaluación de la utilidad y efectividad de diferentes técnicas a-) de
diagnóstico de la sarna sarcóptica (ELISA, histopatología, inmunohistoquímica,
aislamiento in vivo del ácaro, técnicas moleculares…) y b-) de monitorización
epidemiológica del proceso (recogida de datos durante recorridos sanitarios y
censos, datos obtenidos mediante vigilancia sanitaria activa y pasiva, trampeo
fotográfico,…) como herramientas para su estudio y seguimiento en las diferentes
especies de fauna silvestre afectadas.
7. Análisis y caracterización molecular de ácaros S. scabiei aislados en
las diferentes especies afectadas, identificación de los posibles clusters presentes
en fauna silvestre del Principado de Asturias, y determinación de las relaciones
interespecíficas que dichos estudios moleculares pudiesen desvelar.
________________________________________________________________________Introducción
_____________________________________________________________________________ 23
I.4-BIBLIOGRAFÍA
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Rossi, L., Fraquelli, C., Vesco, U., Permunian, R., Sommavilla, G.M.,
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CAPÍTULO 1
SARNA SARCÓPTICA EN CÉRVIDOS SILVESTRES DEL PRINCIPADO
DE ASTURIAS
1.1.- SARNA SARCÓPTICA EN DOS CORZOS (Capreolus capreolus)
DEL NORTE DE ESPAÑA
1.2.- SARNA SARCÓPTICA EN CIERVO IBÉRICO: ¿ENFERMEDAD
EMERGENTE O MEJOR VIGILANCIA SANITARIA?
Foto: María Rosas
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
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________________________________________________________________________ Capítulo 1
_____________________________________________________________________________ 33
1.1.- SARNA SARCÓPTICA EN DOS CORZOS (Capreolus capreolus)
DEL NORTE DE ESPAÑA
Oleaga, A., Balseiro, A., Gortázar, C. (2008) SARCOPTIC MANGE
IN TWO ROE DEER (CAPREOLUS CAPREOLUS) FROM NORTHERN SPAIN. European Journal of Wildlife Research 54, 134-137.
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
34 _____________________________________________________________________________
________________________________________________________________________ Capítulo 1
_____________________________________________________________________________ 35
RESUMEN
La sarna sarcóptica, una enfermedad parasitaria compartida con la cabra doméstica y
la oveja, afecta al rebeco cantábrico (Rupicapra pyrenaica parva) en el Norte de la Península.
Evidencias recientes sugieren que la sarna puede estar emergiendo como enfermedad en
cérvidos de dicha región. Esta comunicación describe dos casos mortales de sarna sarcóptica
en sendos machos de corzo (Capreolus capreolus) en Asturias (Norte de España) durante
2006. Ambos animales presentaron mala condición corporal pero no mostraron lesiones
diferentes de las atribuibles al ácaro. Cabeza, cuello, tronco y extremidades mostraron
alopecia, costras cutáneas e hiperqueratosis afectando a la mayor parte de su superficie
corporal. Numerosos ácaros Sarcoptes scabiei fueron aislados en piel. De acuerdo con
nuestros datos, esta es la primera descripción detallada de sarna sarcóptica mortal en corzos
europeos.
ABSTRACT
Sarcoptic mange, a disease shared with domestic goat and sheep, affects chamois
(Rupicapra pyrenaica parva) in northern Spain. Recent evidences suggest that mange
may be emerging among deer in this region. This communication describes two cases of
fatal sarcoptic mange affecting roe deer (Capreolus capreolus) stags in Asturias
(Northern Spain) in 2006. Both animals were in poor body condition and no significant
lesions other than those caused by the mites were observed. Alopecia, along with
cutaneous crusts and hyperkeratosis were present in head, neck, trunk, and legs,
affecting almost all the body surface. Numerous Sarcoptes scabiei mites were isolated
from the skin. To the best of our knowledge, this is the first detailed description of fatal
sarcoptic mange in European roe deer.
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INTRODUCTION
Sarcoptic mange, commonly referred to as scabies, is a disease affecting seven
different mammalian orders, including humans. It is caused by an obligate parasitic
mite of the skin, Sarcoptes scabiei (Linnaeus 1758), whose burrowing and feeding
activity, in addition to cutaneous hypersensitivity to mite fecal antigens triggers
cutaneous inflammation and pruritus, leading sometimes to excoriation, exudation,
and even hemorrhage (Collebrook and Wall 2004). Severe sarcoptic mange can
cause the death of the host, and is frequently reported in wild Bovidae populations
throughout Europe. This is the case in the chamois (Rupicapra rupicapra) and the
ibex (Capra ibex) in the Alps (e.g., Onderscheka 1982; Schaschl 2003; Rossi et al.
2007), of the Iberian ibex (Capra pyrenaica) in southern and eastern Spain (León
Vizcaíno et al. 1999), and of the introduced Barbary sheep (Ammotragus lervia) in
southeastern Spain (González-Candela et al. 2004). A mange epizootic mainly
affecting Cantabrian chamois (R. pyrenaica parva) in the Cantabrian Mountains,
northern Spain, was first detected in 1993 and is still expanding eastwards nowadays
(Fernandez-Moran et al. 1997 and unpublished reports).
Despite the abundant literature on sarcoptic mange in European wild ungulates,
information and even descriptions of clinical cases in species of deer are scarce.
Sarcoptic mange has sporadically been described in the red deer (Cervus elaphus) in
Alpine countries (e.g., Kutzer 1970; Greßmann 2001; Rossi et al. 2007; and
references therein) and in Spain (León-Vizcaíno et al. 1992; Fernandez-Moran et al.
1997). A single case was reported in a fallow deer (Dama dama) from Spain (León-
Vizcaíno et al. 1992). In the roe deer (Capreolus capreolus), despite being the most
widely distributed cervid in Europe, the reports on sarcoptic mange are also limited.
Several central European reports comment on mange cases in roe deer (e.g., two
cases reported by Kerschagl 1965 and six cases reported by Greßmann 2001, in
Austria), and Sarcoptes mites were identified on the carcass remaining of one roe
deer in the Cantabrian Mountains, Spain (Fernandez-Moran et al. 1997). In Italy, two
roe deer fawns kept together with an infected alpine chamois in a wildlife rescue
center developed clinical mange (Meneguz, personal communication).
________________________________________________________________________ Capítulo 1
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Both density and geographical range of roe deer have increased during the last
decades across Europe, including northern Spain (e.g., Acevedo et al. 2005). These
demographic and geographic changes may increase the risk of acquiring new
diseases through both increased contact rates with other species and density-
dependent impact on individual fitness at higher densities. The aim of this short
communication is to describe two fatal cases of sarcoptic mange in roe deer bucks in
Asturias (northern Spain).
MATERIALS AND METHODS
Two cases of severe sarcoptic mange affecting roe deer males where reported
in Asturias (northern Spain) in April and August 2006. The first one occurred on
21/04/2006 when a sick adult roe deer buck was observed in the Piloña regional
hunting preserve (Asturias, northern Spain, 30T 307337/4801084 UTM). When
approached by the observers, the animal showed an extreme weakness and made
no attempt to escape. Blood was collected from the jugular vein in plain tubes, a fecal
sample was taken from the rectum, and visible ticks were collected. Thereafter, the
animal was sedated with xylazine (Rompun, Bayer) and humanely euthanized with
embutramide and mebezonium (T-61, Intervet). The carcass was weighed,
measured, and taken to the laboratory for a complete necropsy. Skin samples of 5×5
cm2 were taken from the edges of the lesions, including both healthy and altered
tissues, processed in a 10% KOH solution for 60 min at 37°C and examined under a
light microscope. Mites were identified according to Wall and Shearer (1997).
Additional skin sections were resected and fixed in 10% buffered formalin for paraffin
embedding and routine histopathology (hematoxylin–eosine). No other samples were
fixed in formalin.
Blood serum obtained after coagulation was frozen and submitted for serum
biochemistry. This analysis included nine parameters (creatinine, urea UV, albumin,
magnesium, phosphorus, calcium arsenazo, total protein, cholesterol, and glucose)
and was performed using an A25-BioSystems (Biosimex SA) biochemical automatic
analyzer.
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In the second case, the animal was found dead on a path of the Caso regional
hunting preserve (30T 313692/ 4780706 UTM) on 17/08/06. During the first
examination, visible ticks and a fecal sample were collected, and after recording
measurements and weight, the carcass was taken to the laboratory. The necropsy
was made according to the protocol followed in the previous case, paying special
attention to any alteration in connection with the cause of death. Skin samples where
taken and processed for both histological and parasitological examination, and blood
was obtained from the thoracic cavity. Different organ samples (kidney, duodenum,
myocardium, lung, liver, spleen) were taken for histopathology.
Figure 1- Mangy roe deer stag (case 1) with visible alopecia and skin crusts.
RESULTS
The first animal was not completely emaciated and had a body weight of 20 kg,
for a head and body length of 96 cm. The poor condition was confirmed by the
complete absence of kidney fat. No significant traumatic injuries were observed.
Alopecia, along with cutaneous crusts and hyperkeratosis were present in head,
neck, trunk, and legs, affecting almost all the body surface (Fig. 1). A general
lymphadenitis, more evident in the cervical lymph nodes, was noted. All other organs
________________________________________________________________________ Capítulo 1
_____________________________________________________________________________ 39
appeared normal. The animal exhibited a crusting dermatitis characterized by the
presence of large number of mites that were isolated from skin samples. They were
identified as S. scabiei, including both adults and larvae. Histological examination of
skin sections revealed hyperkeratosis with intracorneal tunnels where mites
demonstrated. The hair follicles were clogged with keratin. The epidermis was
hyperplastic with prominent rete ridge formations. The dermis presented an
inflammatory infiltrate consisting of lymphocytes, neutrophiles, and macrophages
(Fig. 2). Eosinophils were rarely seen. Dermal lesions also included variable
vasodilatation; vessels in the superficial dermis were congested, and there was a
predominantly mononuclear cell perivascular infiltrate. No obvious lesions were
observed in the sebaceous or sweat glands. Microscopic inspection of the underlying
muscle revealed the presence of occasional Sarcocystis. Other parasitological
findings included a myasis under the burr of both antlers, nine ticks of the genus
Ixodes, and four lungworms of the genus Dictyocaulus. Biochemical results were
0.72 mg/dl creatinine, 74.51 mg/dl urea, 30.56 g/l albumine, 2.40 mg/dl
magnesium, 10.13 mg/dl phosphorus, 7.59 mg/dl calcium, 72.66 g/l total protein,
69.07 mg/dl cholesterol, and 9.18 mg/dl glucose.
Figure 2- Skin section of roe deer stag 1 showing hyperkeratosis, intracorneal tunnels with mites, and inflammatory infiltrate (H&E stained, ×400).
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
40 _____________________________________________________________________________
The second roe deer was in a worse body condition, with a weight of only 19.5
kg for a length of 120 cm. Neither kidney nor coronary fat was observed. Small
injuries around the snout, probably caused by a fox or a small dog before death,
were observed. No other significant alteration was appreciated. As in the first
animal, there was a general alopecia affecting about 60% of the body surface,
characterized by hyperkeratosis and cutaneous crusts in head, neck, and trunk.
Isolation and identification of mites provided the same results as in the other case.
Only four adult ticks were found in the first examination; however, nine larvae were
recovered during necropsy. Eleven nematodes of the genus Dictyocaulus were found
in the bronchioli. Two Sarcocystis cysts were observed on the myocardial surface,
but no more parasites were isolated in the other organs. As in the first case, a
general lymphadenitis was evident. Histopathologic examination of the skin sections
revealed hyperkeratosis and mites inside intracorneal tunnels, and lymphocytes,
neutrophiles and macrophages appeared as an inflammatory infiltrate in dermis. No
significant alteration, other than renal congestion, was observed.
DISCUSSION
Even if the availability of data for the first buck’s biochemical results comparison
is limited, these results show slightly elevated urea and low creatinine and glucose
levels (Stubbe et al. 1975; Haenichen and Barth 1978; Ursache et al. 1980; Volmer
and Herzog 1995). This can suggest a loss of muscular mass due to chronic disease,
a renal disorder, or a consequence of the persistent stress situation caused by
scabies. The lack of formalin-preserved kidney samples in case 1 prevented a
complete histopathology. In case 2, only renal congestion was observed in kidney
histology.
To the best of our knowledge, this is the first detailed description of fatal
sarcoptic mange in free-living European roe deer. The large proportion of affected
skin, with inflammation and probably secondary infections, along with the poor body
condition of both deer and the absence of significant traumas or organic alterations,
strongly indicates that the death was due to the sarcoptic mange in the second
________________________________________________________________________ Capítulo 1
_____________________________________________________________________________ 41
animal, and it would also have been fatal in the first case in a short time. This
extreme weakness, which can course with anorexia, has been suggested to trigger
the death in severe mange cases (Cordero del Campillo and Rojo Vázquez 1999).
The loss of a large part of a vital organ such as the skin may explain the fatal course
of the described cases (Kerschagl 1965).
The first clinical case was detected only 4 km north of a chamois population,
where the first mange cases were reported in 1994. Nonetheless, chamois
populations in the area are monitored, and a recovery trend is evident since 2002
(Quirós, personal communication). No mange cases have been observed in chamois
from this area since 2002. There were also no reports on mangy domestic animals in
this area.
In contrast, in the area where the second mangy roe deer was found (21 km
southeast of the first one), the chamois population is still affected by mange, and
cases of red deer (Cervus elaphus) mange were observed in previous years.
Because cases of mange are extremely rare in roe deer, the occurrence of
these two cases during the last months in Asturias (northern Spain) may suggest
some kind of variation in sarcoptic mange epidemiology. Careful wildlife disease
surveillance will allow evaluating whether these cases were sporadic, or whether this
was the first sign of a disease emerging in Spanish deer.
ACKNOWLEDGMENT
This is a contribution to the agreement between CSIC and Principado de
Asturias. We thank the rangers of the game preserves and our colleagues from
SERIDA and IREC for their help. The comments from two anonymous reviewers and
valuable literature provided by one of them helped to improve the first version of this
manuscript.
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42 _____________________________________________________________________________
REFERENCES
Acevedo, P., Delibes-Mateo, M., Escudero, M.A., Vicente, J., Marco, J., Gortazar, C., 2005. Environmental constraints in the colonization sequence of roe deer (Capreolus capreolus Linnaeus, 1758) across the Iberian Mountains, Spain. J. Biogeogr. 32, 1671–1680.
Collebrook, E., Wall, R., 2004. Ectoparasites of livestock in Europe and the
Mediterranean region. Vet. Parasitol. 120, 251–274. Cordero del Campillo, M., Rojo Vázquez, F.A., 1999. Parasitología
Veterinaria. McGraw-Hill Interamericana, Madrid. Fernandez-Moran, J., Gomez, S., Ballestreros, F., Quiros, P., Benito, J.L.,
Feliu, C., Nieto, J.M., 1997. Epizootiology of sarcoptic mange in a population of Cantabrian chamois (Rupicapra pyrenaica parva) in Northwestern Spain. Vet. Parasitol. 73, 163–171.
González-Candela, M., León Vizcaíno, L., Cubero Pablo, M.J., 2004.
Population effects of sarcoptic mange in Barbary sheep (Ammotragus lervia) from Sierra Espuña Regional Park, Spain. J. Wildl. Dis. 40, 456–465.
Greßmann, G., 2001. Gamsräude und Gamsblindheit: Auftreten in der
Steiermark zwischen 1952 und 1999 sowie Schlußfolgerungen für Präventionsmaßnahmen im Rahmen der Jagd. Dissertation, Karl-Franzens Universität, Graz.
Haenichen, T., Barth, D., 1978. Observations on a stress-caused renal failure
in captured roe-deer. Proc. Symp. On the Comparative Pathology of Zoo Animals. National Zoological Park Smithsonian Institution, Washington, DC, pp 421–424.
Kerschagl, W., 1965. Wildkrankheiten–Ihre Ursachen, ihre Verhütung und
ihre Bekämpfung. Österreichischer Jagd-und Fischerei- Verlag des N.-Ö. Landesjagdverbandes, Wien.
Kutzer, E., 1970. Hirschräude in den Rottenmanner Tauern. Der Anblick 25,
235–238. León-Vizcaíno, L., Astorga, R., Escos, J., Alonso, F., Alados, C., Contreras,
A., Cubero, M.J., 1992. Epidemiología de la sarna sarcóptica en el Parque Natural de las Sierras de Cazorla, Segura y Las Villas. Actas del Congreso Internacional del Género Capra en Europa, Ronda, España, pp 95–98.
León Vizcaíno, L., Ruiz de Ibañez, M.R., Cubero Pablo, M.J., Ortiz, J.M.,
Espinosa, J., Pérez, L., Simon, M.A., Alonso, P., 1999. Sarcoptic mange in Spanish ibex from Spain. J. Wildl. Dis. 35, 647–659.
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Onderscheka, K., 1982. Etat actuel de la recherche sur la gale du chamois. Proc. Symp. Sur le chamois, Conseil International de la Chase et de la Fauna sauvage, Ljubljana, Yugoslavia, pp 89–108.
Rossi, L., Fraquelli, C., Vesco, U., Permunian, R., Sommavilla, G.M.,
Carmignola, G., Da Pozzo, R., Meneguz, P.G., 2007. Descriptive epidemiology of a scabies epidemic in chamois in the Dolomite Alps, Italy. Eur. J. Wildl. Res. 53, 131–141.
Schaschl, E., 2003. Gamsräude. Österreichischer Jagd-und Fischerei- verlag, Wien.
Stubbe, I., Stubbe, W., Stubbe, G., 1975. Morphologische, chemische und
serologische Blutuntersuchungen bei Reh-und Muffelwild. Beiträge zur Jagd-und Wildforschung 9, 225–266.
Ursache, O., Chevrier, L., Blancou, J.M., Jaouen, M., 1980. Valeur des
paramètres biochimiques et hématologiques chez le chevreuil (Capreolus capreolus). Rev. Med. Vet. 131, 547–552.
Volmer, K., Herzog, A., 1995. Rehwild näher betrachtet–Untersuchungen an
Rehwild. Verlag J. Neumann-Neudamm Melsungen, Germany. Wall, R., Shearer, D., 1997. Veterinary entomology. Chapman and Hall,
London.
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1.2.- SARNA SARCÓPTICA EN CIERVO IBÉRICO: ¿ENFERMEDAD
EMERGENTE O MEJOR VIGILANCIA SANITARIA?
Oleaga, A., Casais, R., González-Quirós, P., Prieto, M., Gortázar, C. (2008) SARCOPTIC MANGE IN RED DEER FROM SPAIN:
IMPROVED SURVEILLANCE OR DISEASE EMERGENCE?. Veterinary
Parasitology 154, 103-113.
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
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________________________________________________________________________ Capítulo 1
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RESUMEN
El interés sobre las enfermedades emergentes ha aumentado durante los últimos
años, y situaciones con múltiples hospedadores resultan cada vez más relevantes para la
gestión y conservación de fauna silvestre. Presentamos datos sobre Asturias, en el Norte de
España, donde se han registrado 80 ciervos (Cervus elaphus) con sarna desde el comienzo de
la epizootia en rebeco (Rupicapra pyrenaica parva) en 1993. Combinamos datos de campo y
de necropsia con los resultados de un estudio serológico efectuado mediante un ELISA de
desarrollo propio para evaluar si la sarna provocada por S. scabiei en ciervos de esta zona es
una enfermedad emergente. El número medio de casos de ciervos con sarna por año fue de
5, con un máximo de 16. No se detectó ninguna relación estadísticamente significativa entre
temperaturas mensuales, precipitaciones o número de días con cobertura de nieve y el
número anual de casos de sarna en ciervos. Sólo 4 ciervos con sarna (5%) fueron detectados
fuera del área de distribución de sarna sarcóptica en rebeco durante ese mismo año, y los 4
aparecieron a menos de 2500 m. de ese límite. La mayor distancia registrada entre
localizaciones de dos casos consecutivos de sarna en ciervo fue de 18 km. Los casos de sarna
fueron significativamente más frecuentes en machos que en hembras y en adultos que en
ciervos jóvenes. El momento de aparición de sarna sarcóptica en rebeco en cada sector, el
año con un menor número de rebecos detectados, el año con un mayor descenso
poblacional en rebeco o la densidad de rebecos en cada sector no ofrecieron relación
significativa alguna con el momento y frecuencia de aparición de casos de sarna en ciervo.
En el área afectada por sarna, el ELISA efectuado sobre 327 muestras de sangre de ciervos
cazados sin lesiones compatibles con sarna detectó sólo 4 animales seropositivos. Los 83
sueros procedentes de Reservas de caza sin casos clínicos ofrecieron resultados ELISA
negativos. Atendiendo a los datos epidemiológicos recogidos, la sarna sarcóptica no parece
suponer una amenaza para las poblaciones asturianas de ciervo. En cualquier caso, resulta
aconsejable continuar con la monitorización sanitaria y serológica de estas poblaciones.
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
48 _____________________________________________________________________________
ABSTRACT
Concern about emerging diseases has risen in recent years, and multihost situations
have become increasingly relevant for wildlife management and conservation. We present
data on Asturias, northern Spain, where 80 mangy red deer (Cervus elaphus) have been
found since the beginning of the epizootic in chamois (Rupicapra pyrenaica parva) in 1993.
We combine field and necropsy data with the results of a serosurvey using an in-house ELISA
test to evaluate if deer mange due to Sarcoptes scabiei is an emerging disease in this area.
The mean number of deer mange cases per year was 5, with a maximum of 16. No significant
relationship was detected between monthly temperatures, rainfall or number of days with
snow cover and the annual number of sarcoptic mange cases in red deer. Only 4 mangy red
deer (5%) were detected outside the limits of scabietic chamois distribution during the same
year, and all were less than 2500 m away from that limit. The longest distance reported
between two consecutive mangy deer locations was 18 km. Mange cases were significantly
more frequent in stags than in hinds and in adults than in juvenile deer. The time of the first
mange detection in chamois in each sector, year with minimum number of chamois
recorded, year with maximum chamois population decline rate and chamois density offered
no significant correlation with red deer mange cases appearance moment and frequency. In
the mange affected area, ELISA testing of 327 blood samples from hunter-harvested deer
without obvious mange-compatible lesions revealed only 4 seropositive animals. All 83 sera
from hunting preserves without clinical cases yielded negative ELISA results. According to
these epidemiological data mange does not seem to threaten red deer populations in
Asturias. However, continued monitoring of deer health and ELISA testing for sarcoptic
mange is advisable.
INTRODUCTION
Concern about emerging diseases has risen in recent years, and multihost
situations have become increasingly relevant for wildlife management and
conservation (Gortazar et al., 2007). Sarcoptic mange is a highly contagious skin
________________________________________________________________________ Capítulo 1
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disease affecting seven different mammalian orders, including humans (Arlian,
1989). The etiological agent is an obligate parasitic mite of the skin, Sarcoptes
scabiei (Linnaeus 1758), that causes skin inflammation, pruritus, and usually
cutaneous hypersensitivity leading sometimes to excoriation, exudation and even
haemorrhage (Collebrook and Wall, 2004). As a consequence of physiological
alterations in skin and different organs (Arlian et al., 1990) the host often
becomes dehydrated and emaciated, and can finally succumb to the infestation
(Pence and Ueckerman, 2002).
In many animal species, the prevalence of scabies is very high and often
causes death if it is untreated (Brotowijoyo, 1987; Kemp et al., 2002). It is an
important disease in cattle, pigs, sheep and goats worldwide, with many
economical implications in prophylaxis and treatment (Alonso de Vega et al., 1998;
Rehbein et al., 2003; Tarigan and Huntley, 2005; Menzano et al., 2007). Sarcoptic
mange seems to show more dramatic effects in wildlife, severely affecting the
population dynamics of different species (Morner, 1992; Balestrieri et al., 2006). This
parasitic disease has frequently been reported in wild Bovidae populations
throughout Europe: chamois (Rupicapra rupicapra) and ibex (Capra ibex) in the Alps
(e.g. Onderscheka, 1982; Schaschl, 2003; Rossi et al., 2007), Iberian ibex (Capra
pyrenaica) in southern and eastern Spain (Leon Vizcaino et al., 1999), and the
introduced Barbary sheep (Ammotragus lervia) in south-eastern Spain (Gonzalez-
Candela et al., 2004). A mange epizootic mainly affecting Southern chamois
(Rupicapra pyrenaica parva) in the Cantabrian Mountains, northern Spain, was first
detected in 1993 and is still expanding eastwards today (Fernandez-Moran et al.,
1997). Since the onset of the outbreak until 2006 a total of 1515 mangy chamois
have been detected in Asturias.
The Iberian red deer (Cervus elaphus hispanicus) populations are increasing
during recent years (Gortazar et al., 2000). In Asturias, northern Spain, the last
autochthonous red deer were hunted in the first decade of 20th century, but red
deer were successfully reintroduced between 1955 and 1974 with animals from the
remaining indigenous populations in Central Spain (Nores and Vazquez, 1987;
Perez et al., 1998; Acevedo, 2006).
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
50 _____________________________________________________________________________
Sarcoptic mange transmission has been documented from chamois to red deer,
roe deer (Capreolus capreolus) and Alpine ibex by Kutzer (1966), and from Iberian ibex
to red deer, fallow deer (Dama dama) and mouflon (Ovis aries musimon) by Leon
Vizcaino (Leon Vizcaino et al., 1992). Sarcoptic mange has sporadically been
described in red deer in European countries, usually as sporadic cases (Boch and
Schneidawind, 1988; Rossi et al., 2007). Outbreaks with more than 4 red deer affected
by sarcoptic mange have only been described in Austria (Kutzer, 1966; Kohler,
1970; Greßmann, 2001) and in Spain during the sarcoptic mange epizootic
affecting Iberian ibex in the Cazorla Natural Park since 1987 (Leon Vizcaino et al.,
1992).
During the last decade an increasing number of mangy red deer have been
observed in south eastern Asturias (Cantabrian Mountains, Northern Spain). Although
other cervid species, such as roe deer, have been affected in the same area (Oleaga et
al., 2008), the number of mangy red deer found since the beginning of the epizootic in
chamois (1993) in Asturias, and the small area where all these cases were reported
prompted a study of the known cases. The aim of this paper is to compile this
information and use an in house ELISA test to establish if sarcoptic mange is an
emerging disease in deer.
MATERIAL AND METHODS
Study area
The Principality of Asturias is a 10,603 km2 autonomous region located in the
North of the Iberian Peninsula, including the central part of the Cantabrian mountain
chain. This area includes 17 public game reserves. These reserves have a total
surface area of 2110 km2 and contain most of the red deer and chamois ranges in
the region (Fig. 1).
One of these 17 reserves is the Regional Game Reserve of Caso (438 060N,
0058 150W; number 10 in Fig. 1), that covers 307 km2 and is located in south-
western Asturias. This is the core area of the deer mange cases reported herein. It is
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surrounded by the game reserves of Aller (223 km2, number 9) and Sobrescobio
(67 km2, number 17) to the West, Piloña (54 km2, number 11) to the North and
Ponga (205 km2, number 13) to the East, and by the province of Leon to the
South. The relief is mountainous, with altitudes ranging from 800 to 2100 m, and the
landscape is the typical of the Orocantabrian region, basically composed of forests
(39%, mainly deciduous woods), shrubs (39%,) and meadows (16%).
This region is included in the Eastern nucleus of the Cantabrian chamois
distribution area in Asturias and houses other ungulate species including roe deer,
red deer and wild boar (Sus scrofa); there can also be found large populations of
red fox (Vulpes vulpes), beech marten (Martes martes), stone marten (Martes
foina), badger (Meles meles) and wolf (Canis lupus), all of them listed among the
hosts of S. scabiei in Europe (Todd et al., 1981; Bornstein et al., 2001; Domınguez
et al., 2008). Cattle are also abundant in the studied area, sharing pasture with wild
ruminants, whereas the presence of sheep and goats is almost anecdotal.
Monitoring of chamois and red deer
As a consequence of the chamois sarcoptic mange outbreak detected in
1993, the distribution area of this bovid has been subjected to a detailed
monitoring of both chamois and red deer populations and health status. For this
purpose, the distribution area of chamois affected by sarcoptic mange was divided
into 15 census sectors (A → O, see Fig. 6), which belong to game reserves
numbers 9, 10, 11, 13 and 17 (Fig. 1).
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
52 _____________________________________________________________________________
Figure 1- Map of Asturias showing bordering provinces and numbered game reserves (1: I´bias, 2: Cangas del Narcea, 3: Degan˜a, 4: Somiedo, 5: Teverga, 6: Proaza, 7: Quiro´s, 8: Lena, 9: Aller, 10: Caso, 11: Pilon˜a, 12: Sueve, 13: Ponga, 14: Picos de Europa, 15: Yernes y Tameza, 16: Belmonte, 17: Sobrescobio). Those reserves in dark gray are affected by sarcoptic mange in both chamois and red deer. Red deer is almost absent from reserves 1, 2 and 3.
Red deer population data were obtained from unpublished reports of the
Asturias Government, in the frame of regular red deer census made by gamekeepers
and technicians in the game reserves. In this case, due to orography and
organizational reasons, the already existing division of game reserves in hunting
areas was used as red deer census sectors, different from those signalled for the
chamois census. According to this information, the red deer population estimated for
the 17 reserves of Asturias was 6360 animals in 2006, although abundance and
distribution were irregular. Along with areas with a very low or even nil density, with
less than 1 individual for 100 ha (game reserves number 1, 2, 3, 7, 8 and 15, Fig. 1),
other reserves showed average densities higher than 6 red deer/100 ha (game
reserves number 9, 10 and 11; Fig. 1). Even within a given reserve, densities vary
locally. For example, deer densities in Caso (reserve number 10) ranged from 1.8 to
14.7/100 ha. The hind to stag ratio in the Caso hunting reserve was 1.62 hinds per
stag in 2006, while the age ratios reported for this reserve were 0.2 fawns per all-age
deer and 0.4 young (less than 2 years) animals per adult.
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Data on clinical mange in deer
Data on mangy red deer were recorded by the technician in charge of the
chamois and deer monitoring in Asturias (Pablo Gonzalez-Quiros, Biogestion). Since
1993 to date, the total number of deer showing visible scabies lesions or any other
anomalies and their location were recorded. For the description and comparison of
sarcoptic mange distribution area in both chamois and red deer we used the minimum
convex polygon (MCP) method, i.e. the smallest (convex) polygon containing all
points where mangy animals where detected during a given period. A selective
culling programme was conducted for animal welfare reasons, shooting animals with
visible scabies. Age, sex and any other information available were recorded from deer
(a) found dead, (b) hunted with scabies-compatible lesions, or (c) observed with
scabies-compatible lesions (Fig. 2). Although sarcoptic mange is not the only
possible cause of alopecia in red deer, no other sanitary problem affecting skin
condition has been described in ungulates in the study area. A confirmatory
laboratorial diagnosis was performed whenever skin sample collection was possible.
Mange diagnosis in clinical cases
In 15 cases, skin samples of 5 cm x 5 cm were taken from the edges of the
lesions, including both healthy and altered tissues, processed in a 10% KOH
solution for 60 min at 37 8C and examined using a light microscope, for the
identification of mites according to Wall and Shearer (1997). Additional skin
sections were fixed in 10% buffered formalin for routine histopathology
(Hematoxyline–Eosine). In three cases it was possible to perform complete
necropsies, paying special attention to any lesions connected with sarcoptic mange.
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
54 _____________________________________________________________________________
Figure 2- Sarcoptic mange lesions in red deer: (a) skin crusts and alopecia in head region; (b) hyperkeratosis, skin crusts and cracking in chest and foreleg; (c) hyperkeratosis, alopecia and poor body condition; (d) histology of the skin revealing hyperkeratosis and intracorneal tunnels containing mites.
ELISA test
A total of 418 red deer serum samples collected from 2002 to 2006 were
available for testing. Blood was taken from the thoracic cavity, and serum was
obtained after coagulation and centrifugation and stored at -20 8C until tested
by ELISA. Most sera (n = 410) were randomly obtained by technicians or
gamekeepers during the regular hunting season from apparently healthy red
deer, while 8 samples were obtained from clinical mange cases. Sera from the
410 apparently healthy red deer were collected in 11 different preserves. Out of
these, 327 sera came from preserves with mange reports (104 sera from game
reserve number 10, 61 from # 17, 74 from #11, 70 from #13 and 18 from #9). The
remaining 83 sera came from reserves with no previous mange reports in red deer
or chamois (41 from game reserve number 12, 4 from #4, 2 from #7, 24 from #5, 10
from #6 and 2 from #2).
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All 418 sera were tested for the detection of specific antibodies to S. scabiei
using a recently developed ELISA test (Casais et al., 2007). This ELISA is based on a
structural antigen of the mite (Ssl20DB3), whose encoding cDNA was identified in
a S. scabiei var. hominis library using the sera from an infected chamois and
expressed in Escherichia coli as a unitary recombinant antigen. The ELISA method,
for its use in the immunodiagnosis of the disease in deer, was validated with a
panel of 41 deer sera (33 from healthy unexposed animals originating from a
scabies-free area outside Asturias and 8 that were obtained from scabieitic
animals) and its cut off level established as a relative optical density (OD) value of
0.43. With this validation panel, the ELISA test was characterized as having 75%
sensitivity and 97% specificity.
Statistics
We used Spearman’s rank correlation test to detect any relationship between
data on monthly temperature, rainfall and snow cover and the number of deer mange
cases from 1995 to 2007 (data provided by ‘‘Instituto Nacional de Meteorologıa’’
I.N.M., Caso station). Prevalence data were compared between age and sex
categories with the Chi square test.
RESULTS
S. scabiei was isolated for the confirmation of mange from 13 of 15 skin
samples. Bacterial infections (secondary to an important injury in one case) were the
cause of cutaneous alteration in the two negative samples.
Detailed necropsies were performed on 3 of the 13 confirmed cases,
corresponding to two adult males and an adult female with different degrees of
disease (Fig. 2). The first animal was an adult male shot in January 2006 by
game keepers after observing skin lesions compatible with sarcoptic mange. The
affected skin area was about 30% of the body surface, and the body condition was
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
56 _____________________________________________________________________________
fair (kidney fat index KFI = 0.79). No other macroscopic lesions or alterations apart
from the injured skin were detected.
The second case was also an adult male found shot by poachers in December
2006. This animal presented a pitiful body condition, with 95% of the body surface
affected by alopecia, cutaneous crusts, hyperkeratosis due to sarcoptic mange and
presence of a large number of ticks (the estimation was more than 200). Apart from
the almost complete absence of fat, confirmed by a KFI value of 0.11, a general
lymphadenitis and many pulmonary and hepatic parasites (Dyctiocaulus sp. and
Fasciola hepatica) were detected.
The last case was an adult female shot in February 2007 in an ordinary
hunt. A small lesion of 10 cm x 15 cm containing mites (isolated in the
laboratory) was detected in the posterior right leg during inspection. Body condition
was fair, with a KFI of 0.92. The animal was pregnant, with a male foetus, and no
significant alteration was detected during necropsy.
The first affected deer appeared in April 1995, when a mangy stag was
detected and subsequently shot by rangers. Sarcoptic mange was confirmed in the
laboratory. Another three affected red deer were described during that year in the
same area, very close to the location of the second outbreak of sarcoptic mange
detected in Asturian chamois in the spring of 1994 (see Fig. 3). Since then, 76
cases have been reported up to March 2007, affecting altogether the game
reserves number 10, with 60 mangy animals, #17 with 3 animals, #13 with 10
animals, #11 with 3 animals, #9 with 3 animals and Laviana Council (1 mangy
animal). Out of these 80 mangy red deer, 12 (15%) were field observations of deer
with lesions, 20 (25%) were shot deer and 48 (60%) were deer that were found dead
in the field.
No significant relationship was detected between monthly temperatures,
rainfall or number of days with snow cover and the annual number of sarcoptic
mange cases in red deer during the period under study (p > 0.05). These
findings may have been due to the lack of a complete series of data.
The first mangy stag appeared in April 1995, inside the area affected by the
second nucleus of the chamois sarcoptic mange outbreak detected in 1994 in
Asturias. Since then, only 4 out of the 80 affected red deer (5%) were detected
________________________________________________________________________ Capítulo 1
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outside the limits of scabietic chamois distribution during the same year, and all of
them were less than 2500 m away from that limit (estimated using the method of the
minimum convex polygon). The longest distance reported between two consecutive
mangy deer locations was 18 km and occurred between 1995 and 1996 (Fig. 3).
These kinds of ‘‘jumps’’ of the disease were observed at least twice more (1997 and
2000). Sporadic cases near to previously reported ones appeared all over the study
area until 2001. Since then, the pattern of appearance of new cases seems to have
changed, with an apparent concentration of mangy animals in the Nalon river
valley in the Caso game reserve. Out of the total 38 mangy animals described
between 2002 (year with the maximum number of scabietic red deer reported, 16
cases) and 2007, 37 were located no farther than 2 km from Nalon river, and only 1
appeared in the Ponga Game Preserve in 2003, 10 km away (Fig. 3).
Four out of the five game reserves where deer mange has been described
(number 9, 10, 11 and 13) have the highest values of red deer density observed in
all Asturian game reserves (there is no data for the fifth affected one, game
reserve number 17, where game keepers also signal a high density rate).
Out of the total 73 adult mangy animals, 48 (67.6%) were stags and only 23
(32.4%) hinds, while in two cases the sex was unknown. Considering the hind to stag
ratio reported for the Caso preserve (1.62), the expected number of cases was 28
stags and 45 hinds. Hence, this difference was statistically significant (Chi2 = 11.2,
1 d.f., p < 0.001). During the study period, from 1995 to 2007 (Fig. 6), mange
cases were not uniformly distributed throughout the year: 56.4% of total
described cases (55.3% stags, 54.1% hinds and 71.4% young animals) were
registered from January to April, with a maximum of 16 mangy animals in
January (10 stags, 3 hinds and 3 young animals) and a minimum of 1 single stag in
October (Fig. 4).
From 1999 to 2007 the number of scabietic young animals (two or less years
old) was 7 (4 males and 3 females) out of a total of 56 (39 males and 17 females).
Considering the age ratio reported for Caso (0.4 young per adult), the expected
proportion was 16 cases in young deer and 39 in adults. The difference was also
statistically significant (Chi2 = 4.45, 1 d.f., p < 0.05).
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
58 _____________________________________________________________________________
Fig. 5 shows the area affected by deer and chamois mange and the number of
cases recorded per year. It becomes evident that, while chamois mange showed a
marked epidemic wave from 1994 to 2001, deer mange showed a rather continuous
graph of 1–16 (mean 5) annual cases. Moreover, while the area of chamois mange
steadily increased, the area of deer mange remained rather constant.
Mange cases in deer appeared most often 1–3 years after the first chamois
mange cases (range 0–4 years; Fig. 6). In 50% of the affected sectors (5 out of
10) mange cases in deer have been reported at least in 4 different years, with a
maximum of 7 years with mangy cervids in one single sector (Fig. 6).
In the mange affected area, ELISA testing of 327 blood samples from
apparently healthy hunter-harvested deer without obvious mange-compatible lesions
revealed only 4 seropositive animals: two in game reserve number 10 (Caso), one in
number 17 (Sobrescobio) and one in number 13 (Ponga). All 83 sera from hunting
preserves without clinical cases yielded negative ELISA results. Of the 8 sera from
clinical mange cases, only 6 tested positive at the established cut-off value. Those
testing negative belonged to a stag and a hind in poor body condition. The
obtained average relative OD value ± standard deviation was: 0.76 ± 0.48 for mangy
animals, 0.01 ± 0.03 for apparently healthy animals from areas without mange, and
0.02 ± 0.06 for apparently healthy animals from areas exposed to sarcoptic mange.
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Figure 3- Detected mangy red deer found dead ( ), shot ( ) and observed with sarcoptic mange compatible lesions ( ) in Asturias during the study period (1995–2007). Dots (*) represent cases in preceding years. The shaded area represents the chamois sarcoptic mange distribution area.
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
60 _____________________________________________________________________________
DISCUSSION
Despite the limited number of cases (80) during a period of 13 years, this is to
our knowledge the highest concentration of mange cases ever reported in European
deer. The present report is the first epidemiological description of sarcoptic mange
in red deer from Spain, and one of the few reports with a high number of
affected deer in Europe.
Figure 4- Distribution of red deer sarcoptic mange cases by age, class and month (Asturias, 1995–2007).
One of the features signalled in the study is the high proportion of dead or
severely affected animals in comparison with those observed as slightly affected or
without clinical signs but confirmed as scabietic in laboratory by isolation of mites
or shown to have been in contact with mange using serological techniques; this
seems to differ from descriptions of other red deer populations affected by
scabies where a higher proportion of slightly affected red deer has been reported
(Leon Vizcaino et al., 1992).
The presence of 2 skin samples negative for mite isolation from the total of
15 taken for detection of S. scabiei confirms that some field reports of affected deer
may not be due to mange. Nevertheless, although the existence of false positives
(due to incorrect diagnostic in the field or lack of laboratorial confirmation) must be
taken into account, the number of mangy animals was probably underestimated
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because of the woody and rocky study area, as described in other scabies affected
wildlife populations (Rossi et al., 2007).
Figure 5- Number of sarcoptic mange cases in chamois and red deer and affected surface (in km2) in Asturias from 1995 to 2007.
Taking into account the references to sarcoptic mange cases reported in red
deer in Austria from 1969 to 1993 (Greßmann, 2001) and in Cazorla (Spain) in
1991 (Leon Vizcaino et al., 1992), this is the largest succession of cases
described in Europe (80 cases so far) and the second longest (with a 13 year
duration up to the present, 1995–2007). Our data confirm the annual trend observed
in other sarcoptic mange epidemics from European ungulates (Fernandez-Moran et
al., 1997; Gortazar et al., 1998; Gonzalez-Quiros et al., 2002; Rossi et al., 2007)
where the largest amount of affected animals was detected at the end of winter and
spring, is due to mite (that seems to prefer humidity and low temperatures
present in this season for satisfactorily completing its life-cycle, Sokolova et al.,
1989), host (whose body condition is usually worse at this time of the year, Loison
and Langvatn, 1998) and ecological characteristics. However, the evolution of illness
during the studied period differs from that observed in chamois. Instead of a long
cycle (about 15 years in Alpine chamois) with a peak in the number of affected
individuals 4–6 years after the beginning of the outbreak and a slow and
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
62 _____________________________________________________________________________
continuous decrease in morbidity and mortality during the following years
(Fernandez-Moran et al., 1997; Gonzalez-Quiros et al., 2002; Rossi et al., 2007), in
the case of red deer we observed sporadic cases for a long time.
The first case in deer always appeared between 0 and 4 years after the arrival of
sarcoptic mange to the local chamois population. Although this value is similar to
that reported for first cases in hosts different than chamois in a scabies epidemic
described in Italy (Rossi et al., 2007), a difference observed in Asturias is that
mangy red deer have been reported 12 years after the first described case.
The detection of the first mangy deer cases just 1 year after the beginning of
the chamois sarcoptic mange outbreak in one of the foci and 3 years after in the
other one, strongly suggests that the origin of red deer mange is chamois. This
hypothesis is supported by the fact that 95% of the affected deer were detected within
the limits of the scabietic chamois distribution area for any given year.
In contrast with the ‘‘oil spot’’ advancement of the disease signalled for other
wild ruminants (Rossi et al., 2007), sarcoptic mange in red deer usually appeared as a
disorganized succession of cases, isolated or in clusters of up to 6 animals per year
(Fig. 3).
No epidemiological explanation has been found for the spatial concentration of
mangy red deer cases reported around the Nalon River in the Caso (#10) game
reserve since 2002. A similar trend in the distribution of mangy red deer detected
was reported in Austria (Kohler, 1970; Greßmann, 2001) where most of the
carcasses and diseased animals appeared near a river or road, or even close to
feeding troughs used in this area during winter. This kind of place is also more
accessible and frequented by people, facilitating detection of animals or carcasses.
However, this pattern of spatial concentration has not been observed along other
roads, rivers or valleys in the sarcoptic mange affected area in Asturias. No
significant variation or difference with other surrounding localities has been
detected in this limited area regarding red deer or chamois densities, scabies
incidence on chamois population, habitat quality or even search effort.
The explanation for the restriction of mange cases in red deer to the signalled
game reserves is unknown. With the advancement of the epidemic in chamois new
sympatric red deer populations have been exposed to S. scabiei not only in Asturias,
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but also in Leon and Cantabria in recent years (Fig. 1), without the detection of new
cases. Density (Rossi et al., 1995; Guberti and Zamboni, 2000), population health
level (Gortazar et al., 1998; Leon Vizcaino et al., 1999), and genetic-based
variations in mite virulence and host resistance to mange (Pence and Windberg, 1994;
Berrilli et al., 2002) have been suggested as possible factors determining
appearance and evolution of sarcoptic mange.
Figure 6- Temporal relationship between the first detection of chamois mange and first detection of scabietic red deer. Gray cells signal presence of sarcoptic mange in chamois. White numbers inside black cells indicate the number of mangy red deer detected for that year in a census sector.
The fact that the affected reserves are of high deer density may indicate the
existence of a relationship between red deer population density, the risk of
sarcoptic mange appearance and the morbidity rate in red deer (suggesting
overabundance, e.g. Gortazar et al., 2006). However, density-reactive indicators
of fitness such as KFI, parasitation levels, trophy and body measurements, or
fertility rates estimated by 138 complete red deer necropsies carried out by the
authors in the affected and unaffected areas in Asturias show no significant
difference, suggesting no relation of these factors with deer mange (data not
shown).
A difference with mange outbreaks in other European ungulates (Fernandez-
Moran et al., 1997; Leon Vizcaino et al., 1999; Greßmann, 2001; Rossi et al.,
2007) was that males showed a higher morbidity than females. Physiological
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
64 _____________________________________________________________________________
characteristics (i.e. the immunocompetence handicap hypothesis) and differences
in behavior between sexes (different roles in activities related to sexual selection,
different exposure to parasites due to feeding habits and habitat use) may explain
this sex related bias (Vicente et al., 2007).
The low proportion of mangy juveniles agrees with previous reports. However
difficulty of observation, higher mortality rates and easier disappearance of the
carcasses must also be taken into account (Fernandez- Moran et al., 1997).
Although the existence of temporal and spatial coincidence between chamois
and deer sarcoptic mange emergence strongly suggests that the origin of red deer
mange is chamois mange, prevalence curves show an independent evolution and
maintenance of disease in both species.
The red deer has been signalled by different authors (Leon Vizcaino et al.,
1992; Rossi et al., 2007) as a possible ‘‘supplementary host’’ for S. scabiei. Even
though this mite shows host specificity, the presence of high densities of red deer in
the habitat of its most important host (namely caprines) in Spain may have made
these parasite strains able to infect this species when chamois densities are low,
potentially improving mite strain survival. This hypothetical ‘‘adaptation’’ to deer is
consistent with the detection of 2 roe deer (a very unusual host of S. scabiei) severely
affected by mange in the same area (Oleaga et al., 2008).
The presence of two confirmed mangy animals with seronegative results in the
ELISA test highlights the probable existence of false negative animals in the
serosurvey. The existence of diseased seronegative individuals is a common
problem in surveillance and control programs of debilitating illnesses, due usually to
the existence of immunocompromised or anergic animals (Pence and Ueckerman,
2002; Fernandez-de- Mera et al., 2006).
All four positive sera of the ELISA testing of 410 blood samples from
apparently healthy hunter-harvested deer belonged to the area where mangy red
deer had already been described, confirming the absence of deer mange outside the
chamois mange range. However, further testing is advisable to continue monitoring
the disease.
Only a small number of seropositive animals were found among randomly shot
deer. This suggests (a) that most deer infected with S. scabiei do develop clinical
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disease, (b) that most mangy red deer die as a consequence of the disease, or
alternatively quickly lose antibodies after overcoming sarcoptic mange, and (c) that
the ELISA test may be unfit to reveal the low mite loads of ‘‘healthy carrier’’ deer.
However, we found no animals in apparent clinical recovery. Our findings
emphasize the need of epidemiological studies and experimental infections to better
understand sarcoptic mange epidemiology in red deer.
CONCLUSIONS
Both serology and recorded clinical cases suggest low morbidity and high
mortality due to sarcoptic mange in studied deer populations. Attending to these
epidemiological characteristics scabies does not seem to threaten red deer
populations in Asturias. However, available data do not allow to predict if scabies
will become extinct or progress, or if it could turn into an endemic problem.
Data recorded in this paper indicate no disease emergence. Thus,
recommending special measures does not appear necessary. Nevertheless,
monitoring of deer sanitary condition and ELISA testing for sarcoptic mange are
advisable.
ACKNOWLEDGEMENTS
This is a contribution to the agreement between CSIC and Principado de
Asturias and MEC research grant AGL2005-07401. We thank the rangers of the
game preserves, technicians working for the Asturias Government and our
colleagues from SERIDA and IREC for their assistance in field and laboratory work.
The authors wish to thank Kevin Dalton for revising the English and Pelayo Acevedo
for his contribution in geographical analysis and maps design. Special thanks to
Patricia A lvarez for her patience.
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66 _____________________________________________________________________________
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CAPÍTULO 2
PREVALENCIA DE ANTICUERPOS FRENTE A DIFERENTES AGENTES
PATÓGENOS COMPARTIDOS ENTRE REBECO CANTÁBRICO Y
CABRA DOMÉSTICA
Falconi, C.*, Oleaga, A.*, López-Olvera, J. R., Casais, R., Prieto,
M., Gortázar, C. (2010) PREVALENCE OF ANTIBODIES AGAINST SELECTED
AGENTS SHARED BETWEEN CANTABRIAN CHAMOIS (RUPICAPRA PYRENAICA
PARVA) AND DOMESTIC GOATS. European Journal of Wildlife Research
56, 319-325.
* Contributed equally
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RESUMEN
El rebeco cantábrico (Rupicapra pyrenaica parva) comparte su hábitat con ungulados
domésticos, y puede por tanto tener relevancia en la epidemiología de agentes patógenos
compartidos. El objetivo de este estudio fue determinar la seroprevalencia frente a Brucella
spp., Mycobacterium avium ssp. paratuberculosis (MAP), pestivirus, y Sarcoptes scabiei en el
rebeco cantábrico (Rupicapra pyrenaica parva) y comparar estos datos con los de cabras
domésticas (Capra hircus) simpátricas. Entre 2005 y 2008 se recogieron muestras de sangre
de 236 rebecos cantábricos adultos en 2 poblaciones diferentes, Este y Oeste de su área de
distribución. La seroprevalencia frente a Brucella spp. y pestivirus se evaluó utilizando kits
ELISA comerciales, mientras que frente a MAP y S. scabiei se emplearon test ELISA
específicamente diseñados. No se detectaron anticuerpos frente a Brucella spp. En cambio sí
se detectaron anticuerpos frente a MAP, pestivirus (rebeco 3.8%; cabra 2.3%), y S. scabiei
(rebeco 11.9%; cabra 12.8%) en ambas especies. La seroprevalencia frente a MAP fue
significativamente mayor en cabra doméstica (26%) que en rebeco (9,7%). En el rebeco
cantábrico, la seroprevalencia frente a pestivirus fue mayor en la población Occidental
(6,5%) que en la Oriental (rango 0-1,8%), mientras que la seroprevalencia frente a S. scabiei
mostró una distribución opuesta (Oeste 4.6%; Este 16.7–21.4%). Sugerimos que ciertas
enfermedades pueden circular entre las poblaciones de rebeco y cabra doméstica, y que el
ganado puede representar una amenaza para el estado sanitario del rebeco cantábrico
simpátrico.
ABSTRACT
Southern chamois (Rupicapra pyrenaica parva) share the habitat with domestic
ungulates, and may, therefore, play a role in the epidemiology of shared agents. The
objective of this study was to determine the seroprevalence for Brucella spp.,
Mycobacterium avium ssp. paratuberculosis (MAP), pestivirus, and Sarcoptes scabiei in
Cantabrian chamois (Rupicapra pyrenaica parva) and compare these data with those of
sympatric domestic goats (Capra hircus). From 2005 to 2008, blood samples were obtained
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
74 _____________________________________________________________________________
from 236 adult Cantabrian chamois in two different populations, the western one and the
eastern one. Seroprevalence for Brucella spp. and pestivirus was assessed using commercial
ELISA kits, whereas specifically designed ELISA tests were used for MAP and S. scabiei.
No antibodies against Brucella spp. were detected. Conversely, antibodies against MAP,
pestivirus (chamois 3.8%; goat 2.3%), and S. scabiei (chamois 11.9%; goat 12.8%) were
detected in both species. Seroprevalence for MAP was significantly higher for domestic goats
(26%) than for chamois (9.7%). In chamois, seroprevalence for pestivirus was higher in the
west (6.5%) than in the east (range 0–1.8%), whereas seroprevalence for S. scabiei followed
the opposite trend (west 4.6%; east 16.7–21.4%). We suggest that certain diseases could
circulate between Cantabrian chamois and domestic goat populations, and domestic
livestock may suppose a threat for the health status of sympatric Cantabrian chamois.
INTRODUCTION
Southern chamois (Rupicapra pyrenaica) is a wild mountain ungulate
belonging to the Bovidae family and Caprinae subfamily. Two subspecies can be
found in Spain: the Pyrenean chamois (Rupicapra pyrenaica pyrenaica) in the
Pyrenees and the Cantabrian chamois (Rupicapra pyrenaica parva), endemic
from the Cantabrian Mountains and occupying the western limit of the Rupicapra
genus distribution area (Fig. 1). A western and an eastern population of Cantabrian
chamois, physically separated by anthropogenic barriers, have been defined
(Catusse et al. 1996; Shackleton and the IUCN/SSC Caprinae Specialist Group
1997; Pérez-Barbería and García-González 2004). For the purpose of this
study, the eastern population is shared by two different political regions: Asturias
and León.
In 1993, the eastern chamois populations were affected by sarcoptic mange,
the disease becoming endemic since then (Fernández-Morán et al. 1997).
Sarcoptic mange is considered absent in the western population, without clinical
cases of mange detected up to date in this area, where Cantabrian chamois
population has continuously increased throughout the last years, reaching a density
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of 14.35 Cantabrian chamois per square kilometer in 2007. This compares to the
6.50 chamois per square kilometer registered for the mange-affected eastern
population in 2007 (Anonymous 2008a, 2008b). Domestic livestock in Asturias and
León includes approximately 80,000 domestic goats (Capra hircus; Anonymous
2008c), some of which share habitat in summer with more than 13,500 Cantabrian
chamois (ca. 3,400 in the western nucleus, ca. 5,300 in the eastern Asturias one,
and ca. 4,800 in the eastern León nucleus) (Pérez-Barbería and García-
González 2004; Anonymous 2008a, 2008b).
This commingling may play a significant role in the epidemiology of several
infectious agents since cross-transmission may occur as livestock graze in
mountain pastures (Gauthier et al. 1991; Cubero-Pablo et al. 2000; Gortázar et al.
2007). Transmission of agents has been suggested in both directions, with
wildlife acting as reservoirs of infectious agents which affect domestic animals
and livestock being a source of infection for wild mountain ruminants (Bengis et al.
2002; Simpson 2002; Gaffuri et al. 2006).
Figure 1- Chamois distribution in Europe and study areas in north– western Spain. Adapted and modified from Catusse et al. 1996 and Fernández-Morán et al. 1997. Gray northern chamois (Rupicapra rupicapra) populations, Black southern chamois (Rupicapra pyrena- ica) populations, 1 western Cantabrian chamois (Rupicapra pyrenaica parva) population, 2a eastern Cantabrian chamois (Rupicapra pyrenaica parva) population in Asturias, 2b eastern Cantabrian chamois (Rupicapra pyrenaica parva) population in León.
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Antibodies against several infectious agents of ruminants have been
detected in chamois (Rupicapra spp.). Antibodies against viral pathogens include
bovine herpesvirus 1, encephalomyocarditis virus (Gentile et al. 2000), bovine
parainfluenza type 3 virus, bovine respiratory syncytial virus (Gaffuri et al. 2006),
and pestivirus (Gentile et al. 2000; Arnal et al. 2004; Hurtado et al. 2004;
Gaffuri et al. 2006; Marco et al. 2007; Pioz et al. 2007; Marco et al. 2008).
Regarding bacterial agents, antibodies have been detected against Brucella
melitensis (Garin-Bastuji et al. 1990), Mycoplasma conjunctivae (Giacometti et al.
2002; Gaffuri et al. 2006), Salmonella enterica, Coxiella burnetii (Pioz et al. 2008),
Leptospira interrogans (Gentile et al. 2000), and Chlamydophila abortus (Pioz et
al. 2008; Salinas et al. 2009). Finally, antibodies against two protozoa, Neospora
caninum and Toxoplama gondii, have also been reported (Gentile et al. 2000;
Gaffuri et al. 2006).
In spite of such information regarding other species or subspecies of
chamois, only limited information on the seroprevalence of infectious agents in
Cantabrian chamois is available (Fernández-Morán et al. 1997; Gauss et al.
2006; Almería et al. 2007). The objective of this study was determining the
seroprevalence for Brucella spp., Mycobacterium avium ssp. paratuberculosis
(MAP), pestivirus, and Sarcoptes scabiei in the Cantabrian chamois. This was
compared with data on sera from sympatric domestic goats sharing summer
pastures with the chamois, in order to detect potentially shared diseases between
both species.
MATERIALS AND METHODS
From 2005 to 2008, blood samples were obtained from 236 adult Cantabrian
chamois, either hunted (N=213) or life captured with drive-nets (N = 23), an
already reported method to capture chamois (López-Olvera et al. 2009a). Two
different chamois populations were defined according to geographical criteria, the
eastern one and the western one, the latter covering an area shared by two
different public administrations, León and Asturias (Fig. 1). The sampled area
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represents more than 75% of the current distribution area of Cantabrian chamois.
Since hunting programs are aimed at maintaining population structure balance,
animals from both sexes and all ages were included in our sample, thus being
representative of the studied population.
Blood samples were collected by jugular venipuncture in the live chamois and
by heart puncture in the hunted ones. Blood was allowed to clot at room
temperature and centrifuged less than 24 h after collection. Sera were also
obtained from 219 domestic goats through the Official Health Testing Programme of
the Spanish government for Brucella abortus. All sera were stored at −20°C
until analyzed.
A commercial competitive enzyme-linked assay (ELISA; INGEZIM BRUCELLA
BOVINE 1.2.BB.K.1, INGENASA, Madrid, Spain) was used for the detection of
antibodies against Brucella spp. according to the manufacturer's recommendations.
This ELISA detects antibodies against the protein–polysaccharide complex LPS-S
of Brucella, and it has been previously used in mouflon (Ovis aries musimon; López-
Olvera et al. 2009b).
An ELISA BVD/Mucosal Disease p80 kit (Institut Pourquier, Montpellier,
France) was used to search for pestivirus antibodies, according to the procedure
described by the manufacturer, as previously described in Pyrenean chamois (Pioz
et al. 2007). The test detects antibodies directed against protein p80/125, common
to all pestivirus strains. Samples with a percentage of inhibition equivalent or less
than 40% were considered positive.
Antibodies to MAP were detected using a specifically designed adsorbed
ELISA test following protocols reported previously for cattle and sheep (Garrido
2001; Sevilla 2007), as previously used and reported for red deer (Cervus elaphus)
(Reyes-García et al. 2008). This test detects antibodies against MAP
protoplasmatic antigen 3. The cutoff was set as the mean optical density value of
the negative controls added to three times the standard deviation of all negative
control.
Antibodies against S. scabiei were determined using an “in house” ELISA test
based in the use of a recombinant antigen. This test detects the Ssλ20 antigen of
the parasite, located in the mite's organs, the integument of the epidermis, and
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
78 _____________________________________________________________________________
the spaces surrounding its vital organs (Casais et al. 2007). This test was
characterized by 100% sensitivity and 97% specificity in both red deer and
southern chamois (Casais et al. 2007), although field trials of the test in red deer
revealed a slight decrease of specificity (97%) but a stronger decrease of
sensitivity, which was 75% (Oleaga et al. 2008).
A chi-square analysis was performed for all agents, using the PROC FREQ
procedure of SAS® System for Windows V8 (SAS Institute Inc., Cary, NC, USA), to
detect seroprevalence differences between species, among areas for both species,
and among years for southern chamois.
RESULTS
Table 1 shows the serologic results for both Cantabrian chamois and
domestic goat in the different study areas. No antibodies were detected against
Brucella spp. either in chamois or goat. Conversely, antibodies against MAP,
pestivirus, and S. scabiei were detected in both species.
DISCUSSION
The absence of antibodies against Brucella spp. in all the animals examined
has been previously reported in the Cantabrian chamois (Fernández-Morán et al.
1997). Our results seem to further confirm that Brucella spp. is apparently not
circulating within the wild chamois population and the domestic goat flocks in the
study area.
Wild ruminants may become MAP-infected by contact with infected domestic
livestock (Riemann et al. 1979; Marco et al. 2002; Deutz et al. 2005; Kopecna et al.
2006), but transmission from wildlife to domestic livestock has also been
suspected (Chiodini and Van Kruiningen 1983; Greig et al. 2003). Absence of
antibodies against MAP had been previously reported in Cantabrian (González-
Quirós et al. 1996; Fernández-Morán et al. 1997) and Abruzzo (Rupicapra
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Mycobacterium avium ssp.
paratuberculosis Pestivirus Sarcoptes scabiei
Sera
analyzed Positive Percentage
Confidence
interval (95%) Positive Percentage
Confidence
interval (95%) Positive Percentage
Confidence
interval (95%)
Chamois Total 236 23 9.7%* 6.0-13.5 9 3.8% 1.4-6.3 28 11.9% 7.7-16.0
Goat Total 219 57 26.0%* 20.2-31.9 5 2.3% 0.3-4.3 28 12.8% 8.4-17.2
Chamois West 108 8 7.4%* 2.5-12.4 7 6.5%a* 1.8.11.2 5 4.6%a 0.7-8.6
East-
Asturias 60 8 13.3% 4.7-22.0 0 0.0%b 10 16.7%b 7.2-26.2
East-
León 56 4 7.1%* 0.4-13.8 1 1.8%b 0-5.3 12 21.4%b 10.7-32.1
Unknown 12 3 25.0% 0-50.6 1 8.3% 0-24.7 1 8.3% 0-24.7
Goat West 68 29 42.6%a* 30.8-54.5 0 0.0%* 8 11.8%ab 4.0-19.5
East-
Asturias 86 11 12.8%b 5.6-20.2 2 2.3% 0-5.5 7 8.1%a 2.2-14.0
East-
León 65 17 26.2%c* 15.6-36.7 3 4.6% 0-9.7 13 20.0%b 10.5-29.5
Table 1- Serologic prevalence of selected infectious diseases in Cantabrian chamois (Rupicapra pyrenaica parva) and domestic goats (Capra hircus) from north–western Spain. Means with different letters are statistically (p <0.05) different among areas for the corresponding species. * p<0.05 (means are statistically different between species for the corresponding area).
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
80 _____________________________________________________________________________
pyrenaica ornata) chamois (Gentile et al. 2000). However, paratuberculosis (or
specific antibodies) has been recently reported in the fallow (Dama dama)
(Marco et al. 2002; Balseiro et al. 2008) and red deer (Reyes-García et al.
2008) in the Cantabrian Mountains. Our results indicate that paratuberculosis is
present in the study area both in Cantabrian chamois and domestic goats, livestock
probably being a source of MAP-infection for Cantabrian chamois due to their
higher seroprevalence of antibodies against MAP. Nevertheless, these results
should be considered with caution, since ELISA has demonstrated to have a low
sensitivity to detect MAP-infected domestic and wild ruminants, with culture and
polymerase chain reaction (PCR) MAP-positive animals showing negative ELISA
results (Weber et al. 1992; Nebbia et al. 2000; Juste et al. 2005). Strain
characterization of MAP isolates from both Cantabrian chamois and domestic
goats, as well as other domestic species such as cattle (Balseiro et al. 2003), could
help to elucidate the epidemiology of this pathogen in the study area.
Year Positive/analyzed Percentage
2005 0/7 0.0%a
2006 1/61 1.6%a
2007 3/153 2.0%a
2008 5/15 33.3%b
Table 2- Pestivirus seroprevalence in Cantabrian chamois (Rupicapra pyrenaica parva) from north–western Spain according to the year. Means with different letters statistically (P < 0.05) different from each other.
To our knowledge, this is the first study to provide data on antibodies against
pestivirus for Cantabrian chamois. A mortality outbreak affecting Pyrenean chamois,
associated to a newly described pestivirus, has been reported in the western
Pyrenees, seroprevalences ranging from 62.8% to 70.3% (Hurtado et al. 2004;
Frolich et al. 2005; Marco et al. 2007; Pioz et al. 2007; Marco et al. 2008). Unlike
the Pyrenees outbreak, no mortality or clinical signs potentially related to pestivirus
infection have been observed in our study area. Lower seroprevalences against
pestivirus (from 5.6% to 25.5%) without associated virus isolation, clinical signs, or
mortality have been previously reported in Pyrenean (Arnal et al. 2004), Abruzzo
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(Gentile et al. 2000), and Alpine chamois (Rupicapra rupicapra) (Baradel et al. 1988;
Olde Riekerink et al. 2005; Gaffuri et al. 2006). The relatively low seroprevalence
for pestivirus and the absence of mortality and clinical signs found in the present
study are similar to this epidemiological situation. Domestic animals (e.g., sheep)
have been considered the origin of pestivirus inducing low seroprevalence in
healthy chamois populations (Olde Riekerink et al. 2005; Gaffuri et al. 2006; Marco et
al. 2009), due to higher prevalence and relatively frequent detection of persistently
infected farm animals (Vilcek and Nettleton 2006), although domestic ungulates can
also be infected from wild ungulates through indirect contact (Uttenthal et al. 2005).
New high pathogenic virus strains could cause a mortality outbreak, as it has
happened in the Pyrenees (Hurtado et al. 2004; Vilcek and Nettleton 2006; Marco et
al. 2007), and pestivirus strains circulating in the wildlife–domestic livestock
interface should be determined to evaluate the potential risk. However, the
phylogenetic grouping of a highly pathogenic pestivirus isolated of Pyrenean
chamois from France suggests that cross-specific transmission of this isolate
from domestic sheep to chamois via shared pastures is unlikely (Frolich et al. 2005).
Statistically, higher pestivirus seroprevalence of Cantabrian chamois as
compared to goats in the western area suggests that the virus could circulate in
the chamois population alone, or that other domestic ungulates (like sheep or
cattle) are participating in the epidemiology of pestivirus. The increase along time
of pestivirus seroprevalence in the chamois population, together with the higher
seroprevalence against pestivirus found in the western population, agree with the
increase in Cantabrian chamois population density in this area, which could ease
transmission and circulation of the virus. However, since the ELISA used is
unspecific and cross-reactions between different pestivirus exist, virus
neutralization tests should be used to determine pestivirus strains circulating in
domestic ruminants and Cantabrian chamois in the study area, in order to evaluate
the potential risk of disease and mortality for both wildlife and livestock.
Sarcoptic mange, caused by the mite S. scabiei, affects both livestock and
wildlife (Menzano et al. 2007; Morner 1992). A sarcoptic mange epizootic affecting
the eastern population of Cantabrian chamois was first detected in 1993 and has
since then become endemic in this area, without detected clinical cases of
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
82 _____________________________________________________________________________
sarcoptic mange in the western chamois population up to date (González-Quirós et
al. 1996; Fernández-Morán et al. 1997). The statistically significant higher
seroprevalence of antibodies against S. scabiei in the eastern population (both in
Asturias and León) when compared with the western population agrees with this
epidemiological situation. Chamois can be infected with S. scabiei mites from
domestic goats and develop disease (Lavín et al. 2000), and domestic ruminants are
the most probable origin of the sarcoptic mange epidemic which affected
Cantabrian chamois in 1993 (Fernández-Morán et al. 1997), as exposed in other
outbreaks of sarcoptic mange in wild Bovidae species (Vyrypaev 1985; León-
Vizcaino et al. 1999; González- Candela et al. 2004; Rossi et al. 2007). Conversely
to Spanish ibex, anti-mange antibodies have a certain protective effect in southern
chamois (Lastras et al. 2000), and therefore, the six seropositive Cantabrian
chamois found in the western population are probably related to contact with mites
from mange-infected domestic goats. The western population can still be
considered mange-free, since S. scabiei causes high morbidity in chamois, and
clinical cases seem to be required to confirm that a chamois population is affected
by mange (Rossi et al. 1995; Fernández-Morán et al. 1997; Rossi et al. 2007).
Statistically, significant higher mange seroprevalence of the goats from León
compared to Asturias within the eastern population is probably related to
differences in the sanitary management of the two administrations involved (Pérez-
Barbería and García-González 2004). However, no similar differences in chamois
seroprevalence were found among areas. This could indicate that sarcoptic mange
has become endemic in the eastern chamois population (both in Asturias and León)
and is self-maintained independently from domestic livestock. The presence of
mange antibodies in the Western population of Cantabrian chamois and the almost
statistically significant (P=0.07) higher seroprevalence of goats in this area suggest
that domestic goats are a threat of a new mange epidemic for the currently unaffected
western chamois population. Measures to control mange, including treating
domestic livestock and monitoring animal movements between affected and
unaffected zones, together with effective measures to reduce Cantabrian chamois
population, especially in the high-density Western area, should be undertaken in
________________________________________________________________________ Capítulo 2
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order to prevent the spread of this disease and the emergence of new mange
epizootics.
To summarize, this is the first study to provide systematic data on
seroprevalence of several infectious diseases in the Cantabrian chamois
population. MAP, pestivirus, and sarcoptic mange are present in the population of
Cantabrian chamois and domestic goats in the Cantabrian Mountains. Although
higher MAP seroprevalence of goats seems to suggest that they could be
maintaining this disease, a long-time MAP surveillance including strain
characterization in wildlife and domestic livestock is needed in order to better
understand the role of Cantabrian chamois and the sympatric domestic population in
the epidemiology of paratuberculosis. Presence of antibodies against pestivirus has
been reported for the first time in Cantabrian chamois. Surveillance of pestivirus and
strain determination in domestic ungulates and Cantabrian chamois is crucial for the
early detection of new or known pestivirus which could cause a mortality outbreak.
Domestic goats could be a risk regarding sarcoptic mange in the currently chamois
mange-free western range. Control measures should be taken to prevent new
epizootics in the chamois population, such as the previously reported ones in the
Cantabrian Mountains and the Pyrenees, caused by mange and pestivirus,
respectively. By contrast, the absence of relevant disease such as Brucella in
chamois and the lower seroprevalence of MAP suggest that chamois do not carry
sanitary risks for domestic goats in the study area.
ACKNOWLEDGMENTS
R. Casais is recipient of a “Ramón y Cajal” contract from the Spanish
Ministerio de Ciencia e Innovación (MCINN) cofinanced by the European Social
Fund. Research involving development of an ELISA for the diagnosis of Sarcoptes
scabiei has been supported by Grant RTA2006-00046 from the Spanish Instituto
Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA). This is a
contribution to grant AGL2008-03875 on paratuberculosis in Spanish wildlife,
awarded by the Spanish (MCINN). Special thanks to rangers, technicians, and
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
84 _____________________________________________________________________________
administration from Principado de Asturias and Regional Hunting Reserves of León.
Wildlife disease surveillance in Asturias is supported by Principado de Asturias and the
Spanish Consejo Superior de Investigaciones Científicas (CSIC).
C. Falconi acknowledges a grant from the Government of Sardegna.
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CAPÍTULO 3
*
SARNA SARCÓPTICA EN CÁNIDOS SILVESTRES DEL PRINCIPADO
DE ASTURIAS
3.2.- CONCOMITANCIA E INTERACCIONES ENTRE PATÓGENOS EN
EL LOBO IBÉRICO (Canis lupus)
3.1.- NUEVAS TÉCNICAS PARA ESTUDIAR UNA VIEJA
ENFERMEDAD: SARNA SARCÓPTICA EN EL LOBO IBÉRICO
€
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Oleaga, A., Casais, R., Balseiro, A., Espí, A., Llaneza, L.,
Hartasánchez, A., Gortázar, C. (2011) NEW TECHNIQUES FOR AN OLD
DISEASE: SARCOPTIC MANGE IN THE IBERIAN WOLF. Veterinary Parasitology 181, 255-266.
3.1.- NUEVAS TÉCNICAS PARA ESTUDIAR UNA VIEJA
ENFERMEDAD: SARNA SARCÓPTICA EN EL LOBO IBÉRICO
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RESUMEN
La sarna sarcóptica, enfermedad parasitaria provocada por el ácaro excavador
Sarcoptes scabiei, ha sido confirmada en más de 100 especies de mamíferos incluyendo al
ser humano. En especies amenazadas la sarna tiene relevancia a nivel de conservación
debido a que puede diezmar poblaciones aisladas contribuyendo incluso a su desaparición.
La Península ibérica alberga una de las mayores poblaciones de lobo (Canis lupus) de toda
Europa. En España, la sarna sarcóptica es endémica en zorro (Vulpes vulpes) y los primeros
casos de sarna sarcóptica en lobo fueron recientemente descritos. No obstante, nuestro
conocimiento sobre S. scabiei en lobos es escaso por las dificultades de muestreo y estudio
inherentes a especies poco abundantes y crípticas como el lobo. Con el ánimo de conocer la
epidemiología de la sarna sarcóptica en lobo e inferir su posible efecto a nivel de
conservación, este trabajo combinó técnicas tradicionales de laboratorio con la revisión de
fotografías de lobos muertos tomadas por biólogos de campo así como con información
original obtenida en campo mediante la técnica de fototrampeo. Un total de 125 necropsias
y un esfuerzo de fototrampeo de 8783 días-cámara trampa permitieron profundizar en la
epidemiología de la sarna en lobo entre 2003 y 2010. Ácaros Sarcoptes vivos fueron
detectados en un 19% de los lobos remitidos en fresco. Se observó a nivel dérmico una
respuesta inmune de tipo alopécico (reacción de hipersensibilidad Tipo IV o retardada),
mientras que las lesiones de tipo paraqueratótico fueron poco frecuentes. El número de
ácaros aislados por lobo varió entre 1 y 78, y mostró una correlación negativa con el
porcentaje de piel lesionada. No se detectó efecto alguno del sexo en la prevalencia de
sarna. Los jóvenes del año mostraron una menor probabilidad de presentar lesiones
compatibles con sarna que cachorros o adultos. Los animales con lesiones compatibles con
sarna tuvieron un menor índice de engrasamiento renal que los aparentemente sanos. El
test ELISA efectuado sobre 88 sueros reveló una seroprevalencia del 20%. La técnica del
fototrampeo registró la presencia de lobos con lesiones compatibles con sarna desde 2003,
con un pico aparente de morbilidad en 2008. El porcentaje de lobos registrados por las
cámaras trampa con lesiones compatibles con sarna durante un año mostró correlación con
el porcentaje de zorros fotografiados con lesiones el año anterior. Este es el primer estudio
amplio centrado en sarna sarcóptica en lobo ibérico. Tanto los datos de necropsia, con
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alopecia como rasgo principal y un efecto limitado sobre condición corporal, como las
tendencias registradas en campo mediante fototrampeo, coinciden en mostrar una
prevalencia relativamente baja y una situación aparentemente estable de la enfermedad en
este hospedador, sugiriendo que este parásito no representa actualmente una amenaza
importante para la población de lobo estudiada. En cualquier caso, es precisa más
información para evaluar el efecto de la sarna en determinados aspectos como la tasa de
supervivencia de cachorros.
ABSTRACT
Sarcoptic mange, a parasitic skin infection caused by the burrowing mite Sarcoptes
scabiei, has been reported in over 100 mammals, including humans. In endangered species,
mange causes conservation concerns because it may decimate isolated populations and
contribute to extinction. The Iberian Peninsula still maintains one of the largest wolf (Canis
lupus) populations in Europe. In Iberia, sarcoptic mange is endemic in red foxes (Vulpes
vulpes) and the first confirmed wolf mange cases were recently reported. However,
knowledge on S. scabiei in wolves is scarce because of the sampling difficulties inherent to
research on scarce species. In order to describe wolf mange epidemiology and to infer
conservation implications, this study combined traditional laboratory techniques with the
revision of wolf carcass pictures taken by field biologists and original information obtained
by camera trapping. A total of 125 necropsies and 8783 camera-trap days allowed insights
into wolf mange epidemiology between 2003 and 2010. Living Sarcoptes mites were
detected in 19% of the fresh carcasses. Alopecic (delayed) type IV hypersensitive response
reactions were observed, while parakeratotic lesions were infrequent. The number of mites
isolated per wolf ranged from 1 to 78, and had a negative correlation with the percentage of
alopecic skin. No effect by sex on mange prevalence was found. Yearlings showed a lower
probability to present mange-compatible lesions than pups or adults. Wolves with mange-
compatible lesions had a lower kidney fat index than apparently healthy ones. ELISA testing
of 88 sera yielded an antibody prevalence of 20%. Photo-trapping recorded mange-
compatible lesions since 2003 with a peak in 2008. The percentage of wolves with mange-
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compatible lesions registered in camera-traps during 1 year correlated with the percentage
of red foxes with lesions in the previous year. This is the first large survey on sarcoptic
mange in the Iberian wolf. Necropsy data, with alopecia as the main feature and a slight
effect on body condition, and trends derived from camera trapping coincided in showing a
rather low prevalence and an apparently stable situation of the disease and its host,
suggesting that this parasite is currently not a major threat for this wolf population.
However, more information is needed in order to assess the effect of mange on aspects such
as pup survival.
INTRODUCTION
Sarcoptic mange is a parasitic skin infection caused by the burrowing
mite Sarcoptes scabiei, whose antigenic material deposited in tunnels dug in
the epidermis causes intense irritation and an inflammatory response producing
characteristic skin lesions. Such lesions are distinguished by dermal
inflammation, alopecia, hyperkeratosis and a characteristic thickened, wrinkled
and slate grey-coloured skin, leading in some cases to dehydration, emaciation
and eventually death of the animal (Mörner and Christensson, 1984).
The disease has been reported for more than 100 mammal species,
including humans and domestic and wild mammals (Bornstein et al., 2001). It
has been proposed that the mite S. scabiei is a single highly variable species
with different strains manifesting physiological specificity in different hosts
(Pence et al., 1975; Arlian et al., 1989, 1996a), with this specificity as a
subject of ongoing debate (Alasaad et al., 2011). Molecular studies carried out
in recent years support this hypothesis (Walton et al., 2004; Rasero et al.,
2010).
Apart from the importance as a zoonotic disease and its economical
relevance in domestic animals, sarcoptic mange may be an important
problem for isolated or small size populations (Henriksen et al., 1993;
Martin et al., 1998), and reaches epizootic proportions in certain wildlife
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populations (Fernández-Moran et al., 1997; Leon Vizcaino et al., 1999; Pence
and Ueckerman, 2002; González-Candela et al., 2004).
The family Canidae (Order Carnivora) includes at least 12 different
species from all over the world described as affected by sarcoptic mange
(Bornstein et al., 2001; Deem et al., 2002). The epidemiology of the process
seems to vary between different areas and host populations, and both
endemic and epidemic situations have been reported in the scientific
literature concerning sarcoptic mange and wild canids (Todd et al., 1981;
Pence et al., 1983; Mörner, 1992; Pence and Windberg, 1994; Gortázar et
al., 1998; Bates, 2003; Davidson et al., 2008). This parasitic disease has
been widely described in American coyotes – Canis latrans – and red foxes
– Vulpes vulpes (Todd et al., 1981; Gosselink et al., 2007) and in European
red foxes (Lindström et al., 1994). In the coyote and wolf (Canis lupus),
mortality due to this parasite has usually been considered a compensatory
cause of death, usually related to lack of hair and cold temperatures (Pence
et al., 1983; Pence and Windberg, 1994). However, knowledge focussed on S.
scabiei in grey wolves is scarce (Todd et al., 1981; Jimenez et al., 2010),
with most references mentioning a small number of individuals presenting
dermal lesions, often without detailed clinical descriptions of the process
(Wobeser, 1992; Je drzejewska et al., 1996; Shelley and Gehring, 2002;
Lovari et al., 2007). Recently, Domínguez et al. (2008) reported the first
confirmed mange cases in Iberian wolves.
Sarcoptic mange can present two different pathological forms: the
parakeratotic form, consistent with a type I (immediate) hypersensitive
response; and the alopecic form, consistent with a type IV (delayed)
hypersensitive response (Skerratt, 2003). Both forms of sarcoptic mange have
been recorded in red foxes in England (Bates, 2003). Clinical manifestations of
sarcoptic mange vary depending on individual variations in the duration and
intensity of the hypersensitivity reaction and on the capacity of each individual
host to limit parasite multiplication (Yager and Scott, 1993).
The grey wolf distribution area includes Europe, Asia and North America,
with many populations fragmented and reduced as a result of human
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persecution and habitat alteration (Boitani, 2003; Fernández and Ruiz de Azua,
2009). The largest population in Western Europe survives in the Iberian
Peninsula, where after the recovery experienced since 1970 the total wolf
population was estimated in more than 2000 wolves in 2008, with 322 packs
occupying a continuous area of 120,000 km2 in the northwest and a small
isolated population in the south of Spain (Álvares et al., 2005; Blanco et al.,
2008).
The confirmation of sarcoptic mange by mite isolation carried out by
authors in 6 wolves submitted for necropsy in 2008 as part of the wildlife disease
surveillance program of Asturias (North Spain) suggested the need of a specific
study to evaluate the importance of a possible threat of the disease for wolf
population. Serological, histopathological and necropsy data were
complemented with the retrospective revision of wolf carcass pictures and
epidemiological information obtained by camera trapping.
The objective in this study is to present the collected information
concerning sarcoptic mange and wolves in Asturias, analyse data reported in
epidemiological surveillance programs carried out since 2003, evaluate the
usefulness of different study methods and discuss the possible causes and
management or conservation implications of mange in Iberian wolves.
MATERIALS AND METHODS
Study area
The Principality of Asturias (Spain) is a 10,603 km2 autonomous region
located on the North coast of the Iberian Peninsula, with an abrupt orography
that includes the central part of the Cantabrian mountain chain and altitudes
ranging from sea level to 2640 m within only 40 km. This region is included in
the Eurosiberian climatic dominion of Atlantic type climate, with cold winters
(minimum of 6 months of potential frosts in non-coastal areas and
temperatures ranging from 3 to 8 ◦C to −4 to 0 ◦C in the coldest months) and
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abundant precipitations (1400–2100 L/m2 per year), with frequent snowfalls in
the winter season in the highest areas (Lines Escardó, 1970). The predominant
landscape is a mixture of deciduous and mixed forests with pastures and
meadows for cattle feeding that share this territory with different wild ungulate
species such as Southern chamois (Rupicapra pyrenaica parva), roe deer
(Capreolus capreolus), red deer (Cervus elaphus), fallow deer (Dama dama) and
wild boar (Sus scrofa), and also with other wildlife species such as the red fox,
beech marten (Martes martes), stone marten (Martes foina), badger (Meles
meles), brown bear (Ursus arctos) and wolf, all of them listed among the hosts of
S. scabiei in Europe (Bornstein et al., 2001), in some cases even being
diagnosed as suffering sarcoptic mange in the study area (Fernández-Moran et
al., 1997; Gortázar et al., 1998; Oleaga et al., 2008a,b).
The distribution area of the wolf in Asturias includes almost the whole
territory except the most populated Central region (Blanco et al., 2008) (Fig. 1).
The wolf population showed a gradual increase in Asturias from the late nineties
up to 2004, with an apparent maintenance of population values in recent
years with an estimation of average density of 3.9–4.4 wolves/100 km2 in 2004
(Llaneza et al., 2004).
For the work presented here, Asturias was divided into three different
geographical areas: Western, Central and Eastern Asturias, in order to group
samples and study animals for analysis.
Studied animals
A total number of 125 wolves were submitted for necropsy between 2003
and March 2010 (Table 1), which included animals collected in population
control hunts carried out by wildlife officers (n = 95; the wolf is not a hunted
species in Asturias) and those found dead (vehicle collision n = 12, illegal kill n =
5, poisoning n = 2 and infectious disease n = 1). In 10 cases it was not possible
to determine the cause of death.
Animals came from almost the whole distribution area of wolves in
Asturias, but they were not homogeneously distributed due to larger hunting
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effort carried out by officers in some areas according to the established Wolf
Management Plan followed in the region (Fig. 1). Based on tooth wear
(Gipson et al., 2000) three age classes were determined for this work: pups
(<1 year), yearlings (between 1 and 2 years) and adults (>2 years). The studied
group was composed of 58 males (11 pups, 10 yearlings and 37 adults) and
61 females (18 pups, 22 yearlings and 21 adults). In 6 cases it was not possible
to register complete data regarding sex and age due to bad preservation.
Figure 1- Distribution in the three different geographic areas considered in Asturias for this work
(Western, Central and Eastern) of wolves submitted for necropsy in 2008: O, 2009: □ and 2010: ∆.
Icon colours represent mangy-confirmed wolves: ●, skin-injured wolves without mite isolation:
and wolves without lesions or mite isolation: O. The icon represents the only wolf without skin
lesions that allowed mite isolation. Grey areas correspond to the three council districts (Proaza,
Belmonte and Somiedo) where the long-term camera-trapping study was carried out.
Samples and pathological study
From January 2003 to December 2007, 78 necropsies were carried out in
SERIDA (Regional Government Animal Health Laboratory, located in Gijón-
Asturias). During necropsies photographs of the general aspect and particular
features of the animals studied were taken, sex, age, weight and different body
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measurements were registered, and whenever possible blood (taken by cardiac
puncture for serology), parasites and tissue samples were collected.
Figure 2- Body scheme indicating the number of confirmed mangy wolves showing alopecia and mite isolation in the 8 different body regions examined during the mite searching protocol, and the percentage of total mites isolated in each studied body region.
The above-described necropsy procedure was improved in order to get
more data about the aetiology and characteristics of skin lesions observed
since January 2008 (Table 1). Thus, in the 47 animals submitted for necropsy
from January 2008 to March 2010 location, extension and characteristics of
dermal and/or internal lesions were recorded; KFI (kidney fat index) and adrenal
glands weight were noted (as body condition and stress degree indicators,
Salak-Johnson and McGlone, 2007); eight 5 cm × 5 cm skin samples (from ears,
head, neck, back, foreleg, rear leg, abdomen and tail, Fig. 2) were resected with
a scalpel and additional skin sections, lymph node pieces and any lesion
detected in other tissues were fixed in 10% buffered formalin, embedded in
paraffin and sectioned at 4 µ,m for histopathological studies whenever
possible.
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Laboratory techniques
Mite isolation/identification
Between January 2008 and March 2010, the eight skin samples taken
from every wolf that arrived for necropsy less than 12 h after death (n = 32,
including 12 wolves with mange-compatible lesions and 20 apparently healthy
animals, Table 1) were submitted to an established ectoparasites search
protocol. Briefly, the skin pieces were placed on Petri dishes and incubated at
37 ◦C for 24 h in order to stimulate mites to leave the skin. Both Petri dishes
and skin samples were then meticulously examined for the presence and
identification of ectoparasites using an Olympus SZX9 (10–57×) magnifier.
Detected mites were identified according to Wall and Shearer (1997),
collected and preserved in 70% ethanol.
Histopathological study
Skin samples and prescapular lymph nodes from the nine wolves with
sarcoptic mange-compatible lesions and S. scabiei isolation were collected,
fixed in 10% neutral-buffered formalin, and dehydrated through graded alcohols
and xylol before being embedded in paraffin wax. Several serial sections, 4 µ,m
thick, were cut from each sample and stained with hematoxylin and eosin (HE)
for histological characterization of detected lesions.
From January 2008 to January 2009, 16 wolves (including 7 confirmed
mangy animals, 4 skin-injured without mite isolation and 5 apparently healthy
animals) were subjected to immunohistochemical examination by means of
the peroxidase anti-peroxidase (PAP) method (Sternberger et al., 1970). The
sections were incubated with a rabbit anti-serum against the S. scabiei
Ssλ20∆B3 antigen (Casais et al., 2007) diluted 1 in 700. Preimmunization
rabbit serum was used as negative control.
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Year Wolves
studied at
necropsy
Wolves with mange-
compatible lesions
at necropsy
Wolves submitted
to mites searching
protocol
Wolves with mange-
compatible lesions submitted
to mites searching protocol
Wolves with mange-
compatible lesions and
mites isolation
2003→2007 78 0 0 0 0
2008 21 9 12 7 6
2009 10 3 5 2 1
2010 16 3 15 3 2
Total 125 15 32 12 9
Table 1- Number of wolves subjected to necropsy during the study period, including wolves with mange-compatible lesions, those subjected to the established mite searching protocol and those with skin lesions and confirmed sarcoptic mange by mite isolation. Data from 2003 to 2007 have been grouped due to the absence of mange-compatible lesions detected at necropsy before 2008 and the lack up to that year of a mite searching protocol.
Serum antibody survey (ELISA test)
The antibody levels against S. scabiei present in blood sera taken from 88
wolves submitted for necropsy between 2004 and 2010 (annual average of 12.6
samples, range 7–19) were assessed using an in-house ELISA. This ELISA
uses as antigen the recombinant protein Ssλ20∆B3 and has been previously
used successfully for diagnosis of sarcoptic mange in other wild species (Casais
et al., 2007; Oleaga et al., 2008a; Falconi et al., 2010). The absorbance at 450
nm was measured in a microplate reader (Sigma, model 680). Results were
expressed as a percentage of the relative optical density (% relative OD450
nm), which was calculated according to the following formula:
Serum samples were tested in duplicated, with control sera being
included in every series. The sera from a S. scabiei-infected wolf and a wolf
without mange-compatible lesions or mite isolation were used as positive and
negative controls, respectively. The mean value from the % relative OD450 nm
of seven scabies-free animals without skin lesions plus three times the standard
deviation (Bornstein and Wallgren, 1997; Hollanders et al., 1997) was defined as
the cut-off between positive and negative serum samples, offering for this
species a cut-off value of 8.9%, indicating that animals with a relative OD450 nm
percentage of 8.9% or above were considered positive.
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Revision of wolf carcass pictures
Due to the execution of necropsies by different technicians (belonging to
ARENA S.L., SERIDA and IREC) during the study period and the lack of
information concerning sarcoptic mange in Spanish wolves before 2008,
revision of pictures obtained from dead animals before that year was used as
a retrospective method for studying the presence of mange-compatible
lesions and to compare the obtained data with those coming from other study
methods.
A total of 1195 photographs (958 from 2003 to 2007 and 237 from 2008)
were taken and provided by ARENA S.L. biologists in order to evaluate the
presence of skin lesions in dead wolves. The first 958 pictures revised included
71 of the 78 animals submitted for necropsy from 2003 to 2007 (Table 1) and
10 additional wolves dead before 2008 where necropsy was not possible but the
external general aspect was recorded (average value = 16.2 animals/year). The
animals studied were 43 males and 50 females, including 17 pups, 31
yearlings and 45 adult wolves. The abovementioned pictures and results
obtained in their revision were compared with 237 photographs and subsequent
necropsy and laboratory data of 15 animals studied in 2008 (when the improved
necropsy protocol for the diagnosis of sarcoptic mange had already been
established). This comparison allowed us to evaluate the suitability of the method
to detect skin lesions and measure its success rate in correctly detecting
sarcoptic mange.
Pictures were obtained using both analogical and digital cameras,
recording external general aspect, particular features of skin and different parts
of the body and paying special attention to the presence of any kind of lesion
or anomaly.
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Camera-trapping data
The evaluation of camera trapping as a potential tool for the epidemiological
study of sarcoptic mange in the Iberian wolf was carried out using two different
approaches:
(a) A long-term evaluation of the detection frequencies of wild canids
and the presence of mange-compatible lesions in their skin was
carried out taking advantage of a camera-trapping monitoring program
established by FAPAS (fund for the protection of wild animals) for the
study of free-ranging brown bear populations in Central Asturias.
Pictures from wolves and red foxes obtained by the eight camera-
traps (4 in Somiedo, 2 in Belmonte and 2 in Proaza’s council district,
Central Asturias – Fig. 1) maintained in the same location for at least
5 consecutive years during the period 2003–2009 were carefully
revised in order to: (i) detect presence and characterize mange-
compatible lesions in free-ranging wolves and red foxes; (ii) signal
possible trends in the frequency of the appearance of these lesions in
nature through time and compare these trends with those reported at
necropsy during the same period; and (iii) detect possible temporal
and spatial agreements between the presence and frequency (as
number of wild canid contacts per 100 camera-trap days of work) of
both apparently healthy and skin-injured red foxes and wolves.
The eight camera traps used in this long-term evaluation were
analogical Canon® camera systems activated by a heat and
movement sensor (external Scanfer sensor connected from 2003 to
2006, and a Trail scan Canon system with incorporated sensor since
2007, in all cases using flash as artificial light source). These
cameras were placed along natural animal paths through beech
woods (n = 3), oak woods (n = 1), grazing land (n = 1) and chestnut
tree woods (n = 3), with an average altitude of 845 m above sea level
(ranging from 480 to 1520 m a.s.l.). Distances from the closest
camera and to the nearest village were 3.49 (ranging from 1.6 to 5.6
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km) and 1.41 (ranging from 0.4 to 3.1) km average values,
respectively.
Cameras were usually operative from March to November (average
value of 179.25 days working/year throughout the study period for the
8 cameras) due to the bad meteorological conditions existing during
the winter season and the lower bear activity during that period.
Revision of cameras and picture collection was done with a
periodicity of 15–30 days.
The analysis of obtained pictures was carried out for each camera,
district and the whole study area. All pictures of a single animal
obtained during a 15-min period were considered and analysed as a
single contact.
(b) In order to get all the information available regarding epidemiology
of the process in particular wolf packs, the careful revision of all
pictures obtained in the camera-trapping long-term study signalled
above was completed with photographs obtained close to carrions or
temporal locations since 2003 by FAPAS and those got by 6
additional digital camera traps (passive systems Leaf River IR-3BU® –
Leaf River Outdoor Products, Taylorsville, Mississippi, USA, with an
infrared light source and an infrared movement and temperature
sensor) set up since January 2008 nearby to suspicious sarcoptic
mange-affected pack reproduction areas.
Statistics
Statistical analysis was performed using the Epi Info 3.5.1 and SPSS
15.0 software.
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RESULTS
Sarcoptic mange confirmation and distribution
Different degrees of alopecia or dermal lesion compatible with
sarcoptic mange were detected in 15 out of the 47 wolves submitted for
necropsy from January 2008 to March 2010. From the 32 fresh submitted
wolves during that period, S. scabiei was isolated from 9 out of 12 animals
showing skin lesions and 1 out of 20 without skin alterations (Table 1 and
Fig. 1). The presence of mange-compatible lesions offered an “apparent”
average prevalence value of 31.91% (19.52–47.25%, 95% confidence interval
– C.I.) for the period 2008–2010, reaching a peak value of 42.86% (22.59–
65.56%, 95% C.I.) in 2008. The real prevalence (wolves with lesions and
confirmed S. scabiei presence) average value was 19.15% (9.65–33.73%, 95%
C.I.) from 2008 to 2010, and 28.57% (12.19–52.31%, 95% C.I.) in 2008. No
data regarding this kind of skin alterations were signalled in necropsies of
animals carried out before 2008 (n = 78).
Sarcoptic mange was confirmed in the three considered geographical
areas of Asturias – Eastern, Central and Western, with 3 (out of 13), 5 (n = 23)
and 1 (n = 11) wolves with confirmed mange by isolation of mites, respectively,
since January 2008 (Fig. 1). The Chi square test revealed no sex-related
difference in the probability to show mange-compatible lesions or even
harbour S. scabiei. Nevertheless, yearling wolves studied according to the
necropsy protocol established in 2008 (n = 11) showed a statistically
significant lower probability to present mange-compatible lesions than pups (n
= 11) or adults (n = 21) (X2 = 8.353, p = 0.015), offering “apparent”
prevalence values of 0%, 54.54% and 42.86%, respectively. While months like
April, May and November had 2 mangy-confirmed wolves (this happened at
least in one of the study years), none of the 15 wolves with sarcoptic mange
or compatible lesions recorded at necropsy was collected in March, August,
September or October.
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Clinical characterization
The number of mites isolated in confirmed parasitized wolves ranged
from 1 to 78 per animal. With the exception of two wolves (with only 1 and 2
detected mites), all animals with lesions and S. scabiei isolation showed mite
presence in at least 2 of the 8 studied skin areas (ears, head, neck, back,
foreleg, rear leg, abdomen and tail), with a maximum of 7 of the 8 body areas
tested with acari detection in one single animal. Skin pieces from tail, ears and
rear legs showed an overall higher number of live mites detected (55, 57 and
47, respectively), and were also the body areas where more wolves (4, 6 and
6, respectively) allowed S. scabiei identification during examination (Fig. 2).
28.35% of detected mites were isolated in areas without dermal lesions.
The most frequent and conspicuous detected macroscopical skin alteration
was alopecia, present in the 9 mangy-confirmed animals in different degrees
(from 10% to 90% injured skin surface); rear leg, abdomen, ears and foreleg
were the most frequently alopecic areas (Figs. 2 and 3a, b and d).
The relationship between the percentage of alopecic skin and the
number of isolated mites for each confirmed mangy wolf presented a linear
distribution (Fig. 4), with a statistically significant decrease in the number of
alive mites as the percentage of affected skin increases (r = −0.7818; p <
0.05).
Both hyperpigmentation and hyperkeratosis were detected in 4 confirmed
mangy wolves, although especially in the case of hyperkeratosis reported
alterations were of a small size and thickness (Fig. 3b and c). Three of the four
animals showing hyperkeratosis were those with the highest percentage of
affected skin surface (90, 80 and 70%, respectively). No recent ulcers or
wounds were detected.
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Figure 3- Macroscopical and microscopical pictures showing characteristic lesions detected in confirmed mangy wolves at necropsy. The most frequently affected skin areas were abdomen (a), rear legs (b), base of tail (c) and ears (d). Cracking and thickened skin was infrequent, very localized when present (b and c). (e) The most frequently reported microscopical alterations were focal hyperkeratosis (A), hyperplasic epidermis (B) and inflammatory infiltrate in the dermis (C). Hypertrophy of the sebaceous glands (D) and hair follicles clogged with keratin (E) can be also observed (HE = 400×, bar = 200 µ,m). (f) Skin. Positive immunostaining in macrophages (arrows) located in the dermis (PAP = 200×, bar = 100 µ,m). (g) Prescapular lymph node. Positive immunostaining in macrophages (arrows) (PAP = 400×, bar = 50 µ,m).
Histologically, an alopecic (delayed) type IV hypersensitive response
reaction was observed in the nine wolves with confirmed sarcoptic mange.
Skin samples from the alopecic areas demonstrated an inflammatory
infiltrate consistent with lymphocytes, macrophages and few neutrophils in
the dermis (Fig. 3e and f). Occasionally, the stratum corneum showed focal
parakeratosis and hyperkeratosis, and the epidermis was hyperplasic. The
sebaceous glands were hypertrophied and some of the hair follicles clogged
with keratin (Fig. 3e). Scab formation was not observed and mites could only be
detected in one case.
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Lymphocytes and macrophages, located both in the dermis (Fig. 3f) and in
the prescapular lymph nodes (Fig. 3g), showed positive immunostaining
against S. scabiei antigen in 4 of the 7 wolves with confirmed sarcoptic
mange subjected to immunohistochemical examination. There was no
statistical relationship between the appearance of immunostaining and the
percentage of affected skin or the number of isolated mites in these
animals. None of the 4 wolves with mange-compatible lesions but without
mite isolation showed positive immunostaining and only 1 of the 5 apparently
healthy animals showed positive immunostaining, located only in the
prescapular lymph node. The preimmune serum controls showed no staining.
With the exception of one wolf with mange-compatible lesions and S. scabiei
isolation where histological analysis suggested co-infection by a virus, no
other pathological alteration was detected in studied animals besides those
attributed to sarcoptic mange.
The KFI gave a statistically significant (t = 3.035, p < 0.005, n = 39)
lower value for wolves with mange-compatible lesions, but showed no
significant relationship (t = 1.637, p = 0.110) with the isolation of mites in
suspicious wolves. The KFI average values were 0.407 ± 0.04, 0.228 ± 0.024
and 0.264 ± 0.04 for apparently healthy wolves (n = 26), animals with
injured skin (n = 13), and confirmed mangy wolves (n = 9), respectively. No
statistical relationship was detected regarding adrenal mass and the presence
of mange-compatible lesions or confirmed sarcoptic mange.
Figure 4- Regression analysis carried out considering the percentage of affected skin versus the number of isolated mites in the nine confirmed mangy wolves (r2 = 0.6112; r = −0.7818, p = 0.0128; y = 55.5391933 − 0.765068493x).
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Figure 5- Temporal evolution during the 7 years of the study period of: (i) seroprevalence, (ii) percentage of wolves with mange-compatible lesions at necropsy, (iii) percentage of wolves with isolated mites confirming sarcoptic mange at necropsy, (iv) percentage of dead wolves with mange-compatible lesions in pictures and (v) percentage of wolves with mange-compatible lesions in camera-trapping pictures. (ii), (iii), (iv) and (v) percentages are in agreement with the apparent morbidity increase reported in Asturian wolves during 2008.
Serology
Considering the 8.9% (percentage of relative OD450 nm) calculated cut off
level, the ELISA test was characterized by 75.0% sensitivity and 87.5%
specificity.
The analysis of 88 sera samples obtained from 2004 to 2008 allowed
the detection of 18 seropositive wolves, offering an average seroprevalence
value of 20.45% (12.59–30.39%, 95% C.I.). The percentages of OD450 nm of
the ELISA positive wolves were below 27.3%, indicating a weak antibody
response.
The first seropositive animal was detected in 2004 (first year with collected
blood samples), with seropositive wolves detected during the whole study
period (Fig. 5). ELISA positive animals appeared equally distributed between
both sexes, with 20.93% (10.04–36.05, 95% C.I.) and 21.43% (10.29–36.82,
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95% C.I.) seropositive males and females, respectively. Regarding age groups,
pups showed a slightly lower percentage of seropositivity (14.28%
seroprevalence average value, 95% C.I. = 3.04–36.35%) than adults (22.5%
average value, 95% C.I. = 10.83–38.46%) and yearlings (25% average value,
95% C.I. = 9.77–46.72%). The effect of sex and age on seroprevalence was
not statistically significant.
The Western area, with a 35% seropositivity (15.39–59.22%, 95% C.I.),
showed the highest seroprevalence average value, whereas similar data of
17.2% (5.84–35.78%, 95% C.I.) and 16.7% (6.37–32.82%, 95% C.I.) were
reported for the Central and Eastern regions, respectively. Differences were
not statistically significant. The first half of the year showed a statistically
significant (X2 = 10.159, p = 0.017) higher seroprevalence value, with 16 out
of 18 seropositive wolves sampled between January and June.
Photographic studies
The revision of pictures of dead wolves obtained from 2003 to 2007 (n =
958 pictures), focussed on the detection of skin lesions and alterations similar
to those described for confirmed mangy wolves since 2008, permitted the
detection of six animals showing mange-compatible lesions. The first
suspicious animal was detected in 2003, and since then, wolves showing
mange-compatible skin alterations were detected in pictures taken in 2005,
2006 and 2007 (with 1, 1 and 3 skin-injured animals, respectively, Fig. 5). The
average global percentage of animals showing mange-compatible lesions was
7.40% between 2003 and 2007 (Fig. 5). Distribution of these animals
included Western (1), Central (2) and Eastern (3) areas. The apparently
affected animals were 2 males and 4 females, including 1 pup, 2 yearlings
and 3 adults.
From the 15 animals photographed by ARENA S.L. biologists in 2008 whose
pictures were revised as an evaluation of this study method, mange-
compatible lesions were detected in 6 (40%) wolves, from which sarcoptic
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mange was confirmed by the subsequent mite searching protocol in 4 cases,
indicating the suitability of the evaluation method used.
Since the beginning of the camera-trapping long-term study (2003), an
8783 camera-trap/day monitoring effort was carried out. A total of 832 and
184 contacts with red foxes and wolves, respectively, were registered,
including 20 contacts with red foxes and 22 with wolves showing mange-
compatible lesions. Both canid species were registered in all 8 camera traps
used in this study, with average values of 16.50 (ranging from 0.29 to 48.1)
and 3.69 (from 0.14 to 8.8) contacts/year/camera for red foxes and wolves,
respectively.
The first pictures showing mange-compatible lesions in red fox and wolf
were obtained in 2003 in Belmonte and Somiedo, respectively. Since then,
both wolves and red foxes with cutaneous alterations were photographed in
2006, 2007 and 2008 (Figs. 6 and 7). Mange-compatible lesions were
detected in the three camera-trapping studied areas in both species with the
exception of Belmonte (Fig. 1), where no apparently affected wolves were
registered (in this area a wolf with dermal lesions was photographed in a
camera trap placed close to a carrion, thus not being included in this
monitoring program). In one single camera-trap (from Somiedo) no canid with
mange-compatible lesions was photographed.
The analysis of recorded data both for each individual camera trap and
for the group of cameras allocated in each area (Somiedo n = 4, Belmonte n
= 2 and Proaza n = 2) showed a different evolution for each camera and
even area, with a great annual variation in detection of red foxes not
observed for wolves (expressed as the number of contacts registered in 100
camera trap days), and often single or very scarce data regarding the
detection of mange-compatible lesions, preventing trend descriptions and
statistical analysis on this scale.
Nevertheless, the study of camera-trapping collected data from the 8
cameras grouped as a whole provided data enough to attempt analysis, which
agreed to a large extent with results obtained by the other study techniques
used in this work (Figs. 5 and 7). A gradual increase in the percentage of skin-
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injured red foxes and consecutive decreases in the number of red fox-
contacts/100 camera-trap days were detected, reaching the lowest detection
rate and the highest percentage of red foxes with mange-compatible lesions in
2007, suggesting a possible effect of the disease on the presence or density
of this species in the studied area.
Figure 6- Example of pictures obtained by the camera-trapping technique in the present work showing different degrees of mange-compatible lesions in wild wolves. The last picture shows a skin-injured animal next to another member of its wolf pack without detectable skin lesions.
Regarding the wolf, an important increase (reaching 27.7% of registered
wolves) in the percentage of wolves photographed with mange-compatible
lesions was observed in 2008, whereas the number of wolf-contacts
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
116 _____________________________________________________________________________
detected for 100 camera-trap days as a measure of detection showed very
small variations during the study period (Fig. 7).
The statistical analysis of obtained pictures, although carried out on a
limited number of years and contacts, showed a close statistically significant
relationship between the percentage of skin-injured wolves registered in
camera traps during 1 year and the percentage of red foxes with mange-
compatible lesions registered in the previous year (Rs = 0.9856, p < 0.0005, n
= 6; Fig. 7).
Figure 7- Comparison between year-on-year variations undergone by the “number of contacts/100 camera-trap days” and the “percentage of contacts showing mange-compatible lesions” on pictures obtained by camera trapping for both red foxes and wolves from the study area.
The examination of all pictures obtained by the 8 camera traps used in
the long-term study plus those placed near carrions or added since 2008
provided valuable information regarding clinical and epidemiological aspects of
sarcoptic mange in wolves: (i) showing the similarity of lesions reported in the
field with those confirmed as a consequence of sarcoptic mange at necropsy; (ii)
allowing the detection of wolf packs with members showing mange-compatible
lesions for two consecutive years or in areas where these skin alterations had
not been reported before; (iii) reporting the presence of skin-injured and
apparently healthy pups from one same pack and reproductive season and
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(iv) showing the apparently slow extension of skin lesions and limited body
condition worsening in individually recognizable wolves (especially compared
to the quicker, often lethal, clinical evolution of the process in red foxes from
the same area – unpublished data). A single wolf with mange-compatible
lesions recognizable in pictures obtained during a long enough time period
(from 14/08/2008 to 31/10/2008 in the Proaza area) showed the maintenance
of a good body condition and even the apparent recovery of hair and skin
condition through the series of pictures, as signalled in particular cases from
other affected wolf populations (Jimenez et al., 2010).
DISCUSSION
This is the first large survey on sarcoptic mange in the Iberian wolf.
Although the number of samples and obtained data available were limited
due to the species’ status, data compiled herein by a combination of
pathological and retrospective studies (including serological and
photographic techniques) allowed valuable insights into the epidemiology of
this parasite–host relationship. Mange was confirmed by isolation of S.
scabiei mites from wolves necropsied between 2008 and 2010. Moreover,
visible lesions recorded on photographs and serum antibody detection by
ELISA coincide, suggesting that the disease was already present at least
since 2003. Lesion distribution was similar to those described in previous
reports in wolves (Todd et al., 1981; Domínguez et al., 2008), and in other
canids such as the red fox and coyote (Trainer and Hale, 1969; Pence et al.,
1983; Mörner, 1992). However, parakeratotic lesions were infrequently
reported in this study.
Regarding the ELISA, the low number of mites usually detected on
infected wolves may have limited the sensitivity of this antibody test. The mean
antibody prevalence was similar to the 21% reported in a Scandinavian study,
where 86 wolves collected from 1998 to 2002 included 6 individuals that died
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
118 _____________________________________________________________________________
of sarcoptic mange (Olsen, 2003). The lack of sex, age or site differences in
antibody prevalence suggests a widespread contact with the parasite.
Visualizing the mite by isolation or histology is the only definitive diagnosis
of sarcoptic mange (Bornstein et al., 1996; Morris and Dunstan, 1996;
Davidson et al., 2008). In the present study, mites were detected in only one
of 20 apparently healthy wolves (with no visible lesions), whereas they were
isolated in 9 of 12 animals with dermal lesions and were also
immunohistochemically detected in 4 of the 7 mangy animals studied at this
level. This, along with an apparent sarcoptic mange/viral co-infection in one
male as the only pathological process different of sarcoptic mange detected by
histology or other applied techniques in the frame of the wildlife disease
surveillance program in studied wolves, signals mange as the cause of the
lesions observed in these animals.
Live mites were detected in only 75% of the properly tested suspect
wolves, and usually in a low number. This agrees with data on coyote and
other studies on sarcoptic mange in wolves (Todd et al., 1981; Domínguez et
al., 2008), and coincides also with low isolation rates obtained by skin scraping
in mange-suspect dogs (Canis familiaris) (Bornstein, 1991; Hill and
Steinberg, 1993; Griffin, 1993; Carlotti and Bensignor, 1997; Beck and Hiepe,
1998; Davidson et al., 2008). These isolation numbers contrast with those
reported for the red fox (up to 5000 mites per cm2, Little et al., 1998a).
Similarly, the inverse relationship observed between the number of mites and
the size of the affected skin (Fig. 4) contrasts with findings in foxes (and wild
ruminants, data not shown), suggesting a different evolution of the process
and an apparent greater ability of wolves to control the number of alive mites
as compared to most of the other susceptible natural S. scabiei hosts in the
study area (different immune responses and efficacy against the mite have
already been signalled for dog and red fox: Arlian et al., 1996b; Little et al.,
1998a). However, caution is required in using the number of isolated mites as
a proxy for parasite burden (Alasaad et al., 2009).
Most wolf lesions were histologically confirmed as alopecic, opposed to
the frequent parakeratotic form (type I “immediate” hypersensitivity response)
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observed in red foxes from the study area by the authors (data not shown) and
most frequently reported in other red fox populations (Little et al., 1998b;
Newman et al., 2002). This alopecic form reported in Asturian wolves, a type IV
hypersensitivity response, is also the most common presentation of sarcoptic
mange in dogs (Paterson et al., 1995), but rarely reported in the fox, where
this presentation has been described as characterized by low mite burdens,
few (if any) crusty lesions and a limited effect on body condition (Bates, 2003).
Experimental studies have shown no significant KFI value differences
between mangy and healthy dogs (Arlian et al., 1995; Little et al., 1998a),
whereas coyotes and wolves tend to show limited effects of mange on
body condition (Todd et al., 1981; Pence et al., 1983; Pence and Windberg,
1994), and this effect is more severe in red foxes (Gortázar et al., 1998; Little
et al., 1998b; Newman et al., 2002; Davidson et al., 2008). In this study
wolves with mange-compatible lesions showed a 50% lower KFI value than
those without visible lesions, but never appeared emaciated or with an
extremely poor body condition at necropsy. These findings coincide with
those signalled in the few cases of red foxes reported by Bates (2003) with
the alopecic presentation of sarcoptic mange, and are lower than the 85%
difference observed for foxes with parakeratotic mange lesions (Little et al.,
1998a; Newman et al., 2002). Nevertheless, the detection of both
parakeratotic and alopecic forms in a red fox population with different effects
on body condition (Bates, 2003) confirms the existence of individual
differences in the response to the mite, warning us about the possible
presence of different disease presentation forms in Asturian wolves not yet
reported at necropsy. In this sense, the authors were able to observe in the
field in 2010 one wolf with extremely poor body condition, abnormal
behaviour and severe parakeratotic mange-compatible lesions, frequent in
red foxes, but not registered in Asturian wolves at necropsy up to date.
Regarding the time trends, both the photographic records and the
necropsy data showed an increased morbidity in 2008. Possible explanations
for this increase include changes in mite pathogenicity (Pence and Windberg,
1994), density dependence in transmission rates (Pence et al., 1983; Pence
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
120 _____________________________________________________________________________
and Windberg, 1994), co-infections or other debilitating factors (Lloyd, 1980)
and a higher mange circulation among sympatric carnivores (Trainer and Hale,
1969; Todd et al., 1981; Clayton, 2003; Mörner et al., 2005; Soulsbury et al.,
2007; Domínguez et al., 2008). Mite pathogenicity variation is difficult to test in
the field. On the subject of density dependence, there are no indications of an
increase in wolf numbers during the study period in Asturias: after a moderate
increase observed from 1998 to 2004, an apparent stabilization in wolf density
was reported from 2005 to 2008 (Llaneza et al., 2004; García and Llaneza,
2009). Regarding co-infections, it is known that Iberian wolves are often
exposed to disease agents including canine distemper virus, canine parvovirus
and Leishmania infantum (Sobrino et al., 2008a,b); the histopathological
detection of a wolf apparently co-infected by S. scabiei and a viral agent
emphasizes the need of further studies about the role that concomitant agents
can play in the development of sarcoptic mange and skin lesions, with ongoing
research in the studied population by the authors. Finally, the statistically
significant relationship signalled between the trends in mange-compatible
lesions detection for red fox and wolf with 1-year delay (Fig. 7) strongly suggests
sarcoptic mange in the red fox as the most likely origin of the observed time
trend in wolf disease. Interspecific transmission of S. scabiei has been
observed between the red fox and coyote (Trainer and Hale, 1969), the red
fox and dog (Soulsbury et al., 2007; Clayton, 2003) and coyote and wolf (Todd
et al., 1981; Jimenez et al., 2010), also with 1-year difference in the morbidity
peak of mange reported between the coyote and wolf by Todd et al. (1981).
Mörner et al. (2005) and Domínguez et al. (2008) indicate that the red fox is
the most probable origin of sarcoptic mange in wolf. The possible role of
sympatric ungulates confirmed as S. scabiei hosts in Asturias (Fernández-
Moran et al., 1997; Oleaga et al., 2008a,b) in the epidemiology of sarcoptic
mange in Iberian wolf is still unclear.
The high percentage of macroscopically affected skin in confirmed mangy
wolves at necropsy suggested the usefulness of photographic techniques for
the detection of sarcoptic mange lesions in the Iberian wolf, which was confirmed
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by data on pictures taken by technicians in 2008 from dead wolves
subsequently studied at necropsy.
Remote photography methods have been increasingly used in recent
years to address a great variety of questions in wildlife biology (Karath and
Nichols, 1998; Cutler and Swann, 1999; Carbone et al., 2001; Grassman et
al., 2006; Lucherini et al., 2009), whereas their use in wildlife sanitary studies
is infrequent, usually related to animal behaviour or biological estimations
with epidemiological interest (Hegglin et al., 2004; VerCauteren et al.,
2007; Jennelle et al., 2009). Even with the limited number of cameras
available and contacts obtained (that prevented weighty conclusions), this
camera-trapping technique has been shown to be useful for the detection of
animals with mange-compatible lesions in the field, providing interesting
information about the presence of injured animals at different times of year
and in different areas and interesting data regarding population trends and the
epidemiology of the disease (confirming in the field the apparent increase in
morbidity reported for sarcoptic mange at necropsy during 2008, Figs. 5 and 7).
To the best of our knowledge, this is the first time photo-trapping has been
used to study disease-compatible lesions and time trends in a wildlife
disease.
The combination of these field techniques with the necropsies and
laboratory results allowed interesting insights of mange epidemiology in
Iberian wolves. Necropsy data (with skin alteration as the only detected
lesion related to S. scabiei and a limited effect on body condition) and
reported trends (showing a rather low prevalence and an apparently stable
distribution of the disease and its host) suggest that this parasite is not a
major threat for wolf populations in Asturias.
However, the possible lack of data and incomplete overview of the
disease in such a scarce species emphasize the need of more information in
order to evaluate its possible effect on fertility and pup survival rates, where
the effect of the parasite seems to be more important (Todd et al., 1981;
Pence and Windberg, 1994; Soulsbury et al., 2007; Jimenez et al., 2010), and
even the long-term effect on the health of adult wolves. Hence, surveillance
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
122 _____________________________________________________________________________
should be maintained and, eventually, more information gathered through larger
surveys (increasing the number of available cameras; including wolf tagging
with epidemiological surveillance purposes) in order to better understand and
characterize sarcoptic mange in the Iberian wolf and assess the epidemiology of
this parasitic disease and its effect on wild canids population trends and wolf
conservation.
ACKNOWLEDGEMENTS
This is a contribution to the agreement between CSIC and Principado de
Asturias. We thank the rangers of the game preserves (specially Jaime Marcos
Beltrán, Francisco Alonso Mier, Fernando Rodríguez Pérez, Pedro González
and Angel Nuñ o) for their help in carcasses submission and camera-trapping,
our colleagues from ARENA S.L. and IREC (Emilio José García, Óscar Rodríguez
and María Suárez) for their assistance in field, necropsy and laboratory work,
and other workmates (Jose Luis García Díaz and Rafael Alba Zarabozo) for
their advice and field collaboration in camera-trapping work. We wish to
thank also Kevin Dalton for revising the English of the manuscript, Pelayo
Acevedo for his contribution in statistical and geographical analysis and
Joaquín Vicente for his advice in the study design and camera trapping
procedures. Projects RTA 2009-00114-00-00 (INIA) and CIT-060000-2009-
0034 (MICINN) contributed to this study.
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3.2.- CONCOMITANCIA E INTERACCIONES ENTRE PATÓGENOS EN
EL LOBO IBÉRICO (Canis lupus)
Oleaga, A., Vicente, J., Casais, R., Espí, A., García, E. J.,
Gortázar, C. CONCOMITANCE AND INTERACTIONS OF PATHOGENS IN THE
IBERIAN WOLF (CANIS LUPUS). Research in Veterinary Science (submitted).
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RESUMEN
Con la intención de profundizar en sus características epidemiológicas, se estudió la
exposición a largo plazo (2004-2010) frente a Parvovirus Canino (CPV), Moquillo Canino
(CDV) y Sarcoptes scabiei en 88 lobos silvestres de Asturias (Norte de España). Para ello se
utilizaron datos serológicos, prestando especial atención a las posibles interacciones
existentes entre los diferentes agentes patógenos así como al posible efecto de diferentes
factores individuales y poblacionales, que fueron evaluados a través de su análisis
estadístico.
Los valores medios de seroprevalencia fueron de 19%, 61% y 20% frente a CDV, CPV y
S. scabiei respectivamente. Los tres agentes patógenos mostraron una mayor
seroprevalencia en aquellas áreas con mayor densidad de lobos, y se detectó una asociación
positiva entre la presencia de anticuerpos frente a S. scabiei y moquillo.
Los datos obtenidos ponen de manifiesto la necesidad de considerar los agentes
patógenos concomitantes y sus posibles interacciones para una mejor comprensión de las
enfermedades y su monitorización y gestión en fauna silvestre.
ABSTRACT
With the aim of deepening in their epidemiological features, exposure to Canine
Parvovirus (CPV), Canine Distemper Virus (CDV) and Sarcoptes scabiei were studied in 88
wild wolves from Asturias (Northern Spain) by means of long-term (2004-2010) serological
data, paying special attention to the possible interactions between them and the effect of
different individual and population factors by means of their statistical analysis.
The overall seroprevalence values were 19%, 61% and 20% for CDV, CPV and S. scabiei,
respectively. Sarcoptic mange, CDV and CPV showed higher seroprevalence values in the
areas with higher wolf densities, and a positive association between CDV and S. scabiei
antibody responses was detected.
Reported data highlight the need of considering concomitant pathogens and their
possible interactions for a better understanding of diseases and their management in
wildlife.
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INTRODUCTION
The understanding of diseases´ ecology and impact on populations is a key
aspect in the conservation and management programs of wildlife species. The
establishment of sanitary monitoring programs is an essential tool on this sense,
providing valuable information such as prevalence and exposure rates along time
(Almberg et al. 2009). The importance of infectious diseases in carnivore
conservation and their role in population dynamics and even declines have already
been described for a wide range of species throughout the world (Pedersen et al.
2007).
The largest population of gray wolves (Canis lupus) in Western Europe survives
in the Iberian Peninsula, where this wild canid has experienced a recovery during last
decades resulting in a total estimate of more than 2000 wolves in 2008 (Blanco et al.
2008). Some diseases are thought to produce changes in population dynamics or
temporal trends in wolves (Murray et al. 1999), a species susceptible to a large
spectrum of canine pathogens commonly found in domestic dogs (Canis familiaris),
including Canine Parvovirus (CPV -Mech et al. 1997), Canine Distemper virus (CDV -
Müller et al. 2011) and the mite Sarcoptes scabiei (Todd et al. 1981). Contact with
these three agents has already been reported in Iberian wolf populations (Sobrino et
al. 2008; Oleaga et al. 2011). Both CDV and CPV can reduce wild canid populations
recruitment by affecting young animals, while their long-term population effect seems
to be moderate (Barker and Parrish 2001; Williams 2001; Almberg et al. 2009). The
effect of sarcoptic mange on wolf population trends has been proposed to be limited
in the study area during recent years (Oleaga et al. 2011).
Host mammals with multiple simultaneous infections (co-infection), or having
been exposed to a number of pathogens, represent the most common situation in the
wild (Petney and Andrews 1998). Nonetheless, pathogens are usually studied
independently, despite the strong potential for interaction. The co-occurrence of
several pathogens can facilitate or hinder other infections (or even their clinical
manifestations), directly by competing for resources or indirectly via the host immune
system (Telfer et al. 2010; Hawley and Altizer, 2011). For example, interactions in
multiple infections become especially evident when some of the involved pathogens
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elicit immunosuppression in the host, as confirmed for CDV and CPV in canids
(Sykes 2010). The survey and monitoring of several pathogens over time in wildlife
provides a more realistic approach for the understanding of disease and sanitary
status in multiple pathogen-host systems, raising management and conservation
implications usually not identified in “single pathogen” studies (Hawley and Altizer,
2011).
We used long-term (2004-2010) serological data to characterize exposure for
CPV, CDV and Sarcoptes scabiei among Iberian wolves in Northern Spain. We
aimed (i) to assess contact rates of wild wolves with the three surveyed pathogens
along the study period, (ii) to study the influence of sex, age, time, geographic area
and exposure to the other pathogens on pathogen exposure for each one so as (iii)
to evaluate the potential interactions occurring among them.
MATERIAL AND METHODS
Study area
The study was carried out in the Principality of Asturias (North-Western Spain),
a 10,603 km2 autonomous region with an abrupt topography, altitudes ranging from
sea level to 2,640 m within only 40 km and Atlantic type climate. The predominant
vegetation consists of deciduous and mixed forests mixed with open pastures and
meadows shared by cattle and wild ungulates. This diverse habitat is suitable for a
wide range of mammalian species, including the endangered brown bear (Ursus
arctos), other carnivore species (red fox -Vulpes vulpes-, badger -Meles meles-, otter
-Lutra lutra-, beech marten -Martes martes-, stone marten -Martes foina-, ferret -
Mustela putorius-, weasel -Mustela nivalis-, ermine -Mustela erminea-, genet -
Genetta genetta- and european wildcat -Felis silvestris-), and several wild ungulates
(Cervus elaphus, Capreolus capreolus, Sus scrofa and Rupricapra pyrenaica parva)
representing the majority of the diet of wolves in the study area (Llaneza et al. 1996).
Due to the frequent presence of sheepdogs, hunting dogs and even those from
villages or belonging to tourists in wolf´s habitat, it can contact domestic dogs
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(visually confirmed by authors in the field and with the hybridization with Iberian
wolves already confirmed in the area: Godinho et al. 2011), that often are not
properly vaccinated in Asturian rural areas and can share several pathogen agents
with wild carnivores (Müller et al. 2011). Domestic livestock, also common in the
study area, is consumed as a frequent alternative prey (Llaneza et al. 1996).
After a gradual increase shown by the Asturian wolf population from late
nineties until 2004 and an apparent population and density maintenance since 2004
to date, its distribution area includes nowadays almost the whole Principality of
Asturias territory except the most human populated Central Region (Blanco et al.
2008; García and Llaneza 2012). The region can be divided into three different
geographical areas: Western, Central and Eastern Asturias, separated by large
north-to-south oriented valleys through the Cantabric mountain range. The number of
confirmed wolf packs and calculated average wolf density were higher in Western
and Central areas than in Eastern region during the study period, with estimated
average densities of 2.2, 2.3 and 1.1 wolves/100km² for Western, Central and
Eastern areas respectively in 2010 (when considering an average winter pack size of
4.93 wolves in the Cantabrian chain - García and Llaneza 2012-). No significant
differences have been described among the three mentioned areas regarding
climate, prey species available, wolves´ diet, contact with people or management
regimes. Nevertheless, the current differences present in human population average
densities in Asturias, showing their highest and smallest values in the Central and
Western areas respectively (source: INE, Instituto Nacional de estadística -
http://www.ine.es/-), can represent unreported differences in certain aspects such us
contact with people and domestic animals or prey species available (Llaneza et al.,
1996) with unknown effects on wolf populations to date.
Sampling
In accordance with a wildlife diseases surveillance program established in
Asturias since 2003, blood was taken by cardiac puncture from 88 wild wolves
submitted for necropsy between May 2004 and April 2010 (annual average of 12.6
samples, range 7–19), which included animals collected in population control hunts
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carried out by wildlife officers (n= 70) and those found dead (vehicle collision n=11,
illegal kill n=2, poisoning n=1 and infectious disease n=1). In 3 cases it was not
possible to determine the cause of death. Serological sampling was performed less
than 32 hours after the death of animals for hunted wolves. Although the exact time
elapsed between death and blood collection in death-found animals is unknown, no
animals found in bad preservation status or putrescent condition where included in
this study. Studied animals were 43 males and 42 females, including 21 pups (< 1
year), 24 yearlings (between 1 and 2 years) and 40 adult (> 2 years) wolves
(classification based on tooth wear). In three cases it was not possible to register
complete data about sex, age and geographical origin due to incomplete carcass or
data submission. Regarding geographical distribution, 20 wolves came from
Western, 29 from Central and 36 from Eastern Asturias. Most of the animals sampled
died acutely without underlying chronic disease processes. Nonetheless a potential
bias due to weaker individuals or in bad sanitary status could occur (especially dead
found wolves), which is considered in the discussion of our findings. In the most
recent 43 wolves submitted for necropsy (from January 2008 to April 2010), the
establishment of an improved necropsy protocol allowed a more complete data and
tissue samples collection: in addition to blood for serological survey, size and
characteristics of dermal and/or internal lesions were recorded and samples were
taken for histopathological examination; lymph node samples were taken both for
molecular analysis and histopathological studies.
Diagnostics
After centrifugation and storage at -20 ºC, the 88 obtained sera were screened
for exposure to canine parvovirus (CPV), canine distemper virus (CDV) and
Sarcoptes scabiei. The technique of indirect enzyme-linked inmunoassay (ELISA)
was applied for the assessment of antibodies to CPV and CDV, using two
commercial kits for the detection of specific antibodies in dog serum or plasma
(15.CPV.K1 and 15.CDG.K.1, Ingenasa, Madrid, Spain) following manufacturer´s
instructions (see Sobrino et al. 2008). The antibody levels against S. scabiei were
assessed by an in-house ELISA previously used successfully for the
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138 _____________________________________________________________________________
immunodiagnosis of this mite in different wild species (including wolves after suitable
adaptation and implementation of the technique for this wild canid, Oleaga et al.
2011).
Statistics
We assessed the factors potentially determining pathogen exposure or
presence (CPV ELISA, CDV ELISA and S. scabiei both ELISA and lesions) so as the
possible relationships among diseases (the remaining diseases included as
explanatory binomial factors) by means of 4 different GLMs. The factors included
were sex, age, time, and geographic area. We used a binomial distributed error with
a logistic link function.
RESULTS
Obtained seroprevalences, including distribution by geographic area, sex and
age, are summarized in Table 1. Overall seroprevalence values were 19%, 61% and
20% for CDV, CPV and Sarcoptes scabiei, respectively.. The co-occurrence of
antibodies against CDV, CPV and sarcoptic mange (% ±S.E.) in wolves as a function
of the age class is shown in Table 2, with a 27,6% of animals seronegative to the
three analyzed agents, and all wolves seropositive to CDV revealing contact with at
least another pathogen agent.
The only macroscopical alteration detected in necropsies carried out during the
study period related to the surveyed pathogens was the presence of skin lesions in
15 wolves (9 in 2008, 3 in 2009 and 3 in 2010, without skin-injured animals from
2004 to 2007), from which sarcoptic mange was confirmed by mite isolation in 9 of
the 12 animals that were submitted unfrozen (Oleaga et al. 2011). Only in one case
among all studied animals (in the wolf with the highest percentage of skin injured -
90% - of those 9 with mange confirmed by Sarcoptes scabiei isolation) was it
possible to confirm the presence of clinical CDV infection, revealed by
histopathological examination of the skin where the Morbillivirus´ presence was
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Age Western
area
Central area Eastern
area
♂ ♀ Total
Canine distemper virus - CDV (ELISA)
Pups 0 (1) 12 (8) 0 (11) 10 (10) 0 (10) 5 (20)
Yearlings 0 (5) 0 (5) 7 (14) 0 (8) 6 (16) 4 (24)
Adults 50(14) 33 (15) 18 (11) 32 (25) 40 (15) 35 (40)
Total 35 (20) 21 (28) 8 (36) 21 (43) 17 (41) 19 (84)
Canine parvovirus - CPV (ELISA)
Pups 0 (1) 25 (8) 27 (11) 10 (10) 40 (10) 25 (20)
Yearlings 60 (5) 100 (5) 43 (14) 62 (8) 56 (16) 58 (24)
Adults 93 (14) 80 (15) 73 (11) 84 (25) 80 (15) 82 (40)
Total 80 (20) 68 (28) 47 (36) 63 (43) 61(41) 62 (84)
Sarcoptic mange (ELISA)
Pups 0 (1) 33 (9) 0 (11) 10 (10) 18 (11) 14 (21)
Yearlings 40 (5) 0 (5) 29 (14) 37 (8) 19 (16) 25 (24)
Adults 36 (14) 13 (15) 18 (11) 20 (25) 27 (15) 22 (40)
Total 35 (20) 17 (29) 17 (36) 21 (43) 21(42) 21 (85)
Table 1- Seroprevalences for the studied pathogens in Asturian wolves (2004-2010), including distribution by geographic area, sex and age. Sample sizes are indicated within parenthesis (n).
confirmed by immunohistochemistry. Neither macroscopical nor microscopical
evidence of lesion or alteration related to CPV were detected in the studied animals.
The inter-annual variation along the study period of antibodies detection for the
three surveyed agents is presented in Figure 1. We detected antibodies against CPV,
CDV and sarcoptic mange since the beginning of the survey (2004), with small peaks
and falls, but an overall stable trend. Only CPV offered annual seroprevalences
higher than 50%, reaching an 87% seroprevalence value in 2009, and no year effect
was detected for any pathogen, whereas mange detection in serum was statistically
higher in the first semester.
Results of the GLMs on diseases are displayed in Table 3. Individual
immunoreactivity varied significantly among age classes for CPV, showing a clear
age-increasing pattern (pups: 25%; yearlings: 58%; adults: 82%, Table 1). A non-
significant trend was found for CDV (pups: 5%; yearlings: 4%; adults: 35%), but when
grouping pups and yearling, an additional model indicated statistical difference
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140 _____________________________________________________________________________
against adults. No sex effect was statistically identified for any pathogen. Although
not statistically significant, an apparent geographical gradient was observed for CDV,
CPV and sarcoptic mange seroprevalences, decreasing from Western to Eastern
Asturias. Regarding the possible existence of relationships among diseases we
detected a statistically significant positive association between CDV and S. scabiei
presence of antibodies (Table 2, Table 3).
Figure 1- Inter-annual variation of average seroprevalence values for sarcoptic mange, CDV and CPV along the study period (2004-2010) in studied wolves from Asturias.
DISCUSSION
Although the species’ status means a limited number of samples and data
available and hampered the obtaining of heavier conclusions, reported results allow
valuable insights into the epidemiology of these three pathogens and their presence
in wild wolves in Northern Spain. In addition to geographical and temporal
distribution, our findings are following discussed in order to better understand the
0
10
20
30
40
50
60
70
80
90
100
2004 2005 2006 2007 2008 2009 2010
Pre
vale
nce (
%)
± S
.E.
Year
Mange
CPV
CDP
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factors potentially determining pathogen exposure as the possible relationships
among them.
The seroprevalence of CDV reported in this work (19%) agrees with data
obtained for the same wolf population between 1997 and 2007 (seroprevalence
average value= 23.3%, Sobrino et al. 2008). Although without statistical significance
(Table 3), an apparent pattern of 2 years with high seroprevalence values (near 30%)
followed by 1 or 2 years with low seroprevalence (close to 10%) was reported (Figure
1). A similar pattern has already been described for CDV in a wolf population in
Alaska, which was interpreted as “short-term epizootics followed by interepizootic
periods” (Zarnke et al. 2004). The lower percentage of CDV seropositive pups and
yearlings (with only 1 of 20 and 1 of 24 sampled pups and yearlings, respectively)
compared to adults has also been observed in other wolf populations (Zarnke et al.
2004), with two main possible explanations. First, an extreme susceptibility of
youngest animals to the virus, dying once exposed to it, and second, the absence of
virus circulation among pack animals since the birth of reported seronegative pups
(CDV and CPV are considered to produce long-lasting immunity -Barker and Parrish
2001; Williams 2001; Greene and Appel 2006). Wolf pups have been identified as the
most sensitive age cohort to CDV, with a poor prognosis for clinically affected
animals (Williams 2001). The apparent maintenance (and even increase) of wolf
populations during recent decades suggests that, as in other wild canid populations
(Zarnke et al. 2004), this possible fatal effect of CDV on pup survival has not
jeopardized, at least thus far, the Asturian wolf population.
Serological data for CPV (61% average seroprevalence) are similar to those
reported in the study carried out in Asturias in 2007 (seroprevalence average value=
53.3%, Sobrino et al. 2008). This relatively high average value, together with the
apparently gradual increase of seroprevalence with age, suggest that CPV circulation
among wolves is frequent and can be considered endemic in Asturias. We
emphasize the need for both long term serological sampling efforts (to identify and
interpret epidemiological patterns of contact with the pathogens) and specific surveys
on pup survival and recruitment rates in order to evaluate the possible threat of these
infectious agents. The use of molecular techniques and non-invasive monitoring
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142 _____________________________________________________________________________
methods such as faecal samples collection (for parasitological and molecular
analysis) during breeding season could be a useful tool for future field studies.
Pathogen agents Pups (n=20) Juveniles
(n=24) Adults (n=40) All (n=87)
No pathogens 55±11.1 25±8.8 12.5±5.2 27.6±4.7
Only mange 15±7.9 16.7±7.6 2.5±2.4 9.2±3.1
Only CDV 0±0 0±0 0±0 0±0
Only CPV 10±6.7 45.8±10.1 32.5±7.4 29.9±4.9
Mange + CDV 5±4.8 0±0 2.5±2.4 2.3±1.6
Mange + CPV 15±7.9 8.3±5.6 17.5±6 13.8±3.7
CDV + CPV 0±0 4.2±4.1 17.5±6 10.3±3.2
All 3 pathogens 0±0 0±0 15±5.6 6.9±2.7
Table 2- Co-occurrence of antibodies against sarcoptic mange, Canine Distemper Virus (CDV) and Canine Parvovirus (CPV) (% ±S.E.) in wolves as a function of the age class.
The absence of differences in seropositivity between age classes in the case of
sarcoptic mange (14%, 25% and 24% for pups, yearlings and adults respectively),
could reflect an early and widespread contact with the mite. Fertility and pup survival
rates have been suggested as severely affected parameters in wolves´ populations
suffering sarcoptic mange (Todd et al. 1981). This fact reveals the importance in wolf
of early contacts with the parasite, and has been confirmed by authors in the study
area by the detection during 2012 of a complete litter (composed by 4 pups) severely
affected by sarcoptic mange, with a much worse body condition – including caquexia,
unpublished data- than that previously reported in adult wolves affected by sarcoptic
mange from Asturias. The marked increase in the number of animals with skin
lesions during 2008 confirmed as mangy wolves at necropsy and detected at the
same time in the field by camera trapping (Oleaga et al. 2011) contrasts with the
apparently stable behaviour of seroprevalence values for sarcoptic mange
throughout time (Figure 1). ELISA seroprevalence did not differ between animals with
and without mange lesions (Fisher test, p=0.495). A sensitivity or specificity of the
ELISA test lower than that reported in laboratory tests, a limited seroconversion due
to the low number of mites detected on studied wolves with confirmed mange
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lesions, the existence of contacts with the mite capable to produce seroconversion
but not skin lesions in wolves, or the disability to mount a detectable antibody
response in the case of immunocompromised wolves could explain this apparent lack
of correlation between annual seroprevalence and detected morbidity of sarcoptic
mange in studied animals. Contacts with Sarcoptes mites derived from other hosts
(namely ungulates as wolf´s preys), probably with a worse performance in canid
hosts but maintaining immunogenicity, cannot be rejected as a possible explanation.
Finally, the apparently different nature of the immune response reported in wolves
from the study area, with prevalence of the hypersensitivity (alopecic) response
against sarcoptic mange to the detriment of the hyperkeratotic form (Oleaga et al.
2012) could explain not only its apparent higher ability to control S. scabiei, but also a
milder production of antibodies and a more important role played by the cellular
immune response against the parasite in this species.
CDV, CPV and sarcoptic mange showed an apparent decrease in
seroprevalence values from West to East (Table 1). The CDV, CPV and sarcoptic
mange higher seroprevalence values reported in Western and Central areas may
relate to the wolf density gradient reported in distribution studies in Asturias (showing
lower wolf density values for the Eastern region -García and Llaneza 2012-). High
host density would increase i) the contact and transmission rates of these three “wolf
to wolf” transmissible pathogens, ii) the pathogen species richness present in a given
host and iii) the susceptibility to different pathogens in natural conditions (by
producing a weaker immunity response due to stress and/or a worse body condition
as a consequence of less resources availability). In addition, other possible factors
influencing the spatial distribution and contact patterns of these pathogens
(geographical differences in wild and domestic sympatric carnivores’ density, in
human presence and activities) cannot be excluded.
Regarding the possible interactions present between the four studied
pathogens, the interesting statistically significant positive association between CDV
and sarcoptic mange seropositivity (Table 2, Table 3) may reflect a common
distribution and contact pattern for both pathogens; nonetheless, CDV is recognised
to produce immunosuppression (Williams 2001; Sykes 2010), thus facilitating
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Table 3- Generalized linear models for sarcoptic mange ELISA, CDV ELISA, CPV ELISA and sarcoptic mange compatible lesions. Parameter estimates for the levels of fixed factors were calculated considering a reference value of 0 for the level positive in the variables on pathogen presence, for the level female in the variable “sex”, for the level adult in the variable “age”, for the level 2010 in the variable “year”, for the level 3 in the variable “Area”, for the level first semester in the variable “semester”. Significant p values are highlighted in bold.
Mange ELISA CDV ELISA CPV ELISA Mange lesions
Explanatory variables
Chi2, d.f.; Param. estimate (± SE); p Chi2, d.f.; Param. estimate (± SE); p Chi2, d.f.; Param. estimate (± SE); p Chi2, d.f.; Param. estimate (± SE); p
Sex 0.29, 1; 0.32±0.60; 0.59
0.0, 1; 0.05±0.68; 0.99
0.06, 1; -0.09±0.38; 0.80
3.23, 1; 1.39±0.77; 0.1
Age 1.30, 2; pups= -0.89±1.17, yearlings= -0.88±0.81;
0.52
3.59, 2; pups=1.70±1.21, yearlings=1.73±1.17;
0.16
10.04, 2; pups=1.81±0.57, yearlings=0.53±0.51;
<0.01
1.06, 2; pups=-1.10±1.06, yearlings=20.47±12413.2;
0.59 Year 1.35, 6;
2004=20.14±27508.11, 2005=0.09±1.22,
2006=-0.52±0.78, 2007=-0.54 ±1.26
2008=-0.73±0.97, 2009=0.63±1.52;
0.96
4.81, 6; 2004=-1.42±1.30, 2005=19.62±18992,30
2006=0.80±1.04, 2007=-1.18±1.18,
2008=0.36±0.99, 2009=1.42±1.48;
0.57
2.88, 6; 2004=0.05±0.95, 2005=0.02±0.65, 2006=0.47±0.60, 2007=0.18±0.82,
2008=0.42±0.70, 2009=-0.69±0.83;
0.82
3.54, 6; 2004=20.86±26993.45, 2005=19.67±18347.78, 2006=20.06±16776.13, 2007=20.40±25233.93,
2008=-1.78±1.02, 2009=-2.19±1.23,
0.74 Area 4.89, 2; Area1=-1.09±0.73,
Area2=0.78±0.83; 0.09 1.68, 2; Area1=-1.24±1.04,
Area2=-1.00±0.87; 0.43 1.00, 2; Area1=-0.43 ±0.49,
Area2=-0.44±0.51; 0.60 0.71, 2; Area1=-0.54±1.16,
Area2=0.17±0.89; 0.7 Semester 4.12, 1; -2.58±1.27; 0.04 0.09, 1 ; -0.26±0.87; 0.76 0.53, 1 ; 0.38±0.52; 0.46 1.15 1, -0.80±0.75; 0.28 Mange 5.47 ; 1, 1.74±0.74; 0.01 0.21, 1; -0.24±0.51; 0.64 0.62, 1; 0.85±1.07; 0.43 CDV 5.03, 1; 1.59±0.71; 0.02 2.11 ; 1, 0.86±0.59; 0.14 0.38, 1 ; -0.67±1.08 ; 0.53 CPV 0.04, 1; -0.13±0.67; 0.83 0.92, 1; 0.89±0.92; 0.33 0.36, 1; -0.50±0.84; 0.55 Mange lesion
0.55, 1; 0.70±0.95; 0.45 0.56, 1; 0.68±0.91; 0.45 0.31, 1; -0.32±0.57; 0.57
________________________________________________________________________ Capítulo 3
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secondary infections by viruses (Holzman et al. 1992), bacteria or protozoa
(Stoffregen and Dubey 1991), and allowing overwhelming infections with co-
pathogens (Munson et al. 2008). An interesting result in this respect is that, as a
difference with the other 3 surveyed pathogen agents, all wolves seropositive to CDV
had evidence of contact with at least another pathogen among those we studied
(Table 2). The detection of an adult wolf with S. scabiei isolation, widespread skin
lesions and the presence of dermal CDV infection meant the only one case of
coinfection with clinical relevance at necropsy reported in the present work.
As suggested for other endangered wild canid species ( Pedersen et al. 2007),
and even wolf (Almberg et al. 2009; Müller et al. 2011), the presence of domestic
(especially dogs) and wild (red foxes) sympatric carnivores periodically contacting
with wolves in the study area can facilitate the presence and flow of different
pathogen agents and their possible interactions. For example, the coincidence of an
apparent sarcoptic mange peak reported in red foxes from the area in 2007 -thus
increasing the probability of mites arrival to wolf- with the CDV peak (and its possible
immunosuppressive effect) detected in wolves during 2007-2008 (Figure 1) could
explain not only the apparent sarcoptic mange morbidity increase in Asturian wolves
during 2008 (Oleaga et al. 2011), but also the absence of previous sarcoptic mange
peaks reported in this wolf population despite the endemic nature of this parasitosis
in sympatric red foxes (with periodical morbidity peaks in this small carnivore in the
study area). These data highlight also the need of further investigation in order to
elucidate the possible relevance of CDV in red fox´s sarcoptic mange epidemiology
and reported periodical morbidity peaks in the study area.
Data reported herein remark the role of wild carnivores as hosts and potential
reservoirs of studied agents. We highlight the need of further studies dealing with the
health status of rural dogs and transmission rates among domestic and wildlife
populations, with CDV and CPV vaccination of dogs as an option to reduce the
infection pressure of circulating pathogens in the wildlife.
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
146 _____________________________________________________________________________
CONCLUSIONS
The presence of antibodies against sarcoptic mange, CDV and CPV in wolves
from Asturias (Northern Spain) revealed a widespread contact with these 3 agents
during the seven years of our study period (2004-2010). Nevertheless, the apparent
population and density maintenance since 2004 to date suggest a moderate long-
term population effect of these pathogens on wolves from the study area during last
years. The pathogen showing a higher average seroprevalence value was CPV
(62%). CDV, CPV and sarcoptic mange (transmitted by direct contact) showed higher
seroprevalence values in the areas with higher wolf densities (West and Central
regions of Asturias).
The statistically significant association detected between sarcoptic mange and
CDV seroprevalence highlights the need of considering multiple pathogen-host
systems in wildlife sanitary studies and surveillance. The monitoring and survey of
concomitant pathogens as a whole provides a more realistic approach for the
understanding of diseases and their management and conservation implications in
wildlife. We therefore emphasize the importance of long-term monitoring of wolf and
sympatric species in relation to sanitary status and environmental change for a better
understanding of disease dynamics and their relevance in the conservation of this
wild carnivore.
ACKNOWLEDGEMENTS
This is a contribution to the agreement between CSIC and Principado de
Asturias. We thank the rangers of the game preserves (specially Jaime Marcos
Beltrán an Francisco Alonso Mier) for their help in carcasses submission, Jose Luis
García Díaz and Rafael Alba Zarabozo for their help in field work, and our colleagues
from SERIDA (Ana Balseiro, J. Miguel Prieto), ARENA S.L. (Luis llaneza),
TRAGSATEC (Ginés Expósito) and IREC (Óscar Rodríguez, María Suárez and
Mariana Boadella) for their assistance in necropsy and laboratory work. We wish to
________________________________________________________________________ Capítulo 3
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thank also Catherine Lutton for revising the English of the manuscript. Project RTA
2009-00114-00-00 (INIA) contributed to this study.
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Almberg, E.S., Mech, L.D., Smith, D.W., Sheldon, J.W., Crabtree, R.L., 2009. A
Serological Survey of Infectious Disease in Yellowstone National Park’s Canid Community. PLoS ONE 4(9), e7042. doi:10.1371/journal.pone.0007042.
Barker, I.K., Parrish, C.R., 2001. Parvovirus infections. In: Williams, E.S.,
Barker, I.K. (Eds), Infectious diseases of wild mammals, 3rd edn, 131-146. Iowa State University Press, Ames, Iowa, pp. 113-146.
Blanco, J.C., Sáenz de Buruaga, M., Llaneza, L., 2008. Canis Lupus. In:
Palomo, L.J., Gisbert, J., Blanco, J.C. (Eds), Atlas y Libro Rojo de los Mamíferos Terrestres de España. SECEM-ICONA, Madrid.
García, E. J., Llaneza, L., 2012. Situación del lobo en Asturias, 2011. (Informe
inédito) Consejería de Medio Ambiente, Ordenación del Territorio e Infraestructuras del Principado de Asturias. Oviedo.
Godinho, R., Llaneza, L., Blanco, J.C., Lopes, S., Álvares, F., García, E.J.,
Palacios, V., Cortés, Y., Talegón, J., Ferrand, N., 2011. Genetic evidence for multiple events of hybridization between wolves and domestic dogs in the Iberian Peninsula. Molecular Ecology 20, 5154-5166.
Greene, C.E., Appel, M.J., 2006. Canine distemper. In: Greene, C.E. (Ed),
Infectious diseases of the dog and cat. Saunders/Elsevier: Philadelphia; London. Hawley, D.M., Altizer, S.M., 2011. Disease ecology meets ecological
immunology: understanding the links between organismal immunity and infection dynamics in natural populations. Functional Ecology 25, 48–60.
Holzman, S., Conroy, M.J., Davidson, W.R., 1992. Diseases, parasites and
survival of coyotes in south-central Georgia. Journal of Wildlife Diseases 28, 572-580.
Llaneza, L., Fernández, A., Nores, C., 1996. Dieta del lobo en dos zonas de
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Mech, L.D., Kurtz, H.J., Goyal, S., 1997. Death of a wild wolf from canine parvoviral enteritis. Journal of Wildlife Diseases 33, 321–322.
Müller, A., Silva, E., Santos, N., Thompson, G., 2011. Domestic dog origin of
canine distemper virus in free-ranging wolves in Portugal as revealed by hemagglutinin gene characterization. Journal of Wildlife Diseases 47, 725-9.
Munson, L., Terio, K.A., Kock, R., Mlengeya, T., Roelke, M.E., Dubovi, E.,
Summers, B., Sinclair, A.R., Packer, C., 2008. Climate Extremes Promote Fatal Co-Infections during Canine Distemper Epidemics in African Lions. PLoS ONE 3(6), e2545. doi: 10.1371/journal.pone.0002545.
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Todd, A.W., Gunson, J.R., Samuel, W.M., 1981. Sarcoptic mange: an important disease of coyotes and wolves of Alberta, Canada. In: Chapman, J.A. and Pursley, D. (Eds), Worldwide Furbearer Conference Proceedings. Frostburg, MD, pp. 706-729.
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CAPÍTULO 4
COMPARACIÓN DE LAS CARACTERÍSTICAS PATOLÓGICAS E
INMUNOHISTOQUÍMICAS DE LA SARNA SARCÓPTICA EN CINCO
ESPECIES SIMPÁTRICAS DE FAUNA SILVESTRE DEL PRINCIPADO
DE ASTURIAS
Oleaga, A., Casais, R., Prieto, J. M., Gortázar, C., Balseiro, A.
(2012) COMPARATIVE PATHOLOGICAL AND IMMUNOHISTOCHEMICAL
FEATURES OF SARCOPTIC MANGE IN FIVE SYMPATRIC WILDLIFE SPECIES IN
NORTHERN SPAIN. European Journal of Wildlife Research 58, 997-
1000.
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152 _____________________________________________________________________________
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RESUMEN
El presente estudio recopila información sobre patología de la sarna sarcóptica en
ciervo, corzo, rebeco, zorro y lobo simpátricos del Norte de España, y discute las variaciones
observadas en las respuesta desplegada por las diferentes especies frente a Sarcoptes
scabiei. El lobo fue la única especie estudiada en mostrar prevalencia de la forma alopécica
de hipersensibilidad en detrimento de la forma hiperqueratótica de respuesta,
generalmente observada en herbívoros y zorro en el área de estudio. Este trabajo muestra la
inmunohistoquímica como una valiosa herramienta para el estudio y diagnóstico de sarna
sarcóptica en aquellas especies cuya respuesta inmunitaria o grado de afección limita el
número de ácaros detectables, uno de los problemas con que podemos enfrentarnos al
trabajar con sarna sarcóptica en fauna silvestre.
ABSTRACT
This study collects pathological information concerning sarcoptic mange in sympatric
red deer, roe deer, chamois, wolf and red fox from Northern Spain, and discusses reported
variations in the response from the different species against Sarcoptes scabiei. Wolf is the
only studied species which shows prevalence of the hypersensitivity (alopecic) response to
the detriment of the hyperkeratotic form, usually observed in ungulates and fox. The
present work shows the use of immunohistochemistry as a valuable tool for the study and
diagnosis of sarcoptic mange in those species whose immune response or stage of
infestation limits the number of detectable mites, not an unusual difficulty faced when
working with sarcoptic mange in wildlife species.
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
154 _____________________________________________________________________________
INTRODUCTION
Sarcoptic mange is a common, widespread, highly contagious skin disease of
mammals caused by the mite Sarcoptes scabiei. This parasitosis has been reported
in many species of wild mammals worldwide (Pence and Ueckerman 2002). In
Spain, epizootic sarcoptic mange has been described in Cantabrian chamois and
Spanish ibex with high mortality rates and population declines during the first
years after appearance (Fernández-Morán et al. 1997; León Vizcaino et al. 1999),
whereas an enzootic status of the disease has been reported in red foxes (Gortázar
et al. 1998) and little information is still available regarding the effects of this
parasite on wolf populations (Oleaga et al. 2011). The epidemiology and
pathology of sarcoptic mange in wildlife populations seems to differ between
different areas of the world and animal species (Pence and Ueckerman 2002),
with two pathological forms described: a parakeratotic form, consistent with a type I
(immediate) hypersensitive response and an alopecic form, consistent with a type IV
(delayed) hypersensitive response (Bates 2003; Skerrat 2003).
The aim of this study is to collect pathological information concerning sarcoptic
mange lesions in different sympatric wild species and to discuss their possible
variation in the response against S. scabiei.
MATERIAL AND METHODS
From 2003 to 2010, 22 red deer (Cervus elaphus), 10 chamois (Rupycapra
pyrenaica parva), 9 wolves (Canis lupus), 8 red foxes (Vulpes vulpes) and 2 roe deer
(Capreolus capreolus) with sarcoptic mange compatible lesions and mite isolation
(see Alasaad et al. 2011) were necropsied in the frame of the wildlife disease
surveillance programme of Asturias (Northern Spain). Animals were collected in
population control hunts by wildlife officers or found dead. Macroscopic lesions were
recorded and for histopathological studies skin samples from wolves (n=9), foxes
(n=8), red deer (n=6), chamois (n=3) and roe deer (n=2) were collected and
submitted to standard histological procedures. Several serial sections, 4 μm thick were
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cut from each sample and stained with hematoxylin and eosin (HE).
Immunohistochemical examination was performed by means of the peroxidase
anti-peroxidase (PAP) method and the sections incubated with a rabbit antiserum
against the S. scaibei Ssλ20ΔB3 antigen (Casais et al. 2007) diluted 1 in 700.
Preimmunization rabbit serum was used as negative control. Positive and negative
control tissues were also included.
RESULTS
Macroscopically, lesions in the ungulate species (red deer, roe deer and
chamois) consisted of crusty skin lesions with abundant mites presented during
isolation procedures. The location and progression of detected lesions agreed on
the whole with those described for another ungulate affected by sarcoptic mange as
the Spanish ibex (León Vizcaino et al. 1999). In wild canids, two different
presentations were reported: the most frequently detected in red fox included
crusty lesions leading to an extremely thickened and fissured skin, especially in the
back, head, rear legs and base of tail. As reported in affected ungulates, red foxes
with this kind of lesions showed an extremely poor body condition and a large
number of mites in studied skin pieces. The second sarcoptic mange presentation
detected in two red foxes but representing the only form reported in the nine
studied wolves, included alopecia as the main lesion, mildly thickened skin often with
a slate-grey colour in affected areas and almost complete absence of crusts or
fissuring. This “alopecic” presentation hardly allowed isolation of S. scabiei, while
seemed to affect body condition less than the “parakeratotic” form described above.
Thus, while red fox, red deer, roe deer and chamois often succumb to the disease in
the study area, mangy wolves have not been found in such poor body condition.
Microscopically, the histological study revealed ungulates and foxes exhibiting a
crusting dermatitis characterised by hyperkeratosis, acanthosis, severe degeneration
of epidermal cells, presence of large number of mites and an inflammatory infiltrate
consisting mainly of lymphocytes, macrophages and eosinophils (Table 1, Fig. 1). The
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
156 _____________________________________________________________________________
Lesions/features
Red deer
(Cervus
elaphus)
Chamois
(Rupycapra
pyrenaica parva)
Roe deer
(Capreolus
capreolus)
Wolf (Canis lupus) Red fox (Vulpes vulpes)
Hyperplasia epidermis Yes Yes Yes Yes Yes
Hyperplasia of
sebaceous glands
Yes Yes Yes Yes Yes
Hyperkeratotic form Yes Yes Yes No Yes
Alopecic form Low Low Low Yes Yes
Abundance of mites Numerous Numerous Numerous Scarce/absence Hyperkeratotic form: Numerous
Alopecic form: scarce
Inflammatory
infiltrate
Macrophages, plasma cells, lymphocytes, histiocytes and
eosinophils
Macrophages, lymphocytes
and neutrophils
Hyperkeratotic form: macrophages,
lymphocytes and eosinophils
Alopecic form: macrophages,
lymphocytes and neutrophils
Immunohistochemistry Positivity mainly in mites Positivity in macrophages
in 5 wolves
Hyperkeratotic form: positivity
mainly in mites
Alopecic form: positivity in
macrophages
Mortality Yes Yes Yes No Yes
Table 1- Comparative histopathological and immunohistochemical features in five sympatric wildlife species affected by sarcoptic mange.
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predominant alteration in wolves was an alopecic hypersensitive reaction with almost
complete lack of mites and the presence of an infiltrate consisting of macrophages,
lymphocytes and few neutrophils (Table 1, Fig. 1). In two foxes, both hyperkeratotic
and alopecic forms were observed (Table 1, Fig. 1). Sebaceous gland hyperplasia
and hair follicles clogged with keratin were observed in all studied species.
Positive immunostaining was detected in 5/9 wolves, 8/ 8 foxes, 6/6 red deer,
3/3 chamois and 2/2 roe deer. This reaction was noted in the integument of the
epidermis and in the cavities surrounding vital organs of the mites and in the cells
forming the inflammatory infiltrate (Table 1, Fig. 1). Interestingly, the positive
immunolabelling in ungulates and foxes showing the hyperkeratotic form was
predominantly noted in mites, whereas wolves and foxes affected by the
hypersensitivity (alopecic) form (virtually without mites in skin sections) showed a
strong positive immu-nolabelling mainly located in macrophages in the dermis
(see Fig. 1). This feature seems to reflect the important role that macrophages
can play in the apparently more effective alopecic form against S. scabiei
developed by wolves in the study area.
DISCUSSION
The scarce number of mites detected in wolves and red foxes with the
hypersensitivity (alopecic) form contrasts with the numerous mites observed in
ungulates and red fox (hyperkeratotic form). As the hypersensitivity reaction
progresses mites decrease in the lesions (Arlian 1996). Numbers of eosinophils
in the dermis are correlated with the density of mites, suggesting that recruitment of
eosinophils is influenced by mites or their products (Skerrat 2003). In animals
showing the alopecic form, neutrophils were observed forming the inflammatory
infiltrate. Although neutrophils can be related to secondary infections or skin damage,
the oxidation burst of neutrophils may be important in eliminating mites (Arlian
1996). Although not all mangy confirmed wolves offered immunohistochemical
positive result, the extremely low number of live mites detected in studied
mangy wolves ( ranging from 1 to 78 mites, Oleaga et al. 2011 ) confers
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158 _____________________________________________________________________________
Figure 1- Comparative analysis of pathological features of the skin from wildlife species using hematoxylin–eosin stain (HE) and immunohistochemistry (PAP). a Red deer showing sarcoptic mange in neck, legs and ventral surface of the body. b Severe hyperkeratosis and presence of mites. HE; bar0100 μm. c Inflammatory infiltrate consisted of macrophages, lymphocytes and eosinophils. HE; bar 0100 μm. d Mites in stratum corneous with intense immunolabelling. PAP; bar0 100 μm. e Chamois showing sarcoptic mange. f Hyperkeratosis, acanthosis and numerous mites in the epidermis. HE; bar 0100 μm. g Inflammatory infiltrate in the dermis consisted of macrophages, lymphocytes and eosinophils. HE; bar 0100 μm. h Positive immunolabelling against S. scabiei in mites. PAP; bar 0100 μm. i Roe deer showing sarcoptic mange. j Skin showing hyperplasia in the epidermis, one mite and an inflammatory infiltrate in the dermis. HE; bar 0100 μm. k Inflammatory infiltrate in the dermis consisted of macrophages, lymphocytes and eosinophils. HE; bar 0100 μm. l Positive immunolabelling in a mite. PAP; bar 0100 μm. m Wolf with sarcoptic mange showing alopecic areas in the body. n Alopecic form of sarcoptic mange. A mite can be seen located in the stratum corneous. HE; bar 0100 μm. o Inflammatory infiltrate in the dermis consisted of macrophages, lymphocytes and neutrophils. HE; bar0100 μm. p Positive immunolabelling in macrophages in the dermis. PAP; bar 0 100 μm. q Red fox showing sarcoptic mange. r Hyperkeratotic form of sarcoptic mange in the fox. HE; bar0100 μm. s Alopecic form of sarcoptic mange in the fox. HE; bar 0100 μm. t Alopecic form of sarcoptic mange in the fox. Positive immunolabelling in macrophages in the dermis. PAP; bar 0100 μm.
immunohistochemistry a special value as survey and diagnostic technique of
sarcoptic mange in this species. When the low number of mites prevents their
detection in lesions or when samples cannot be immediately submitted for live mites
isolation, and histopathology cannot be used as a reliable marker of infestation,
immunohistochemistry has proven to be a useful tool in order to discern sarcoptic
mange from other pathologies.
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Both macroscopical and microscopical skin lesions in wolves (at least in the
nine wolves surveyed in the present study) suggest a prevalence of the
hypersensitivity (alopecic) response to the detriment of the hyperkeratotic form
against sarcoptic mange in this wild canid. The coincidence in wolves of this
presentation with the apparent higher ability of the species to control the parasite
(Oleaga et al. 2011), not reported in the rest of species surveyed in the present
work, could indicate that this form of immune response is a more effective defense
mechanism against S. scabiei. The intimate immunological mechanisms developed
by wolf should be better studied and compared with species “taxonomically close”
like red fox, where the same mite seems to trigger different pathological features
and population effects.
A correct interpretation of observed pathological findings of sarcoptic mange
and an appropriate understanding of the immune response mechanisms and
their effectiveness are key pieces in surveillance programmes dealing with wildlife
species and also in their populations' management. The present work shows the use
of immunohistochemistry as a valuable tool for the study and diagnosis of sarcoptic
mange in those species whose immune response or stage of infestation limits the
number of detectable mites, not an unusual finding when working with sarcoptic
mange in wildlife species.
ACKNOWLEDGMENTS
The authors thank Cotos de Caza, Veterinary Services of the Sección de Caza de
la Consejería de Medio Ambiente del Principado de Asturias, and our colleagues of
IREC Oscar Rodríguez and María Suárez for helping with the sample collection. Dr.
Marta Muñoz is thanked for critically reviewing the manuscript. Dr. Ana Balseiro is
recipient of a “Contrato de Investigación para Doctores” from the Instituto
Nacional de Investigación Agraria y Agroalimentaria (INIA). This project is
supported by INIA RTA2008-00041-00-00 and is a contribution to the agreement
between CSIC and Principado de Asturias.
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160 _____________________________________________________________________________
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Benito, J.L., Feliu, C., Nieto, J.M., 1997. Epizootiology of sarcoptic mange in a population of Cantabrian chamois (Rupicapra rupicapra parva) in northwestern Spain. Vet. Parasitol. 73, 163–171.
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Enzootic sarcoptic mange in red foxes in Spain. Z. Jagdwiss. 44, 251–256. León Vizcaino, L., Ruiz de Ibáñez, M.R., Cubero-Pablo, M.J., Ortiz, J.M.,
Espinosa, J., Pérez, L., Simón, M.A., Alonso, F., 1999. Sarcoptic mange in Iberian ibex from Spain. J. Wild. Dis. 35, 647–659.
Oleaga, A., Casais, R., Balseiro, A., Espí, A., Llaneza, L., Hartasánchez, A.,
Gortázar, C., 2011. New techniques for an old disease: sarcoptic mange in the Iberian wolf. Vet. Parasitol. 181, 255–266.
Pence, D.B., Ueckerman, E., 2002. Sarcoptic mange in wildlife. Rev. Sci. Tech.
OIE 21, 385–398. Skerrat, L.F., 2003. Cellular response in the dermis of common wombats
(Vombatus ursinus) infected with Sarcoptes scabiei var. wombati. J. Wildl. Dis. 39, 193–202.
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CAPÍTULO 5
CARACTERIZACIÓN Y COMPARACIÓN A NIVEL MOLECULAR DE
ÁCAROS Sarcoptes scabiei PRESENTES EN CINCO ESPECIES
SIMPÁTRICAS DE FAUNA SILVESTRE DEL PRINCIPADO DE
ASTURIAS
5.1.- ESTABILIDAD TEMPORAL EN LA ESTRUCTURA GENÉTICA DE
Sarcoptes scabiei: EVIDENCIAS EMPÍRICAS EN FAUNA SILVESTRE
DE ASTURIAS
5.2.- EPIDEMIOLOGÍA GENÉTICA DE Sarcoptes scabiei EN LOBO
IBÉRICO EN ASTURIAS
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
162 _____________________________________________________________________________
_______________________________________________________ _ Capítulo 5
_____________________________________________________________________________ 163
5.1.- ESTABILIDAD TEMPORAL EN LA ESTRUCTURA GENÉTICA DE
Sarcoptes scabiei: EVIDENCIAS EMPÍRICAS EN FAUNA SILVESTRE
DE ASTURIAS
Alasaad, S.*, Oleaga, A.*, Casais, R., Rossi, L., Molinar Min, A., Soriguer, R.C., Gortázar, C. (2011) TEMPORAL STABILITY IN THE
GENETIC STRUCTURE OF SARCOPTES SCABIEI UNDER THE HOST-TAXON LAW:
EMPIRICAL EVIDENCES FROM WILDLIFE-DERIVED SARCOPTES MITE IN
ASTURIAS, SPAIN. Parasites & Vectors 4, 151.
* Contributed equally
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
164 _____________________________________________________________________________
_______________________________________________________ _ Capítulo 5
_____________________________________________________________________________ 165
RESUMEN
Antecedentes: De manera implícita, los estudios moleculares de parásitos asumen
estabilidad genética a lo largo del tiempo. En este estudio evaluamos, por primera vez
según nuestros datos, la importancia de los posibles cambios presentes en la diversidad y
estructura genéticas de poblaciones del ácaro Sarcoptes aislados en rebecos (Rupicapra
pyrenaica) de Asturias (España), utilizando una PCR multiple diseñada para la detección de
9 marcadores moleculares microsatélite y muestras de Sarcoptes procedentes de rebeco,
ciervo (Cervus elaphus), corzo (Capreolus capreolus) y zorro (Vulpes vulpes) simpátricos.
Resultados: El análisis de ácaros recogidos con un intervalo de 11 años mostró escasos
cambios en la diversidad genética (diversidad de alelos, y heterocigosidad observada y
esperada). Esta estabilidad temporal de la diversidad genética fue confirmada mediante el
análisis de estructura de poblaciones, que no resultó variable de forma significativa a lo
largo del tiempo. El análisis de estructura de poblaciones reveló estabilidad temporal en la
diversidad genética del ácaro Sarcoptes bajo la ley del “host-taxon” (con ácaros Sarcoptes
diferenciados entre herbívoros y carnívoros) en fauna silvestre simpátrica de Asturias.
Conclusiones: La confirmación de estabilidad genética a lo largo del tiempo es de vital
interés para permitir la realización de generalizaciones, con notables implicaciones en
cuanto a estructura genética, epidemiología y protocolos de seguimiento y estudio del
ácaro Sarcoptes. Este hecho podría eventualmente ser aplicado a otros parásitos.
ABSTRACT
Background: Implicitly, parasite molecular studies assume temporal genetic stability. In
this study we tested, for the first time to our knowledge, the extent of changes in genetic
diversity and structure of Sarcoptes mite populations from Pyrenean chamois (Rupicapra
pyrenaica) in Asturias (Spain), using one multiplex of 9 microsatellite markers and
Sarcoptes samples from sympatric Pyrenean chamois, red deer (Cervus elaphus), roe deer
(Capreolus capreolus) and red fox (Vulpes vulpes).
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
166 _____________________________________________________________________________
Results: The analysis of an 11-years interval period found little change in the genetic
diversity (allelic diversity, and observed and expected heterozygosity). The temporal
stability in the genetic diversity was confirmed by population structure analysis, which was
not significantly variable over time. Population structure analysis revealed temporal
stability in the genetic diversity of Sarcoptes mite under the host-taxon law (herbivore
derived- and carnivore-derived Sarcoptes mite) among the sympatric wild animals from
Asturias.
Conclusions: The confirmation of parasite temporal genetic stability is of vital interest
to allow generalizations to be made, which have further implications regarding the genetic
structure, epidemiology and monitoring protocols of the ubiquitous Sarcoptes mite. This
could eventually be applied to other parasite species.
INTRODUCTION
In the field of parasitology, different molecular markers have been used for
parasite genetic characterization and genetic population studies. All molecular
studies assume that genetic structure and diversity is relatively stable over time
(Garant et al., 2000; Health et al., 2002). Since allele presence and frequency
change over time due to genetic drift, and because of the gene flow between
parasite populations from sympatric host species, the assumption of genetic
stability may not be accurate (Lessios et al., 1994). Here we describe, for the
first time to our knowledge, a temporal analysis of microsatellite alleles and
genetic structure at nine polymorphic loci to examine changes in genetic
diversity of Sarcoptes mite over time.
Sarcoptes mite continues to affect humans and a wide range of mammalian
hosts worldwide (Alasaad et al., 2011), while the debate about its specificity by
the host is still the subject of ongoing debate (Walton et al., 2004a). An epidemic
can result, just from the introduction of a single case of scabies into crowded
living conditions (Obasanjo et al., 2001), which could entail devastating mortality
in wild and domestic animals (Heukelbach & Feldmeier, 2006; Soulsbury et al.,
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2007). Moreover, recent biochemical and molecular approaches highlighted the
threat of emerging acaricide resistance to the treatment of scabies worldwide
(Mounsey et al., 2010).
Sarcoptes mite infections are endemic in many European wild animals and
may cause devastating mortality, which has been reported in the Alpine
(Rupicapra rupicapra) and Pyrenean chamois (Rupicapra pyrenaica parva),
Iberian ibex (Capra pyrenaica), aoudad (Ammotragus lervia) and red fox
(Vulpes vulpes) (Fandos 1991; Mörner 1992; Pérez et al., 1997; León-Vizcaíno
et al., 1999; González-Candela et al., 2004; Rossi et al, 2007; Alasaad et al.,
2009a). Notwithstanding, in other sympatric hosts only a few cases have ever
been reported such as stone marten (Martes foina), badger (Meles meles),
lynx (Lynx lynx), roe deer (Capreolus capreolus) and Iberian wolf (Canis lupus)
(Ryser-Degiorgis et al., 2002; Oleaga et al. 2008a; 2011).
Pyrenean chamois (Rupicapra pyrenaica parva) population in Asturias
(Northern Spain) was affected by a sarcoptic mange epizootic, first detected
in 1993. Although the origin of the parasitosis could not be demonstrated,
infected domestic goats sharing pastures with wild bovids were suspected to
be the source of mites, with subsequent evidence of this cross-infection
possibility (Lavín et al., 2000; Menzano et al., 2007). As reported in epidemics
affecting other wild ungulate populations (León-Vizcaíno et al., 1999;
González-Candela et al., 2004; Rossi et al, 2007), Sarcoptes scabiei produced
an extremely severe effect on chamois population during the first years
after eruption (Fernández-Moran et al., 1997). Nowadays the disease can be
considered endemic and is still the main health issue affecting Southern
chamois.
The number of Sarcoptes generations is influenced by the short
generation interval, as well as by the infected host’s susceptibility and life
expectancy, and hence Sarcoptes mites on an individual host may in fact form
an ‘infra-population’ (Bush et al., 1997) that has a number of recurrent
generations (Alasaad et al., 2008a). Sarcoptes population structure is probably
that of a species subdivided into genetically small populations with restricted
gene flow between local demes (Martínez et al., 1999). Strong specialisation
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
168 _____________________________________________________________________________
could be the result of a host taxon-derived shift and, even if two host taxon-
derived species are sympatric for their host species, they should be considered
as allopatric, if the parasites have no possibility of host choice (Rasero et al.,
2010).
The aim of the present study was to test the extent of possible changes in
the genetic diversity and structure of Sarcoptes mite population from
Pyrenean chamois in Asturias within an 11-years interval period (from the
epidemic wave in 1997 to the endemic situation in in 2008), and to compare
reported molecular data with samples from mangy sympatric red deer, roe
deer and red foxes.
METHODS
Specimen collection and DNA extraction
Using postponed isolation and direct isolation (with aqueous potassium
hydroxide digestion) techniques (Alasaad et al., 2009b) sixty representative
adult mites were collected during two different periods, 1997 and 2008: (i) in
1997, twenty Sarcoptes mite were collected from the skin crust of ten infected
Pyrenean chamois, and (ii) in 2008, fourteen parasites were collected from
the skin crust of seven infected Pyrenean chamois, two mites from two
mangy roe deer, thirteen from eight infected red deer, and twelve from six
red fox.
All mites were identified as S. scabiei on the basis of known
morphological criteria (Fain 1968). The DNA of individual Sarcoptes mites was
extracted using the HotSHOT Plus ThermalSHOCK technique (Alasaad et al.,
2008b) as following: 25 μl of an alkaline lysis reagent (25 mM NaOH, 0.2 mM
disodium EDTA; pH = 12) was used as a substrate for individual
Sarcoptes mite DNA extraction by three cycles of thermal shock (2 min at -
80°C, freezing step, and 15 s at +70°C, thawing step), followed by a short
incubation (30 min at 95°C) and pH adjustment with 25 μl of a neutralizing
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reagent (40 mM Tris-HCl; pH = 5). Two blanks (reagents only) were included
in each extraction to monitor for contamination.
Fluorescent-based polymerase chain reaction analysis of microsatellite
DNA
As described by Alasaad et al. (2008a), nine specific Sarcoptes mite
microsatellites (Sarms 33-38, 40, 41 and 44) were used with one 9× multiplex
PCR. One primer from each set was 5’ labelled with 6-FAM, VIC, NED or
PET® fluorescent dye tag (Applied Biosystems, Foster City, CA, USA). Each
15 μl PCR reaction mixture consisted of 3 μl of the single mite DNA, together
with the PCR mixture containing all primer pairs (ranged from 0.04 to 0.1 μM
per primer), 200 μM of each dNTP, 1.5 μl of 10× PCR buffer (200 mM KCl and
100 mM Tris-HCl, pH 8.0), 1.5 mM MgCl2 and 0.15 μl (0.5 U/reaction) HotStar
Taq (QIAGEN, Milano, Italy). The thermal profile in a 2720 thermal cycler
(Applied Biosystems, Foster City, CA, USA) was as following: 15 min at 95°C
(initial denaturing), followed by 37 cycles of three steps of 30 s at 94°C
(denaturation), 45 s at 55°C (annealing) and 1.5 min at 72°C (extension),
before a final elongation of 7 min at 72°C. Fluorescent PCR amplification
products were analyzed using formamide with Size Standard 500 Liz (Applied
Biosystems, Foster City, CA, USA) by ABI PRISM 310 Genetic Analyzer with
pop4. Allele calling was performed using the Gene-Mapper v. 4.0 software
(Applied Biosystems, Foster City, CA, USA).
Molecular analyses
Expected (HE) and observed (HO) heterozygosity, linkage disequilibria
(LD), and Hardy-Weinberg equilibrium (HWE) tests were calculated using
GENEPOP (v.3.4; Raymond & Rousset, 1995). Deviations from HWE and tests
for LD were evaluated using Fisher’s exact tests and sequential Bonferroni
corrections. We estimated genetic diversity using three values; mean
number of alleles, expected, and observed heterozygosity based on data
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
170 _____________________________________________________________________________
from all nine loci. Possible genotyping mistakes (scoring error due to
stuttering, large allele dropout) were estimated using MICROCHECKER
(Oosterhout et al., 2004).
The heterogeneity of genetic diversity among the different Sarcoptes mite
populations was estimated by the partition of variance components (AMOVA)
applying conventional FST statistics using allele frequencies as implemented
in Arlequin 3.11 (Excoffier 2006). The analysis of relationships between mites
was carried out by the Bayesian assignment test of the software STRUCTURE
(v.2.3.3; Pritchard et al., 2000). Burn-in and run lengths of Markov chains
were both 100000. We ran 30 independent runs for each K (for K = 1-10).
The most likely number of clusters was determined using the method of
Evanno et al. (Evanno et al., 2005). Finally, each of the inferred clusters was
associated with the component populations of its mites.
The degree of genetic relationship among populations was further
investigated with FCA (Factorial Component Analysis) as implemented in
Genetix v.4.05.2 (Belkhir 1999).
RESULTS
Twenty-nine alleles were detected from the nine microsatellite loci. The
allele count for each of the 9 loci ranged from two (Sarms41) to four (Sarms35,
Sarms37 and Sarms38). Sixteen private alleles (alleles present in only one
population) were detected; all of them were from red fox populations, while no
private alleles were detected from the other populations (Table 1). The number
of private alleles ranged between one (Sarms34, Sarms36 and Sarms41) and
three (Sarms35 and Sarms37).
The missing data from all the used microsatellite loci was 0.0315, ranging
between 0 (for Sarms33, Sarms 37, Sarms38 and Sarms41) and 0.13 (for
Sarms36). For all loci examined there was no evidence of LD [linkage
disequilibria] (P > 0.05), and no deviation from HWE [Hardy-Weinberg
equilibrium] was detected from all loci in all the studied population except
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Sarms34 and Sarms38 in Pyrenean chamois collected in 1997, and Sarms33,
Sarms35-38, and Sarms44 in red fox population.
Allele diversity was identical in all loci from both Pyrenean chamois
populations from 1997 and 2008, with the exception of Sarms34 and Sarms38:
Sarms34 was monomorphic with only 176 bp allele present in Pyrenean
chamois from 1997, while Pyrenean chamois from 2008 has two alleles, 176 bp
and 198 bp. The new allele (198 bp) is present in all the other herbivore
sympatric populations (red deer and roe deer), but not in the carnivore-derived
Sarcoptes population (red fox). Sarms38 was monomorphic with only 215 bp
allele present in Pyrenean chamois from 1997, while Pyrenean chamois from
2008 has two alleles, 213 bp and 215 bp. Again, the new allele (213 bp) is
present in all the other herbivore sympatric populations, but not in the carnivore
red fox population.
Intra-host variation was detected in six individuals: one Pyrenean
chamois from 1997 (variation in Sarms34), three red deer (variation in
Sarms34), and two red foxes (variations in Sarms35 and Sarms40).
Locus Allele Frequency
Sarms 33 232 0.5833 240 0.4167
Sarms 34 174 1
Sarms 35 148 0.7222
152 0.1111
156 0.1667
Sarms 36 283 0.6000
Sarms 37 164 0.1667
170
170
1700.5833
0.5833
178 0.2500 Sarms 38 209 0.4167
211 0.5833
Sarms 40 217 0.7222
243 0.2778
Sarms 41 234 1
Sarms 44 270 0.3636
272 0.0909
Table 1- Private alleles
detected at the 9
microsatellite loci of the red
fox-associated mite
population, together with
their frequencies.
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172 _____________________________________________________________________________
R. pyrenaica-1997 R. pyrenaica-2008 C. elaphus-2008 V. vulpes-2008
R. pyrenaica-1997 - 0.054 < 0.001* < 0.001*
R. pyrenaica-2008 0.1919 - 0.099 < 0.001*
C. elaphus-2008 0.4564 0.1179 - < 0.001*
V. vulpes-2008 0.8542 0.7869 0.7556 -
Table 2- Matrix of significant FST P values, with significance level = 0.05 (above diagonal), and population pairwise FST (below diagonal) for each pairwise comparison of four Sarcoptes mite populations collected in 1997 and 2008 from Asturias, Spain (C. capreolus-2008 was not included because of the low sampling size).
AMOVA analysis showed differentiation among populations (FST =
0.74808; P < 0.001), which indicates that the mite component populations
differed greatly. FST value between both chamois-derived Sarcoptes mite
populations was not statically supported (FST = 0.1919; p = 0.054), while red
fox-derived Sarcoptes mite population was statistically (P < 0.001) different from
all other herbivore-derived Sarcoptes mite populations (Table 2).
These results were confirmed by the average number of pairwise
differences between Sarcoptes populations: the lowest differentiation was
between the two chamois-derived Sarcoptes populations, and the highest
was between red fox-derived Sarcoptes population and the other herbivore-
derived Sarcoptes mite populations (Table 3). No pairwise differences were
detected within R. pyrenaica (1997)-derived Sarcoptes mite population, while
the highest value of the average number of pairwise differences was detected
within V. vulpes-derived Sarcoptes mite population.
The modal value of the statistic ΔK (Evanno et al., 2005) for the whole
dataset showed that the uppermost cluster value was K = 2 (Figure 1).
When K = 2, all cluster assignments were consistent with the population of
origin. Sarcoptes mites from all herbivore hosts were consistently grouped in
one cluster, while Sarcoptes mite from red fox (carnivore host) formed another
well-supported cluster (Figure 2).
The posterior probability analyses supported our results, since we
obtained similar grouping when applying K = 3, 4, 5 and 6, which demonstrates
the complete resolution of populations into distinct clusters (herbivore- and
carnivore-derived Sarcoptes mite populations), and illustrates the
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subpopulations within R. pyrenaica-2008 and V. vulpes-derived Sarcoptes mite
populations (Figure 2).
The scatter plot of the FCA, for individuals (data not shown) and
populations (Figure 3) of the microsatellite genotypes using Sarcoptes mite
collected from the sympatric wild animals from Asturias, confirmed the results
obtained by the Bayesian assignment test. The two chamois-derived
Sarcoptes populations were similar and close to the other herbivore-derived
populations (red deer and roe deer), and well-differentiated from the carnivore
(red fox)-derived Sarcoptes population.
R. pyrenaica-1997 R. pyrenaica-2008 C. elaphus-2008 V. vulpes-2008
R. pyrenaica-1997 0.00000 0.23077 0.69231 6.91667
R. pyrenaica-2008 0.03385 0.39385 0.70118 6.83974
C.elaphus-2008 0.27077 0.08272 0.84308 6.91667
V. vulpes-2008 5.61957 5.34572 5.19803 2.59420
Table 3- Population average pairwise differences between four Sarcoptes mite derived populations from Asturias, Spain. Above diagonal: Average number of pairwise differences between populations (PiXY). Diagonal elements: Average number of pairwise differences within population (PiX). Below diagonal: Corrected average pairwise difference (PiXY-(PiX+PiY)/2). C. capreolus-2008 was not included because of the low sampling size.
Figure 1- Results of STRUCTURE analysis showing Δ K as proposed by Evanno et al. [27] method. The best fit of the data was two clusters.
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
174 _____________________________________________________________________________
DISCUSSION
As with other highly divergent taxa, with Sarcoptes scabiei few loci and
low sample sizes are sufficient to find strong population differentiation
between host species (Walton et al., 2004b; Tadano et al., 2008). The
unusually high number of private alleles in red fox population, was the first
indicator of the genetic separation and lack of gene flow between Sarcoptes
mite from this carnivore animal and the sympatric herbivores (roe deer, red
deer and Pyrenean chamois), which is in concordance with the host-taxon
effect among Sarcoptes populations from different sympatric wild animals
(Rasero et al., 2010).
The few detected cases of intra-host variations could be attributed to
the skin-scale phenomenon (Alasaad et al., 2008a), while the deviation from
HWE presented in some loci from the red fox population could be attributed
to possible subpopulations within Sarcoptes mites from this host (Hardy
1908; Stern 1943). Sarcoptes mites lack free-living stages, and individual hosts,
depending on their susceptibility and behaviour, are essentially ephemeral
habitats providing patchy environments that hamper random mating (Price
1980; Criscione et al., 2005).
Two new alleles were detected in two different loci from the 2008-
Pyrenean chamois population comparing with the 1997-population. Both new
alleles are present in all the other herbivore sympatric populations, but not in
the carnivore red fox population. This could be understood as small
change in allele diversity within chamois-derived Sarcoptes population
during the 11- years interval period, which could be attributed, simply to non-
random sampling, or to little gene flow from the other sympatric herbivore
(red deer and roe deer)-derived Sarcoptes mite, (since our first 1997-
sampling coincided with the start of Sarcoptes outbreak wave in chamois, and
it is possible that more waves of Sarcoptes transmission with the other
sympatric herbivore hosts took place until the current endemic situation of
Sarcoptes mite in this 2008-chamois population) but never from the
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carnivore sympatric host (red fox), following the host-taxon law (Rasero et al.,
2010).
Figure 2. Bar plotting of the proportion of individual variation of 60 Sarcoptes mite from different host species in Asturias (Spain) collected with 11-years interval, assigned to a given genetic clusters in STRUCTURE, when two (A: K = 2), three (B: K = 3), four (C: K = 4), five (D: K = 5), and six (E: K = 6) populations are assumed in the dataset. Each cluster is represented by a different colour. 1: R. pyrenaica-1997. 2: R. pyrenaica-2008. 3. C. capreolus-2008. 4: C. elaphus-2008. 5: V. vulpes-2008.
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
176 _____________________________________________________________________________
All AMOVA analysis (showing differentiation among populations), the
Bayesian assignment test (between mites), and the scatter plot of the FCA
(for individuals and populations) confirmed the absence of genetic
differentiation between the two chamois-derived Sarcoptes mite populations
collected in 11-year interval period. On the other hand, the results
corroborate the presence of a host-taxon phenomenon and lack of gene flow
or recent admixture between carnivore- and herbivore-derived Sarcoptes
populations, among Sarcoptes mites from wild animals in Asturias, in
concordance with other European wild hosts (Rasero et al., 2010).
Mite transmission may occur within each host taxon-derived Sarcoptes
mite population (explaining temporal and geographical coincidences reported
between chamois and red deer sarcoptic mange cases in the studied area
and confirming their suspected common origin; Oleaga et al., 2008), but it
seems to be extremely rare or absent between them (Rasero et al., 2010).
Figure 3- Factorial Component Analysis (FCA) of the proportion of variation of five Sarcoptes mite populations from Asturias (Spain) assigned to a given genetic clusters in Genetix.
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CONCLUSIONS
The analysis of 11-year interval period found little change in the
genetic diversity and showed clear temporal stability in the genetic structure of
Sarcoptes mite population under the host-taxon law. The understanding of this
factor is crucial, if generalizations are to be made concerning temporal genetic
stability. Besides the genetic implications of our results, this study could have
further ramification in the epidemiological studies and the monitoring
protocols of the neglected Sarcoptes mite, and could have further
applications in other parasite species.
ACKNOWLEDGEMENTS
We would like to thank S. Maione, R. Rasero and D. Soglia (Università
degli Studi di Torino, Italy) for offering laboratory infrastructure. Thanks are
also due to Jaime Marcos Beltrán, Francisco Alonso Mier (Gobierno del
Principado de Asturias), Óscar Rodríguez, María Suárez (IREC) and Alberto
Espí (SERIDA) for their assistance in field, necropsy and laboratory work. The
experiments comply with the current laws of the countries in which the
experiments were performed. The research was supported by a RTA 2009-
00114-00-00 (INIA) project, RNM-6400, Proyecto de Excelencia (Junta de
Andalucia, Spain) and Juan de la Cierva grant. This work is a contribution to
the agreement between IREC-CSIC and Principado de Asturias.
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Oleaga, A.*, Alasaad, S.*, Rossi, L., Casais, R., Vicente, J.,
Maione, S., Soriguer, R.C., Gortázar, C. (2013) GENETIC
EPIDEMIOLOGY OF SARCOPTES SCABIEI IN THE IBERIAN WOLF IN ASTURIAS,
SPAIN. Veterinary Parasitology 196, 453-459.
5.2.- EPIDEMIOLOGÍA GENÉTICA DE Sarcoptes scabiei EN LOBO
IBÉRICO EN ASTURIAS
* Contributed equally
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RESUMEN
Antecedentes: Durante las últimas décadas se vienen produciendo intentos para tratar
de entender la epidemiología genética de Sarcoptes scabiei y de detectar y clarificar las
diferencias existentes entre ácaros de diferentes hospedadores y regiones geográficas. Han
sido descritos dos fenómenos principales: (i) la infección por variedades de Sarcoptes
determinadas por el taxón al que pertenece su hospedador (revelando la existencia de tres
grupos separados de Sarcoptes, pertenecientes a poblaciones europeas de herbívoros,
carnívoros y omnívoros -enunciada como “host-taxon law”-) y (ii) la infección presa-predador
de Sarcoptes descrita en el ecosistema de Masai Mara (“prey-to-predator effect”).
Resultados: utilizando una PCR multiple diseñada para la detección de 9 marcadores
moleculares microsatélite y muestras de Sarcoptes procedentes de rebeco, ciervo, zorro y
lobo simpátricos, diferentes análisis de estructura de poblaciones revelaron concordancia
con la ley “host-taxon” descrita en fauna silvestre europea, con dos poblaciones principales
de ácaros Sarcoptes diferentes, una aislada en herbívoros y la otra en carnívoros.
Sorprendentemente, las poblaciones de ácaros aisladas en lobo mostraron la mayor
diversidad genética de entre todas las analizadas, incluyendo dos subpoblaciones diferentes:
una similar a las poblaciones propias de herbívoros, y otra similar a la derivada de carnívoros
(zorro).
Conclusiones: la ley del “host-taxon” en fauna silvestre se confirma al mantenerse la
separación entre un grupo derivado de carnívoros y otro de herbívoros en los ácaros
analizados. Sin embargo, este fenómeno parece influido por la inclusión de un gran predador
como el lobo en este trabajo, revelando infección de Sarcoptes vía presa-predador entre los
taxones estudiados y sugiriendo la importancia del sistema inmune del lobo a la hora de
explicar la gran variabilidad registrada en ácaros aislados de Canis lupus. Más estudios
acerca de la dieta, comportamiento y movilidad, así como sobre el papel desempeñado por
el sistema inmune del lobo, resultarían muy interesantes para tratar de esclarecer las
posibles interacciones entre ambas hipótesis descritas, “host-taxon law” y “prey-to-predator
infection”.
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186 _____________________________________________________________________________
ABSTRACT
Background: During the last decades, attempts have been made to understand the
molecular epidemiology of Sarcoptes scabiei, and to detect and clarify the differences
between isolates from different hosts and geographic regions. Two main phenomena have
been described: (i) host-taxon derived-Sarcoptes mite infection in European wild animals
(revealing the presence of three separate clusters, namely herbivore-, carnivore- and
omnivore-derived Sarcoptes populations in Europe) and (ii) prey-to-predator Sarcoptes mite
infection in the Masai Mara ecosystem.
Results: Using one multiplex of 9 microsatellite markers and Sarcoptes mite samples
from sympatric Pyrenean chamois, red deer, red fox and Iberian wolf, different population
structure analyses revealed concordance with the host-taxon law described for wild animals
in Europe, with two main host-derived Sarcoptes mite populations, herbivore- and carnivore-
derived. Surprisingly, Iberian wolf derived Sarcoptes populations had the highest genetic
diversity among the other populations, including two different subpopulations: one similar
to the herbivore-derived Sarcoptes populations, and another similar to carnivore (fox)-
derived Sarcoptes mite population.
Conclusions: The host-taxon effect in wild animals is still supported with the
maintenance of carnivore- and herbivore-derived Sarcoptes clusters’ separation in analyzed
mites. However, this phenomenon could be modified with the inclusion of a large predator
as wolf in the present work, revealing prey-to-predator Sarcoptes mite infection between
the studied host-taxa and suggesting the importance of wolf’s immune system for explaining
the high variability reported in C. lupus derived mites. Further studies of host diet, behavior
and movement, and regarding the role played by its immune system, would be of great help
to clarify interactions between the two hypotheses, host-taxon and prey-to-predator.
INTRODUCTION
Despite representing the first human illness with a known etiologic
agent (Montesu et al., 1991; Gakuya et al., 2012a) and affecting more than
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100 mammal species all over the world (Bornstein et al., 2001; Pence and
Ueckerman, 2002), the taxonomical status of the ectoparasitic burrowing mite
Sarcoptes scabiei has been the subject of continuous debate for decades
(Pence et al., 1975; Fain, 1978; Andrews, 1983). The development of
molecular techniques has provided new approaches and novel data on this
subject (Zahler et al., 1999; Walton et al., 2004a; Gu and Yang, 2008); in
particular, microsatellites are a valuable technique for the analysis of S.
scabiei and the relationship between hosts. Microsatellite analysis supports
the standing of S. scabiei as a single highly variable species with different
strains manifesting physiological host-specificity (Walton et al., 2004b;
Alasaad et al., 2008b, 2011b, 2012).
A mange epizootic first detected in 1993 in the southern border of
Asturias severely affected Southern chamois (Rupicapra pyrenaica parva) from
the area (Fernández- Moran et al., 1997), leading to an overall 61.3% reduction
of their population during the first years after the outbreak (González-Quirós et
al., 2002). Although the origin of the outbreak could not be demonstrated, it has
been attributed to mangy domestic goats sharing pastures with chamois in the
study area, with subsequent cross-infection (Lavín et al., 2000; Menzano et
al., 2007). The epizootic is still expanding eastwards today with sporadic
cases of sarcoptic mange in chamois, red deer (Cervus elaphus) and roe deer
(Capreolus capreolus – Oleaga et al., 2008a,b) from the area. Red foxes
(Vulpes vulpes) and wolves (Canis lupus) have also been diagnosed with
sarcoptic mange in Western, Central and Eastern Regions of Asturias, and
disease is now considered endemic in red foxes.
Three main groups of S. scabiei mites have been genetically identified in
European wildlife studied so far, namely herbivore, carnivore and omnivore-
derived Sarcoptes clusters (Rasero et al., 2010). These mite “strains” occur in
sympatric as well as geographically distant populations, including Southern
chamois, red deer, roe deer and red fox in our study area in Asturias, Northern
Spain (Alasaad et al., 2011b). In sympatric mammals, differentiation of mites
into “strains” may result from null or limited interspecific transmission, due to
low frequency of direct contacts among hosts, low densities of one or more
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188 _____________________________________________________________________________
hosts, or different immunological response to the invading mites, limiting their
population size, hence their number on the skin surface and their
transmissibility to other individuals of the same or a different species. On the
other hand, genetically based preference for a particular host (leading to host-
associated mating) or the evolution of higher performance or viability on that
host (usually entailing a worse performance on the others, Kassen, 2002) have
been proposed as possible mite-dependent mechanisms limiting gene flow
(Magalhães et al., 2007). The apparent lack of gene flow or recent admixture
between carnivore-, herbivore-, and omnivore-derived Sarcoptes populations
reported in European studied species, including sympatric animals, led to the
so called “host-taxon law” formulation (Rasero et al., 2010; Alasaad et
al.,2011b).
Parasites are of pivotal importance in food webs. Moreover food webs
are very incomplete without parasites (Lafferty et al., 2006), with
endoparasites (Sukhdeo, 2012) as the most frequently described trophically
transmitted parasites (Luong et al., 2013). The only report, to our knowledge,
about ecto-parasites transmission was on Sarcoptes mite transmission in
Masai Mara ecosystem (Gakuya et al., 2011). Theoretically, the higher
frequency of direct contacts among hosts within trophic chains (especially
when acting as predator, considering that prey are not a “long-lasting
available habitat” after predation for ectoparasitic mites) should favor a more
efficient gene flow between different mite “strains”. Nevertheless, the study of
S. scabiei molecular features in a predator/prey ecosystem has not been
carried out in any European model, so far. Recently, molecular and
epidemiological studies including wild felids and ungulates in Africa have
signaled the existence of a potential predator/prey bond in sarcoptic mange
transmission, the so called prey-to-predator parasitic infection (Gakuya et al.,
2011, 2012b).
The recent isolation of S. scabiei mites in 9 out of 12 wolves bearing
skin lesion in Asturias (Northern Spain) suggested an increase in morbidity of
sarcoptic mange in this wild canid population (Domínguez et al., 2008; Oleaga
et al., 2011) and allowed a genetic study of mites in this host for the first time.
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This work aims to study the genetic structure of S. scabiei mites affecting
wolves in Northern Spain, and to genetically compare them with mites
originating from sympatric wild herbivores (red deer and Southern chamois)
and carnivores (red fox). Both wild and domestic ungulates are common prey
of the wolf in the study area (Meriggi and Lovari, 1996; Barja, 2009), whereas
predation and scavenging on mangy foxes have been suspected as a source
of infection in this top chain carnivore (Bornstein et al., 1995; Mörner et al.,
2005; Domínguez et al., 2008). Such molecular analyses could hep in
determining the extent to which sarcoptic mites may be interspecifically
transmitted in one of the few available large predator-prey food chains in
Europe.
MATERIALS AND METHODS
Study area
The study was carried out in the Principality of Asturias, a 10,603 km2
autonomous region located in North-Western Spain. The area, with a mixture
of open pastures and meadows with deciduous and mixed forests, is home of
many different wildlife species, including carnivores like the endangered
brown bear (Ursus arctos), wolf and red fox, and several wild ungulates (red
deer, roe deer, wild boar – Sus scrofa – and Cantabrian chamois) that
represent a high percentage of the diet of wolves (Llaneza et al., 1996).
Sampling and mite isolation
The study of wolves submitted for necropsy in Asturias (Northern Spain)
as part of a wildlife diseases surveillance program allowed the confirmation of
sarcoptic mange by mite isolation in 9 out of 47 wolves from 2008 to 2010
(Oleaga et al., 2011), and the extraction of genetic material from mites
belonging to 8 of these 9 mangy wolves (seven of them were collected in
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190 _____________________________________________________________________________
population control hunts carried out by wildlife officers, while one wolf died as
a consequence of vehicle collision). Acari were collected after incubation of 8
skin pieces from each animal on Petri dishes for 24 h at 37 ◦C and meticulous
examination for the detection and identification of ectoparasites using an
Olympus SZX9 (10–57×) magnifier (Alasaad et al., 2009). Collected mites
were identified as S. scabiei according to Wall and Shearer (1997) and
preserved in 70% ethanol until DNA extraction. DNA was extracted from 19
mites belonging to 8 mangy confirmed wolves (seven adults and two 6 month
old pups).
Mites obtained from 7 chamois (n = 14), 8 red deer (n = 13) and 6 red
foxes (n = 12) with confirmed sarcoptic mange were also collected in Asturias
from 2006 to 2010, and analyzed in order to compare molecular features with
those reported in Iberian wolves.
DNA extraction and fluorescent-based polymerase chain reaction (PCR)
analysis of microsatellite DNA
The DNA of individual Sarcoptes mites was extracted using the HotSHOT
Plus ThermalSHOCK technique (Alasaad et al., 2008a). Two blanks (reagents
only) were included in each extraction to monitor for contamination.
As described by Alasaad et al. (2008b), nine specific Sarcoptes mite
microsatellites (Sarms 33–38, 40, 41, and 44) were used with one 9×
multiplex PCR. One primer from each set was 5’ labeled with 6-FAM, VIC, NED
or PET® fluorescent dye tag (Applied Biosystems, Foster City, CA, USA). Each
15 µl PCR reaction mixture consisted of 3 µl of the single mite DNA, together
with the PCR mixture containing all primer pairs (ranging from 0.04 to 0.1 µM per
primer), 200 µM of each dNTP, 1.5 µl of 10× PCR buffer (200 mM KCl and 100 mM
Tris–HCl, pH 8.0), 1.5 mM MgCl2 and 0.15 µl (0.5 U/reaction) HotStar Taq
(QIAGEN, Milano, Italy). The thermal profile in a 2720 thermal cycler (Applied
Biosystems, Foster City, CA, USA) was as follows: 15 min at 95 ◦C (initial
denaturing), followed by 37 cycles of three steps of 30 s at 94 ◦C
(denaturation), 45 s at 55 ◦C (annealing) and 1.5 min at 72 ◦C (extension),
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before a final elongation of 7 min at 72 ◦C. Fluorescent PCR amplification
products were analyzed using formamide with Size Standard 500 Liz (Applied
Biosystems, Foster City, CA, USA) by ABI PRISM 310 Genetic Analyser with
pop4. Allele calling was performed using the GeneMapper v. 4.0 software
(Applied Biosystems, Foster City, CA, USA). Possible genotyping mistakes
(scoring error due to stuttering, large allele dropout) were estimated using
MICROCHECKER (Oosterhout et al., 2004).
Molecular analyses
Expected (HE) and observed (HO) heterozygosity, linkage disequilibria (LD),
and HWE tests were calculated using Genepop (v.3.4 [Raymond and Rousset,
1995]). Deviations from HWE and tests for LD were evaluated using Fisher’s
exact tests and sequential Bonferroni corrections.
The heterogeneity of genetic diversity among the different Sarcoptes
mite populations was estimated by the partition of variance components
(AMOVA) applying conventional FST statistics using allele’s frequencies as
implemented in Arlequin 3.11 (Excoffier, 2006). The analysis of relationships
between mites was carried out by the Bayesian assignment test of the
software Structure (v.2.3.3 [Pritchard et al., 2000]). Burn-in and run lengths of
Markov chains were both 100,000. We ran 20 independent runs for each K
(for K = 1–10). The most likely number of clusters was determined using two
approaches; by estimating the posterior probability for each K, using the
method of Evanno et al. (2005). Finally, each of the inferred clusters was
associated with the component populations of its mites.
The degree of genetic relationship among populations was further
investigated with FCA as implemented in Genetix v.4.05.2 (Belkhir, 1999).
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192 _____________________________________________________________________________
RESULTS
Twenty-eight alleles were detected in the 9 microsatellite loci studied in
wolf-derived Sarcoptes mites, including one private allele (present only in the
wolf population, Sarms 44). The number of alleles for each locus ranged in
the wolf population from two (Sarms40 and Sarms 41) to four (Sarms35,
Sarms37 and Sarms38). All the alleles detected in studied ungulate species
(n = 12, with 11, 12 and 9 alleles for chamois, red deer and roe deer,
respectively, Alasaad et al., 2011b) and 18 out of the 20 reported in red foxes in
Asturias were also present in the wolf population, whereas the only two alleles
Locus# Chamois Red deer Red fox Wolf
Sarms33 226 226 226 232 232
240 240
Sarms34 176 176 176 198 198 198
174 174
Sarms35 162 162 162
148 148
152 152 156 156
Sarms36 279 279 279 279 281 281 281
283 283
Sarms37 172 172 172 164 164
170 170 178 178
Sarms38 213 213 213
215 215 215 209 209
211 211
Sarms40 215 215 215 217 217
243
Sarms41 236 236 236
234 234
Sarms44 262 262 262 262 270 270
272 268
Total number
of alleles 11 12 20 28
Table 1- Comparison of allele size
and total number of detected
alleles in the four sympatric
species-derived mite populations
studied in Asturias at the 9
microsatellite loci. Private alleles
are shown in black, while alleles
present both in ungulates and red
fox (that are also present in wolf)
are underlined and in italics.
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previously detected in Asturian wildlife but absent in the wolf were two private
alleles from red fox (Sarms40 and Sarms44, Table 1).
While mites isolated from wolves contained 27 of the 29 alleles previously
detected in S. scabiei mites derived from sympatric ungulates and red fox
from Asturias, only 3 of these 29 alleles were present both in red fox and
ungulate derived mites. This fact allowed the consideration of 9 (out of 12) and
17 (out of 20) alleles as “exclusive” for ungulate species and red fox derived
mites respectively, before including C. lupus mites in the study.
There was no evidence of linkage disequilibrium for any of the loci
examined (P > 0.05). The analysis of molecular data showed deviation from
Hardy-Weinberg equilibrium (HWE) in all the 9 microsatellite loci studied in wolf
mites, the highest value from studied wildlife species in Asturias (HWE had been
detected in two and six loci from Pyrenean chamois and red fox respectively,
Alasaad et al., 2011b).
Several mite specimens were collected from each of 4 wolves, with intra-
host variation detected in three of them. Variation was reported in all loci with
the exception of Sarms44. Sarms36 showed variation in the 3 wolves where
intra-host variation was detected, and Sarms34, Sarms37 and Sarms38
presented variation in two wolves.
Locus # Rupic. 2010 Cervus Vulpes Canis Mean s.d. Total
number
Sarms33 1 1 2 3 1.750 0.957 3
Sarms34 2 2 1 3 2.000 0.816 3
Sarms35 1 1 3 4 2.250 1.500 4
Sarms36 1 2 3 3 2.250 0.957 3
Sarms37 1 1 3 4 2.250 1.500 4
Sarms38 2 2 2 4 2.500 1.000 4
Sarms40 1 1 2 2 1.500 0.577 3
Sarms41 1 1 1 2 1.250 0.500 2
Sarms44 1 1 3 3 2.000 1.555 4
Mean 0.444 0.667 2.000 3.111 1.556 1.244 3.333
s.d. 0.882 1.000 1.225 0.782 0.972 0.191 0.667
Table 2- Number of alleles detected in mites belonging to the four sympatric species studied in Asturias.
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194 _____________________________________________________________________________
Mites isolated from wolves offered the highest mean number of alleles
(3.111 ± 0.782) from the four studied wild mammal species in Asturias, while
herbivore-derived populations showed a lower mean number of alleles (0.444 ±
0.882 in Pyrenean chamois and 0.667 ± 1.000 in red deer) than red
fox-derived mites (2.000 ± 1.225) (Table 2).
Mean expected heterozygosity was 0.049 ± 0.097 for chamois, 0.117 ±
0.181 for red deer, 0.416 ± 0.232 for red fox and 0.571 ± 0.140 for wolf.
The Bayesian assignment test of the software STRUCTURE, ln Pr(X|K)
for the likely number of populations, indicated K = 2 as the uppermost
cluster value (Fig. 1). This differentiation of two clusters as the best fit
grouped Sarcoptes mites from all herbivore hosts in one cluster, while
Sarcoptes mites from red fox formed another cluster and wolves showed mites
similar to one or the other cluster (Fig. 2).
AMOVA analysis showed differentiation among populations (FST =
0.48352; P < 0.001), signaling that the mite component populations differed
greatly. The wolf-derived Sarcoptes mite population was statistically (P < 0.001)
different from those living in the two studied herbivore species (chamois and
red deer) and from red fox-derived Sarcoptes mite populations. Herbivore-
derived mite populations formed only one group (P = 0.081). There were
statistically supported (P < 0.001) genetic differentiations between the other
Figure 1- Results of STRUCTURE analysis showing ∆K as proposed by Evanno et al. (2005) method. The best fit of the data was two clusters.
_______________________________________________________ _ Capítulo 5
_____________________________________________________________________________ 195
Figure 2- Bar plot of the degree of individual variation between 58 S. scabiei from different host species in Asturias (Spain) assigned to given genetic clusters in STRUCTURE, when two (K = 2) populations are assumed in the dataset. Each cluster is represented by a different color.
species-derived Sarcoptes mite populations. FST between wolf-derived and
herbivore-derived Sarcoptes mite populations was lower than that between fox-
derived and herbivore-derived Sarcoptes mite populations (Table 3).
The scatter plot of the Factorial Component Analysis (FCA) for the
individual mites collected on chamois, red deer, red fox and wolf, agreed with
the results obtained by the Bayesian assignment test: while red deer and
chamois derived mites were similar to each other and appeared grouped, red
fox mites gathered separately, and mites collected on wolves were grouped in
part with the ungulate-derived mites and in part with the red fox-derived mites
(Fig. 3).
Table 3. Matrix of fixation index (FST) significant P values, with significance level P=0.05 (above diagonal), and population pairwise FST (below diagonal) for each pairwise comparison of four Sarcoptes mite populations from Asturias, Spain. (* Significance level p = 0.05.)
Rupicapra
pyrenaica
Cervus
elaphus
Vulpes
vulpes
Canis
lupus
Rupicapra pyrenaica
Cervus elaphus
0.118
0.081 <0.001*
<0.001*
<0.001*
<0.001*
Vulpes vulpes 0.742 0.709 <0.001*
Canis lupus 0.374 0.332 0.236
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
196 _____________________________________________________________________________
DISCUSSION
The present study reveals that wolf mites show the highest number of
different alleles (n = 28) out of the four wild species surveyed (n = 20 alleles in
red fox, with none of the ungulate species housing mites with more than 12
detected alleles [Alasaad et al., 2011b – Table 1]). Wolf mites contained 27 of the
29 alleles previously detected in S. scabiei originating from sympatric ungulates
and red fox in Asturias, thus explaining the low number of private alleles reported
in this work and suggesting limited genetic separation and relatively high gene
flow between Sarcoptes mite populations when including wolf in the analysis. The
higher variability reported in C. lupus mites agrees with results dealing with the
mean number of alleles, deviation from Hardy-Weinberg equilibrium (HWE),
intra-host variation and mean expected heterozygosity, all of them showing in
wolf mites the highest values found in the four analyzed host species.
Regarding the genetic diversity among the different Sarcoptes mite populations,
the obtained FST values are to be considered high, but they are similar to
those obtained in previous studies on S. scabiei (e.g. Gakuya et al., 2011). We
have no clear explanation of such high FST values, however the questionable
taxonomic status of this parasite could be behind this high FST values, and
hence further molecular studies with wider range of molecular markers are
needed.
In the Masai Mara (Kenya) ecosystem (Gakuya et al., 2011), the genetic
study of mites belonging to different species revealed “prey-to-predator”
Sarcoptes gene flow leading to deviations from the host taxon phenomenon
formulated by Rasero et al. (2010). This work is the first one, in Europe, in
which a large predator and the corresponding main sylvatic prey are included in
a study aimed to investigate the molecular diversity and gene flow of Sarcoptes
mites. As in the case of other large predators in Masai Mara, the inclusion of
wolf permitted to investigate the outcome of the enhanced opportunities of
Sarcoptes mite transmission between host taxa within a consolidated
“predator/prey” chain, with the eventual evasion of the genetic separation of
host-associated mites. In Gakuya’s work (2011) the “favorite prey” effect led
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to the identification of four different clusters and highlighted inconsistencies with
the host-taxon law. As an interesting difference, in our case data showed the
maintenance of the two previously described host-associated clusters (red fox
vs. herbivore derived mite populations, Figs. 1–3, Alasaad et al., 2011b) and
an apparently milder “prey-to-predator” effect associated with the presence of
wolf, whose mites belonged to both clusters with scarce variations. The
molecular results in the present work can be better assessed when
considering FCA information (showing mites distributed within those from red
fox or ungulates, Fig. 3) and the Bayesian assignment test for the likely number
of populations K (with K = 2 as the uppermost cluster value, Fig. 1). These data
confirm a substantial compliance with the “host taxon law”, with maintenance of
a differentiation of mites in two populations (ungulate and red fox derived),
and the appearance in wolf of mites belonging to one of these two recognized
clusters in Asturias (thus revealing certain “prey to predator” effect). The bar plot
representing the degree of individual variation between S. scabiei from the
different studied host species (Fig. 2) agrees with this view of wolf being able to
“collect” mites both from red fox and from ungulates.
Fig. 3. Factorial Component Analysis (FCA) of the proportion of variation of Sarcoptes mite populations of red deer, chamois, red fox and wolf from Asturias (Northern Spain) assigned to given genetic clusters in Genetix.
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
198 _____________________________________________________________________________
In the “prey-to-predator” Sarcoptes gene flow reported in wolves in
Asturias, two main differences with the Masai Mara ecosystem results can be
found: (i) a more evident “carnivore to carnivore” (red fox-wolf) gene flow than
observed between lion and cheetah and (ii) an apparent lack of real gene flow,
“evolution” or mixture between red fox- and herbivore-derived mite populations,
even though wolves may harbor mites belonging to both. The first difference is
probably related to the different ecological role that the red fox plays compared
to the wolf, lion or cheetah, implying a different nature and frequency of
contacts between wolf and red fox in comparison with the two African predators.
On the other hand, further research is necessary to clarify the reasons for
the apparent lack of gene admixture and scarce “variation-evolution”
suggested by molecular data in wolf derived mites.
It is the Authors’ opinion that peculiarities of the immune response
developed by the wolf against invading Sarcoptes mites may be a key in
the understanding of reported molecular data. As shown by Oleaga et al.
(2012), in scabietic wolves the hypersensitivity-associated alopecic form is
prevalent on the aneargy-associated hyperkeratotic form which is usually
observed in ungulates and red fox. Demographic and necropsy data suggest
that this predator is able to exert a certain control over infestation with S.
scabiei (Oleaga et al., 2011), in contrast with the usually deadly development
of scabies in the other studied sympatric species in Asturias (Oleaga et al.,
2012). The apparent efficiency of the immune system of the wolf against mite
proliferation may suggest (i) the development of a limited number of mite
generations on it and (ii) the possibility of repeated infestations with mites from
any of the sympatric species this canid may get into contact (as suggested by
the detection, in the present work, of one wolf infested by both “ungulate-like”
and “red-fox-like” mites). These two features may hamper host adaptation and
facilitate gene flow among mite populations, thus preventing genetic selection
and favoring the high variability reported in C. lupus derived mites. A similar
situation has been suggested in wild boars, which show greater resistance to
these parasites than other mammalian species and whose mites showed high
variability and heterozygosity parameters (Rasero et al., 2010). In the case of
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wolf, the wide range of geographical movement and the large number of
species this canid can get into contact (both as predator and as scavenger) are
additional arguments favoring repeated infestations with genetically distinct
mites from different species.
To complement this type of study it would be interesting to obtain mites
isolated from sympatric domestic animals, such as dogs or goats as a possible
source of sarcoptic mange for ungulates and even wolf in Asturias. The
implementation of tools like the Sarcoptes-World Molecular Network
(Sarcoptes- WMN, Alasaad et al., 2011a) can improve collaboration between
researchers and allow more profound studies in the future.
Taking into account these and previous results dealing with hosts
(namely wild boars) which have not been considered in this study (Rasero et
al., 2010), it seems necessary focus on the pathobiology of the mites and the
efficiency of the immune response developed by the different host species,
as key aspects for a better interpretation and tuning of the “host-taxon law”
when studying gene flow and evolution of parasitic mite populations. On the
other hand, the role that host species play in studied ecosystems, especially
when including susceptible carnivores displaying preying behavior on mangy
prey, must be also considered for a correct evaluation of a possible “prey-to-
predator” effect. The consideration of mites and their hosts as an ecosystem
in constant change and adaptation process requires due attention not only to
the biology, ecology and behavior of host species, but also to their immune
system performance and the possibly host species population-specific
variability of clinical presentations of sarcoptic mange.
ACKNOWLEDGEMENTS
This is a contribution to the agreement between CSIC and Principado de
Asturias. The research was partially supported by RNM-6400, Proyecto de
Excelencia (Junta de Andalucia, Spain), Project RTA 2011-00087-00-00 (INIA),
and Juan de la Cierva Grant to Dr S. Alasaad. We thank the rangers of the game
preserves (especially Jaime Marcos Beltrán and Francisco Alonso Mier) for their
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
200 _____________________________________________________________________________
help in carcass submission and our colleagues from IREC and SERIDA
(Óscar Rodríguez, María Suárez and Alberto Espí) for their assistance in
necropsy and laboratory work. We wish to thank also Kevin Dalton for
revising the English of the manuscript.
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DISCUSIÓN GENERAL
D.1- SÍNTESIS DE LOS HALLAZGOS MÁS RELEVANTES
D.2- LÍNEAS DE INVESTIGACIÓN Y TRABAJOS PENDIENTES
D.3- CONCLUSIONES
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DISCUSIÓN GENERAL
Puesto que todos los artículos que conforman este trabajo incluyen una
discusión pormenorizada de los resultados parciales más relevantes expuestos en
cada uno de los mismos, hemos considerado oportuno tratar de evitar reiteraciones
organizando la discusión final de la tesis en dos apartados generales: D.1-) una
síntesis y enfoque integral de aquellos hallazgos más relevantes aportados por la
presente tesis, y D.2-) una somera descripción de las posibles líneas de
investigación sobre sarna sarcóptica y de los trabajos pendientes de conclusión
abordables en el futuro. Finalmente se exponen las principales conclusiones
extraídas como resultado de los trabajos desarrollados (D.3).
D.1- SÍNTESIS DE LOS HALLAZGOS MÁS RELEVANTES
D.1.a-) Avances en la caracterización clínica de la sarna sarcóptica en las
diferentes especies estudiadas
La caracterización de la sarna sarcóptica resultó de especial interés en corzo,
ciervo y lobo, especies sobre las que la bibliografía e información disponibles en
cuanto a su afección por S. scabiei son escasas. Se trata de especies poco
frecuentes o bien raramente afectadas por este proceso (al menos atendiendo a la
bibliografía científica existente), y por tanto difíciles de estudiar a este nivel. Los
datos presentados caben por tanto ser considerados de ayuda en la identificación y
diagnóstico de este proceso por parte de otros investigadores.
De este modo, el capítulo 1 constituye la primera descripción detallada
recogida en la bibliografía científica sobre las características clínicas macro y
microscópicas del proceso en el caso concreto del corzo, una especie muy
raramente descrita como víctima de esta enfermedad (Menzano et al., 2008). El
estudio de un corzo hallado aún con vida severamente afectado por sarna
sarcóptica, sin otras lesiones o afecciones distintas de las provocadas por S. scabiei
(como pudo comprobarse en la posterior necropsia) permitió valorar los síntomas y
alteraciones de comportamiento que pueden llegar a producirse en las fases
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
208 _____________________________________________________________________________
terminales del proceso en este pequeño cérvido. Por otro lado, permitió evaluar los
efectos de esta parasitación sobre diferentes parámetros bioquímicos sanguíneos en
esta especie.
El segundo trabajo presentado en el capítulo 1 supone el primer estudio
publicado centrado en sarna sarcóptica en el ciervo ibérico. Tanto las lesiones
macroscópicas observadas en los ciervos estudiados (corroboradas mediante un
buen número de necropsias adicionales efectuadas tras la publicación del artículo,
figura S.1) como los signos apreciados en los ciervos con sarna observados con
vida, presentaron notables similitudes con los descritos para otras especies de
ungulados más habitualmente afectadas por el mismo agente, como el rebeco o la
cabra montés.
Al igual que en el caso del corzo y el ciervo, los registros existentes sobre las
características clínicas del proceso en lobos afectados por sarna son escasos (Todd
et al., 1981; Mörner, 1992; Domínguez et al., 2008). Esto sucede a pesar de su gran
área de distribución geográfica y de que en el caso de este cánido, la parasitosis por
S. scabiei ha sido identificada en un buen número de poblaciones de diferentes
partes del mundo. Sin embargo, es probable que el escaso número de efectivos que
suele constituir sus poblaciones, su carácter huidizo y las aparentes dificultades
existentes para efectuar un correcto diagnóstico etiológico de sarna sarcóptica en
esta especie, hayan limitado el número de trabajos realizados hasta la fecha al
respecto. El capítulo 3 constituye el primer trabajo a nivel europeo centrado en
aspectos clínicos y epidemiológicos de la sarna sarcóptica en lobo.
Diferentes hallazgos sugieren que el proceso parasitario en este cánido cursa
de un modo diferente al resto de especies estudiadas en esta tesis: (i) las lesiones
macroscópicas observadas (con alopecia como principal alteración apreciable en
piel, escaso engrosamiento dérmico y sólo en contadas ocasiones presencia de
costras o agrietamiento reseñables), (ii) el limitado efecto sobre condición corporal
registrado en los animales afectados, (iii) el escaso número de ácaros vivos aislados
en lobos afectados, así como (iv) la relación (inversamente proporcional) existente
entre dicho número de ácaros aislados y el porcentaje de superficie corporal
afectada.Estos hallazgos pueden guardar relación con un tipo diferente de respuesta
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Figura S.1- Trabajo de campo en la toma de muestras de un ciervo hallado severamente afectado por sarna en el Parque Natural de Redes (Asturias).
inmune y una aparente mayor capacidad de la misma para controlar y eludir la
parasitación por S. scabiei por parte de Canis lupus. Todos estos datos obtenidos a
partir de necropsia, así como los obtenidos mediante fototrampeo (con aparente
recuperación registrada en campo de al menos 2 individuos), parecen indicar que el
lobo presenta cierta capacidad para recuperarse y superar la sarna sarcóptica
(extremo recogido en bibliografía científica para otras poblaciones de lobo afectadas
por este parásito [Jimenez et al., 2010; Almberg et al., 2012]), a diferencia de lo
generalmente observado en las otras 4 especies objeto de estudio en Asturias.
Las dificultades descritas para el aislamiento e identificación del ácaro en lobos
supuestamente afectados por sarna sarcóptica (puesto en evidencia por el escaso
número de ácaros vivos aislados en los lobos estudiados), ha sido descrita también
como una severa dificultad en el diagnóstico de este proceso en el caso del perro
(Curtis, 2012). Esta dificultad nos llevó a la implantación de un estricto protocolo de
búsqueda y aislamiento de S. scabiei en todos los lobos remitidos en fresco que nos
permitió (i) identificar las regiones anatómicas en que resultó más probable
encontrar ácaros (orejas, extremidades posteriores y base de la cola), (ii) comparar
la cantidad de los mismos detectada con la apreciada en otras especies y (iii)
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
210 _____________________________________________________________________________
confirmar la sarna sarcóptica como auténtico agente etiológico de las lesiones
observadas (al encontrar ácaros sólo en uno de los 20 lobos remitidos en fresco que
no presentaban lesiones dérmicas y sobre los que se empleó el mismo protocolo de
búsqueda de S. scabiei).
El estudio y comparación histológicos llevados a cabo en el capítulo 4 de las
lesiones apreciadas en piel de ciervo, rebeco, corzo, zorro y lobo permitió una mejor
caracterización del tipo de lesiones detectadas, la identificación de las células
presentes en las mismas y la descripción y comparación del tipo de respuesta
inmune celular desencadenada a nivel dérmico por cada una de estas especies. Las
cinco especies comparten un mismo hábitat y condiciones ecológicas, de modo que
las características propias del ácaro y de la especie hospedadora, así como su
sistema inmune, pueden ser consideradas como los principales factores
determinantes del tipo de lesiones presentes.
La principal diferencia puesta en evidencia a nivel histológico entre las 5
especies simpátricas estudiadas fue el predominio de una reacción de
hipersensibilidad tipo IV o retardada (forma alopécica) en el lobo en contraste con la
respuesta de hipersensibilidad inmediata o tipo I (forma paraqueratótica) en todos
los casos de cérvidos y rebeco. Esta última también es la más frecuentemente
registrada en zorros afectados por sarna sarcóptica en Asturias. Los trabajos
llevados a cabo han permitido asimismo poner de manifiesto la utilidad de la
inmunohistoquímica (en este caso usando un suero policlonal frente al antígeno
Ssλ20ΔB3 de S. scaibei -Casais et al. 2007- ) como herramienta complementaria en
el diagnóstico y estudio de la sarna sarcóptica. Esta técnica puede resultar
especialmente útil en aquellos casos en que el número de ácaros es escaso o éstos
no se encuentran íntegros en la piel. Mediante esta técnica ha sido también posible
detectar diferencias relevantes en la respuesta inmune celular de las distintas
especies. Mientras que en herbívoros y la mayor parte de zorros afectados (aquellos
que presentaron la forma paraqueratótica) los propios ácaros eran prácticamente los
únicos en que la inmunotinción ponía de manifiesto la presencia de antígeno de S.
scabiei, buena parte de los animales que desarrollaron una reacción de
hipersensibilidad tipo IV o retardada (forma alopécica: todos los lobos y alguno de
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Figura S.2- Lesiones macroscópicas (A) y microscópicas (B) detectadas en el único lobo incluído en el presente trabajo en que pudo confirmarse infección mixta por sarna sarcóptica y moquillo a nivel dérmico. En la imagen microscópica (B) la inmunohistoquímica pone de manifiesto la presencia del virus del moquillo canino.
los zorros estudiados) mostraron presencia de antígeno de S. scabiei en
macrófagos. Este resultado sugiere la importancia que la respuesta inmune de tipo
celular puede tener en la aparentemente más efectiva defensa activada frente a la
sarna sarcóptica por los lobos de nuestra área de estudio (Arlian, 1996; Bates,
2003).
Finalmente la inmunohistoquímica permitió también, mediante la identificación
del virus del moquillo canino en piel de un lobo con lesiones compatibles con sarna
y confirmación de S. scabiei mediante aislamiento, la detección del único caso
clínico de coinfección a nivel dérmico detectado en sala de necropsia durante el
presente trabajo (figura S.2).
D.1.b-) Avances en el conocimiento de los efectos de la sarna sarcóptica
sobre fauna silvestre a nivel de población
Una vez caracterizado clínicamente el proceso en las diferentes especies, el
siguiente propósito de nuestro trabajo fue tratar de evaluar los efectos que la
enfermedad pudiese ocasionar en las poblaciones asturianas de fauna silvestre
afectadas por sarna sarcóptica. Nuestros esfuerzos se centraron en evaluar su
importancia a nivel sanitario y de conservación, especialmente en especies como el
ciervo o el lobo, sobre las que la información acerca de esta parasitosis resulta
mucho más escasa a nivel europeo.
A
B A
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
212 _____________________________________________________________________________
El primer capítulo aborda el brote de sarna sarcóptica con una mayor
concentración de Cervus elaphus afectados descrita hasta la fecha en Europa (con
un mínimo de 80 casos entre 1995 y 2008). Dicho capítulo incluyó el estudio clínico
efectuado sobre 2 corzos severamente afectados por sarna sarcóptica en Asturias
en 2006. A nivel europeo los casos registrados en ciervo son algo más frecuentes
que en corzo, pero generalmente en forma de brotes locales con un escaso número
de individuos implicados (Kutzer, 1966; Greßmann, 2001; Leon Vizcaino et al.,
1992). A pesar de las similitudes existentes entre el tipo de lesiones y hallazgos
clínicos apreciados en ciervo con los registrados en otras especies más
habitualmente afectadas, como bóvidos silvestres, los datos recogidos mostraron
una diferente evolución del proceso en esta especie a nivel de población. En
especies como el rebeco o la cabra montés los primeros años tras la aparición de la
onda epidémica de enfermedad se caracterizan por la detección de llamativos picos
de mortalidad y severos efectos poblacionales a corto-medio plazo (Fernández-
Moran et al., 1997; Rossi et al., 2007). En el caso del ciervo pudo registrarse un
goteo continuo de animales, con pequeños altibajos anuales en el número de bajas
registradas, que sin embargo no llegó a alcanzar las dramáticas proporciones
descritas en bóvidos silvestres afectados por sarna sarcóptica.
La estrecha relación espacio-temporal apreciada entre los casos de sarna
registrados en ciervo y en rebeco sugiere un mismo origen del proceso para ambas
especies. Sin embargo, este trabajo muestra una evolución posterior y
mantenimiento de la enfermedad en apariencia independientes para ambas
especies. Los datos recogidos sugieren asimismo una baja tasa de morbilidad y alta
de mortalidad en ciervos afectados por S. scabiei, que no parece suponer una
amenaza para la conservación de sus poblaciones en Asturias hasta la fecha.
Las Reservas Regionales de Caza de Asturias donde se detectaron casos de
sarna sarcóptica en ciervo (Caso, Sobrescobio, Infiesto, Aller y Ponga) presentaban,
durante la realización de este trabajo, las mayores densidades de este ungulado
para el Principado de Asturias. Esto sugiere la importancia de la densidad en la
presencia de la sarna en cérvidos, posibilitando i-) un contacto frecuente con el
rebeco, y ii-) una posible capacidad de la propia población de ciervos para vehicular
_______________________________________________________ _ Discusión
_____________________________________________________________________________ 213
y mantener el ácaro sin necesidad de recibirlo de dicho bóvido a partir de un
determinado umbral de densidad. En este aspecto, cabe señalar el posible papel
desempeñado por individuos sanos en el mantenimiento y transmisión del ácaro.
Así, de 8 hembras aparentemente sanas abatidas en cacerías ordinarias en el
Concejo de Caso y muestreadas al azar, S. scabiei fue detectado en piel de
extremidades de 2 de ellas, que sin embargo no presentaron lesiones apreciables
macroscópicamente, ni ningún tipo de clínica compatible con el proceso (datos no
publicados). El aparente incremento en el número de ciervos detectados con sarna
en las Reservas de Caso y Sobrescobio durante 2013 (con un número de individuos
registrados con lesiones que duplicó el mayor registro anual obtenido para el período
1995-2008) ha de servir como piedra de toque para continuar con el estudio de este
proceso y su efecto en las poblaciones locales de cérvidos. En este sentido, la
monitorización y seguimiento del número de animales presentes, el número de
individuos afectados y su evolución interanual en series suficientemente prolongadas
de tiempo resultan imprescindibles para una adecuada valoración de la sarna
sarcóptica, de su importancia en esta especie y de la posible aparición de cambios a
nivel epidemiológico.
Esta misma relevancia de la densidad en la transmisión y mantenimiento de la
parasitosis podría existir también en el caso del corzo, del que en 2012-2013 se
tiene constancia de al menos media docena de casos sospechosos (no fueron
remitidos a laboratorio) en uno de los Cotos Regionales de Caza del Principado con
una mayor densidad de este pequeño cérvido en Asturias (Pablo Quirós,
comunicación personal).
La detección de rebecos seropositivos a S. scabiei en su núcleo Occidental de
distribución en Asturias (sin casos de sarna registrados en el periodo bajo estudio,
capítulo 2) sugiere la existencia de contactos con el ácaro sin desarrollo de la
enfermedad en algunos individuos. Este fenómeno, de difícil interpretación, ya fue
descrito en rebecos no afectados de otras regiones europeas (Rambozzi et al.
2004), y ha de ponernos sobre aviso del riesgo que las cabras suponen a nivel
sanitario en esta población de rebeco cantábrico a día de hoy libre de sarna. Si bien
no es descartable el contacto de un rebeco con ácaros de otro hospedador (zorro o
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
214 _____________________________________________________________________________
incluso perro) capaces tal vez de provocar seroconversión pero no enfermedad en
el mismo, la ecología y etología de este bóvido hacen el contacto e intercambio de
ácaros con estas otras especies muy poco probable (salvo tal vez los derivados de
su presencia en fómites o en el medio, fuera del hospedador).
Los datos recopilados en el capítulo 3 han permitido indagar en la afección del
lobo por S. scabiei y la posible amenaza que pudiera suponer para la conservación
de sus poblaciones en Asturias, así como comparar las tendencias recogidas en
campo para este cánido con las registradas en zorros simpátricos. Los resultados
serológicos obtenidos señalan la existencia en Asturias de contacto del lobo con S.
scabiei al menos desde 2004, mientras que los datos de fototrampeo sugieren que la
sarna sarcóptica está presente en las poblaciones asturianas de lobo desde al
menos 2003. El aparente “pico” de morbilidad registrado en sala de necropsias
durante 2008 pudo ser confirmado en campo mediante la técnica del fototrampeo.
Sin embargo, los resultados globales obtenidos señalan que a día de hoy esta
parasitosis no parece representar una amenaza para la conservación de la especie
en Asturias. Se trata del primer trabajo publicado a nivel mundial donde se emplea la
técnica del fototrampeo como herramienta para la detección de lesiones compatibles
con una enfermedad y su estudio epidemiológico en fauna silvestre. Para ello, se
aprovechó la caracterización clínica del proceso efectuada previamente en sala de
necropsias. Esta aproximación mostró la utilidad del trampeo fotográfico para el
estudio y seguimiento de enfermedades que cursan con lesiones macroscópicas a
nivel cutáneo en poblaciones silvestres (S.3).
Ampliando en este caso el abanico de agentes patógenos estudiados, el
segundo trabajo del capítulo 3 analiza mediante serología la exposición de 88 lobos
al virus de la parvovirosis (CPV), al virus del moquillo (CDV) y al ácaro S. scabiei, y
su concomitancia como uno de los posibles factores relacionados con la
epidemiología y morbilidad de la sarna sarcóptica. En consonancia con estudios
previos llevados a cabo en la zona (Sobrino et al., 2008), de los tres agentes
estudiados, parvovirus fue el agente con una mayor seroprevalencia (61%), mientras
que moquillo y S. scabiei presentaron seroprevalencias medias en torno al 20%.
Resulta interesante que para estos tres agentes transmitidos por contacto directo, se
_______________________________________________________ _ Discusión
_____________________________________________________________________________ 215
detectaron más animales seropositivos en aquellas zonas con una mayor estimación
de densidad de lobos (y por tanto una probable mayor tasa de contacto entre
individuos). Esta hipótesis obvia sin embargo el importante papel, pendiente de
estudio, que tanto perros como el abundante zorro pueden jugar en la transmisión y
epidemiología de estos procesos en lobos asturianos.
La detección de una relación estadísticamente significativa entre la seropositividad a
moquillo y a sarna sarcóptica representa un resultado muy interesante, cuya
interpretación no resulta sin embargo sencilla. El posible efecto inmunosupresor del
virus, tal vez limitando la aparente competencia de la respuesta inmune celular
lupina frente al ácaro y facilitando su multiplicación, podría explicar una mayor
aparición de anticuerpos ante un mayor número de ácaros actuando como antígeno.
Este fenómeno permitiría relacionar el aparente pico de seroprevalencia frente a
moquillo detectado en lobos en 2007 con el llamativo aumento de lobos con lesiones
detectado durante 2008. Sin embargo, existen otras explicaciones, tal vez más
simples, que pueden esclarecer también esta relación detectada entre virus y
parásito, como una posible coincidencia en las condiciones de exposición a ambos
agentes o incluso una seroconversión más probable en animales con una mayor
capacidad de respuesta inmune humoral.
D.1.c-) Avances en el conocimiento de las relaciones interespecíficas de
la sarna sarcóptica a través de datos epidemiológicos
Otro de los objetivos marcados durante el diseño inicial de esta tesis fue el de tratar
de identificar (o descartar) la existencia de posibles relaciones interespecíficas
en la transmisión y epidemiología de esta enfermedad entre los diferentes
hospedadores analizados en Asturias. Para indagar en estas posibles relaciones,
recurrimos en primera instancia a los datos disponibles sobre distribución de las
diferentes especies y el número, momento y localización de casos de sarna
registrados en cada una de ellas (figuras S.4 y S.5) en el Principado de Asturias.
Pudo comprobarse (capítulo 1) una clara coincidencia en tiempo y espacio
entre los casos de sarna registrados en cérvidos con la presencia de esta parasitosis
en rebeco para los distintos períodos y áreas de censo en Asturias (figura S.4). Esta
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
216 _____________________________________________________________________________
Figura S.3- Imágenes de lobos en libertad obtenidas mediante fototrampeo utilizadas en la elaboración de esta tesis. Las fotografías A y B pertenecen al estudio a largo plazo efectuado mediante cámaras trampa fijas colocadas al paso (sin cebos ni atrayentes) de forma permanente entre 2003-2009. Estas 2 primeras imágenes permiten valorar la evolución a lo largo de un mes de las lesiones dérmicas presentes en un mismo individuo. Las fotografías C y D fueron tomadas por una de las 6 cámaras adicionales colocadas desde 2008 en las proximidades de áreas de reproducción confirmada para profundizar en aspectos clínicos y epidemiológicos del proceso.
coincidencia evidencia la posibilidad de transmisión interespecífica de S. scabiei
entre dichas especies, y sugiere al rebeco como origen de los casos de sarna
sarcóptica registrados en cérvidos. El análisis de los casos registrados entre 1994 y
2008 parece indicar también un mantenimiento y evolución independiente del
proceso entre bóvidos y cérvidos una vez instaurada la infección en estos últimos
A B
D C
_______________________________________________________ _ Discusión
_____________________________________________________________________________ 217
(figura S.5), en los que podría ser considerada endémica -con bajas tasas anuales
de morbilidad- actualmente.
La sospecha de que las cabras domésticas fuesen el origen del nefasto brote
inicial de sarna sarcóptica registrado en rebeco en 1993 planteó la necesidad de
incorporar al ganado doméstico en el estudio de las relaciones interespecíficas de la
enfermedad en Asturias. En el capítulo 2 se utiliza el estudio de sueros de cabra y
su comparación con datos serológicos obtenidos de rebeco de las mismas áreas
geográficas para tratar de evaluar las posibles relaciones existentes en su
exposición a S. scabiei, Pestivirus, Brucella sp. y MAP (paratuberculosis). Los datos
obtenidos señalan la presencia de anticuerpos frente a MAP, Pestivirus y S. scabiei
en ambas especies, mientras que ninguno de los sueros obtenidos en el período
2005-2008 presentó anticuerpos frente a Brucella. Los datos globales de
seroprevalencia frente a S. scabiei fueron similares en cabra doméstica y rebeco, a
diferencia de lo apreciado para la seroprevalencia de MAP, significativamente mayor
en cabras. Los datos registrados en el núcleo Oriental sugieren que la sarna se
mantiene en la población de rebecos independientemente de las cabras en esta
zona. Esto resulta compatible con el carácter endémico que la parasitosis parece
Figura S.4- Mapa del Principado de Asturias mostrando las áreas en que la sarna sarcóptica ha sido diagnosticada en las especies de fauna silvestre incluídas en esta tesis hasta 2010. : sarna sarcóptica en zorro (considerada endémica en la Península); : distribución sarna sarcóptica en rebeco; . : distribución sarna sarcóptica en cérvidos; : lobo sometido a necropsia con sarna sarcóptica diagnosticada; : lobo sometido a necropsia con lesiones compatibles con sarna pero sin confirmación mediante aislamiento; : Concejos (Proaza, Belmonte y Somiedo) en los que se empleó la técnica del fototrampeo en este trabajo, habiendo sido fotografiados en los 3 territorios tanto lobos como zorros con lesiones compatibles con sarna.
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
218 _____________________________________________________________________________
haber adquirido en las poblaciones de este bóvido silvestre. En el Núcleo Occidental,
por su parte, estos mismos datos nos alertan del riesgo que las cabras (con una
seroprevalencia frente al ácaro del 11.8%) suponen para la población local de
rebeco, no afectada por sarna sarcóptica aún, pero con preocupantes datos de
seropositividad (de difícil interpretación) en 5 individuos muestreados en esta zona.
En el capítulo 3 se planteó la posibilidad de evaluar el fototrampeo como
técnica de utilidad para el estudio epidemiológico de la sarna sarcóptica en lobo y
zorro y las posibles relaciones existentes a nivel epidemiológico entre ambas
especies de cánidos silvestres. Los resultados obtenidos ponen de manifiesto la
utilidad de cámaras de fototrampeo fijas (mantenidas durante prolongados períodos
de tiempo) colocadas para su disparo “al paso” (sin atrayentes o cebos de ningún
tipo) para evaluar presencia y variaciones en la detectabilidad de lobo y zorro, así
como para determinar el porcentaje de los mismos fotografiados con lesiones
compatibles con sarna y su variación a lo largo del tiempo. Se detectó que el
porcentaje anual de lobos con lesiones fotografiados y su evolución temporal
repitieron el mismo patrón registrado por las cámaras para el caso del zorro, pero
con 1 año de retraso. Por otro lado, mientras que los aumentos registrados en el
porcentaje de zorros fotografiados con lesiones dérmicas coincidieron con
descensos anuales del número total de zorros detectados (no necesariamente
debido a mortalidad, tal vez simplemente consecuencia de su descenso a zonas
más bajas), la detectabilidad del lobo a lo largo del período de estudio (2003-2009)
permaneció constante independientemente del porcentaje anual de lobos con
lesiones fotografiados. Nuestros resultados sugieren la capacidad del ácaro para
circular entre ambas especies y la importancia de la transmisión interespecífica en el
caso del lobo, en cuyas poblaciones el proceso no parece sin embargo tener efectos
notables a corto plazo ni suponer un problema importante de conservación hasta la
fecha en Asturias. Cabe reseñar la gran escala geográfica que fue necesario
emplear para poner de manifiesto estas relaciones epidemiológicas entre ambas
especies (incluyendo los 3 Concejos –Proaza, Belmonte y Somiedo- de los que se
disponía de información). La gran movilidad propia del lobo (especialmente
significativa en el caso de jóvenes en dispersión, que pueden desempeñar un
_______________________________________________________ _ Discusión
_____________________________________________________________________________ 219
importante papel en la transmisión de esta y otras enfermedades) así como el
tamaño de los territorios que ocupa pueden estar relacionados con esta gran escala
necesaria para evidenciar dichas relaciones.
Figura S.5- Distribución temporal (arriba) y evolución del número anual de casos (abajo) de sarna sarcóptica registrados en Asturias en las 2 últimas décadas para las diferentes especies de fauna silvestre incluidas en este trabajo. Las barras de distribución temporal (arriba) representan los años en los que la sarna sarcóptica ha sido confirmada para cada una de las especies en el Principado, mientras que las líneas de colores (abajo) indican el número de animales (eje Y) de cada especie registrados con sarna sarcóptica al año durante el desarrollo de la presente tesis. En el caso del zorro, la escasez y parcialidad de los datos disponibles (correspondientes únicamente a tres Concejos donde se empleó fototrampeo desde 2003) impidió su inclusión en la gráfica de evolución temporal del número de casos, considerando que se trata de una especie en la que el proceso es endémico desde antes de 1992 en toda Asturias (ver barra de distribución temporal).
-5
0
5
10
15
20
25
30
35
40
1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009
CIERVO
REBECO (/10)
ZORROS
LOBO (fototrampeo)
LOBO (necropsia)
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€€
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
220 _____________________________________________________________________________
D.1.d-) Avances en el conocimiento de las relaciones interespecíficas de
la sarna sarcóptica a través de la biología molecular
El capítulo 5 permitió profundizar en nuestro conocimiento acerca de la
circulación del ácaro en el medio, de su capacidad de transmisión entre diferentes
taxones y con ello de la epidemiología de la sarna sarcóptica en la fauna silvestre
por medio del estudio genético de ácaros aislados de las 5 especies afectadas en
Asturias.
Se evidenció en primer lugar la ausencia de variaciones moleculares
significativas a lo largo del tiempo (en un intervalo de 11 años) en la estructura
genética de los ácaros presentes en la población asturiana de rebecos afectada.
Este resultado tiene relevancia considerando la sospecha de que los ácaros que
parasitan al rebeco proceden de cabras domésticas afectadas con que estuvieron en
contacto antes del nefasto brote de 1993 (Fernández-Morán et al., 1997; Lavín et al.,
2000), y sugiere un origen único del brote. No resultaba por tanto descartable algún
tipo de “adaptación” a una especie diferente de hospedador como el rebeco para
optimizar su capacidad de reproducción y persistencia en el mismo de forma
independiente a la cabra, algo que sin embargo parece no haber ocurrido durante
los últimos 11 años en Asturias a tenor de la aparente ausencia registrada de
variaciones moleculares.
El estudio en este mismo capítulo de ácaros de rebeco, ciervo, corzo y zorro
simpátricos confirmó lo esperable de acuerdo con la “ley del host-taxon” (Rasero et
al., 2010), según la cual las características genéticas de los ácaros presentes en un
hospedador vienen determinadas por la especie a que pertenece. Así, y en
congruencia con dicha ley, los ácaros de rebeco, ciervo y corzo recogidos en
Asturias se agruparon en un mismo “cluster” o variedad propia de herbívoros,
mientras que los ácaros presentes en zorro mostraron una clara diferenciación
quedando incluidos en la variedad propia de carnívoros descrita en dicho trabajo
desarrollado a nivel europeo. Las características y similitudes moleculares de ácaros
de ciervo, corzo y rebeco registradas en este capítulo nos permiten confirmar la
existencia de una relación epidemiológica entre la sarna sarcóptica de cérvidos y la
_______________________________________________________ _ Discusión
_____________________________________________________________________________ 221
de rebecos (ver capítulo 1), revelada por la confirmación de flujo genético entre
ácaros de estas 3 especies en el Principado de Asturias. Por otra parte, la ausencia
de ácaros de “herbívoros” en el zorro pone de manifiesto la falta de flujo de S.
scabiei entre zorro y ungulados. A pesar de los frecuentes hábitos carroñeros de
este pequeño cánido -pudiendo por tanto entrar en contacto con ácaros de
herbívoros recién muertos por sarna-, éstos no parecen jugar un papel relevante en
la epidemiología y afección del zorro por sarna sarcóptica. Del mismo modo, el zorro
no parece representar una fuente de ácaros ni por tanto un riesgo para la sanidad de
las especies asturianas de ungulados silvestres.
La inclusión en el capítulo 5 por primera vez en Europa del estudio genético de
ácaros pertenecientes a un gran depredador como el lobo y la cadena trófica en que
participa permitió obtener interesantes datos acerca de la sarna sarcóptica en el
mismo y su relación con las otras especies afectadas por esta enfermedad en
Asturias. El lobo, en la cúspide de la cadena trófica ibérica, fue la especie con una
mayor diversidad genética de sus ácaros, que albergaron 28 de los 30 alelos
detectados en los 9 microsatélites estudiados en S. scabiei de la fauna silvestre
asturiana. Este resultado contrasta sobremanera con la detección de sólo 3 alelos
compartidos entre herbívoros y zorro, y la presencia en ácaros de este último de
hasta 17 alelos que no presentaban los ácaros de ninguna de las especies de
herbívoros estudiadas (de acuerdo con la mencionada ley del “host-taxon”).
En el caso del lobo se detectó presencia de ácaros pertenecientes a las dos
variedades previamente descritas en Asturias, la descrita en cérvidos-rebeco y la
variedad propia de zorro, sin apenas variaciones o alteraciones que hayan permitido
identificar una “variedad lobo” de S. scabiei o una evolución hacia su formación en
este gran carnívoro. Este resultado puede ser interpretado como la confirmación en
Asturias de otro fenómeno de transmisión de S. scabiei descrito, en este caso en
cadenas tróficas estudiadas en África, y conocido como el efecto “prey to predator”
(Gakuya et al., 2011). Según este efecto, existe un aparente vínculo presa/predador
en la transmisión de la sarna sarcóptica entre especies incluídas en una
determinada cadena trófica. La presencia en lobo de ácaros de zorro (al que parece
depredar con relativa frecuencia bien como alimento o como competidor) y de
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
222 _____________________________________________________________________________
herbívoros (presas y/o carroñas habituales del lobo en Asturias, probablemente con
más facilidad si se encuentran afectados por sarna) pone de manifiesto la existencia
en Asturias de este tipo de transmisión “de presa a predador”. El capítulo 5 supone
por tanto la primera descripción del efecto “prey to predator” (por primera vez en
Europa) y “host-taxon law” en una misma cadena trófica.
La ausencia en los ácaros de lobo estudiados de “mezcla” o “variación
genética” con respecto a los identificados en sus especies presa analizadas
(herbívoros y zorro) podría ser explicada atendiendo al tipo de respuesta inmune
desencadenada por este cánido. Esta respuesta parece diferente a la del resto de
especies estudiadas (en apariencia más eficaz) de acuerdo con el capítulo 4, y
podría suponer: i-) el desarrollo de un número limitado de generaciones del ácaro
sobre él, así como ii) la posibilidad de infecciones repetidas con ácaros de
cualquiera de las especies simpátricas con que este cánido puede entrar en
contacto. Estas dos características podrían explicar la ausencia de un proceso
efectivo de selección genética y la alta variabilidad reportada en los ácaros aislados
en lobo. Una vez confirmada la presencia en lobo de los mismos ácaros aislados en
herbívoros y zorros, queda descartada la existencia de una variedad diferente del
ácaro (propia del lobo) como causa de las diferencias registradas en las
características clínicas y evolución del proceso en este cánido.
Los trabajos desarrollados en la presente tesis ponen de manifiesto la
conveniencia de estudiar la sarna sarcóptica a nivel de poblaciones, y no sólo de
una especie hospedadora, sino de todas las especies simpátricas afectadas en una
determinada región. Este enfoque permite evaluar la capacidad de transmisión del
parásito entre diferentes poblaciones y especies así como determinar las posibles
relaciones existentes a nivel epidemiológico entre las mismas, permitiéndonos
comprender mejor el “funcionamiento” y epidemiología del proceso parasitario a nivel
global. La combinación de aspectos clínicos, epidemiológicos, moleculares y
ecológicos ha demostrado ser un método apropiado de estudio de la sarna
sarcóptica en fauna silvestre y nos ha permitido comprender mejor la enfermedad en
sus diferentes manifestaciones (cada una de las especies simpátricas afectadas) y
en el conjunto de Asturias como “paciente” afectado por S. scabiei.
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D.2- LÍNEAS DE INVESTIGACIÓN Y TRABAJOS PENDIENTES
Como complemento a la discusión de la presente tesis, pasamos a
continuación a enumerar una serie de tareas, posibles líneas de investigación
futuras o trabajos pendientes de conclusión relacionados con la sarna sarcóptica y
su estudio en fauna silvestre en Asturias. En cada uno de los puntos se aporta una
breve justificación o explicación de su interés e implicaciones:
D.2.a-) Establecimiento (mantenimiento en su caso) de protocolos de censo /
estimas de abundancia repetibles y comparables para las diferentes especies
afectadas por S. scabiei:
- El capítulo 1 pone de manifiesto la necesidad de obtener datos fiables,
continuados y comparables a lo largo del tiempo, acerca del número total de
individuos presentes en un área -tanto sanos como enfermos- y las posibles
variaciones experimentadas por las poblaciones de ungulados a lo largo del tiempo.
Este tipo de información permite evaluar, comprender y tal vez predecir la evolución
de la sarna en función de los datos recogidos y sus variaciones, así como comparar
la evolución del proceso entre las diferentes especies simpátricas susceptibles. El
establecimiento y repetición periódica de los mismos itinerarios de censo se ha
demostrado un método factible a nivel de esfuerzo y notablemente efectivo para la
valoración de tendencias poblaciones en el caso del rebeco cantábrico, así como
para la detección y comparación interanual del número de rebecos y ciervos
afectados por sarna o la identificación de los grupos de sexo y/o edad más afectados
durante dichos itinerarios. Así por ejemplo, un exhaustivo seguimiento de la
población Oriental de rebeco cantábrico -afectado por S. scabiei desde 1993-
resultaría imprescindible para detectar hipotéticos rebrotes de la enfermedad como
los descritos en Alemania o Italia 15 años tras su irrupción (Miller, 1985, Rossi et al.
1995).
- Este tipo de datos reviste la misma importancia a la hora de evaluar el
impacto de la sarna en especies como el lobo o el zorro (Capítulo 3), en las que sin
embargo dicha información ha de ser obtenida por otros medios. Aparte de los
trabajos específicos de censo y/o estimas de abundancia de una especie elusiva y
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
224 _____________________________________________________________________________
escasa como el lobo, notablemente costosos a nivel de esfuerzo desarrollado, el
capítulo 3 pone de manifiesto la utilidad de una técnica relativamente barata como el
fototrampeo a este nivel. Esta herramienta puede proporcionar datos representativos
prolongados en el tiempo y comparables (siempre que se mantengan un número
suficiente de cámaras el número suficiente de años) sobre la presencia y
detectabilidad del lobo, así como sobre la presencia de lesiones compatibles con
sarna en los mismos.
- El zorro constituye una pieza clave del ecosistema por su gran importancia
ecológica como predador y regulador de otras especies, pero también como
hospedador y posible fuente de muchas enfermedades presentes en lobo y otros
carnívoros (Lindström et al., 1994). Su seguimiento (poblacional, sanitario,…) es
importante para comprender lo que ocurre con otras muchas especies a su
alrededor, pero por desgracia parece que su abundancia y escaso interés cinegético
la hacen una especie a menudo subestimada a nivel de investigación y gestión. En
la presente tesis la técnica del fototrampeo ha mostrado también su utilidad para el
estudio de la sarna sarcóptica en zorro, en el que incomprensiblemente no había
sido utilizada hasta la fecha con este propósito y cuyo empleo puede resultar una
alternativa barata y efectiva en muy diferentes hábitats, atendiendo al carácter
ubicuo y adaptable de este pequeño cánido (Sobrino et al., 2009). Debido a su
mayor abundancia y sus hábitos, el zorro podría también ser estudiado a este nivel
mediante el uso y repetición periódica de itinerarios nocturnos de fareo.
D.2.b-) Determinación de la posible relación existente entre densidad
poblacional y sarna sarcóptica en las diferentes especies:
- Los datos recogidos durante la realización de esta tesis, y especialmente
durante los 3 últimos años (no incluídos en el Capítulo 1), señalan un aumento en el
número de casos de sarna detectados en corzo en algunas de las regiones con
mayor densidad registrada en Asturias (Pablo Quirós, comunicación personal). Por
tanto, parece recomendable estudiar el aparente efecto de la densidad del corzo en
la aparición y mantenimiento de la sarna sarcóptica y la posible existencia de un
“umbral mínimo” de densidad para que esta especie (poco gregaria) pueda presentar
casos clínicos con la frecuencia observada en los últimos años.
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- El primer capítulo señala la presencia, entre 1995 y 2008, de sarna sarcóptica
en ciervos de las 4 Reservas Regionales de Caza del Principado con una mayor
densidad de este ungulado durante el desarrollo de dicho estudio. Es por ello que
parece también recomendable tratar de valorar la existencia de un posible umbral de
densidad de este ungulado para el mantenimiento de S. scabiei como “hospedador
alternativo” y su circulación independiente del considerado “hospedador principal” u
“original” en Asturias (el rebeco).
- Del mismo modo, cuanto mayor sea la frecuencia y precisión con que
dispongamos de datos de distribución y abundancia lupinas, más fácil será
determinar qué efectos tiene la densidad de este cánido (y otros, como el zorro…)
en la velocidad y capacidad de mantenimiento y transmisión de la sarna sarcóptica
en la comunidad de carnívoros. Esta información permitiría también conocer con
mayor exactitud los efectos que esta enfermedad (y otras) puede acabar teniendo a
nivel de supervivencia (especialmente en cachorros) o como factor limitante de
densidad en las poblaciones locales de lobo.
D.2.c-) Evaluación de las relaciones entre los parámetros fisiológicos, la
capacidad del sistema inmunitario del hospedador y la afección por S. scabiei:
- La valoración y seguimiento del estado sanitario , mediante la caracterización
objetiva de parámetros como condición corporal de individuos abatidos (a través del
índice de engrasamiento renal, por ejemplo -Lajeunesse & Peterson, 1993; Vicente
et al., 2007- tanto en individuos sanos como enfermos, y tanto en zonas con sarna
como en otras libres) podría aportar información acerca de posibles condiciones
predisponentes y efectos del proceso, así como sobre las aparentes diferencias
existentes entre las especies estudiadas.
- El desarrollo de infecciones experimentales (ver apartado D.2.e, a
continuación) correctamente diseñadas permitiría indagar en la verdadera relación
existente entre condición corporal-sistema inmune-enfermedad.
- Otro aspecto de interés es la evaluación del posible efecto que situaciones de
estrés (a nivel de individuo o población, tal vez valorables mediante el uso de
adrenales como índice fisiológico objetivo de estrés crónico [Estivariz et al., 1992])
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
226 _____________________________________________________________________________
pueden acabar ocasionando en la respuesta inmune frente a la sarna y su
efectividad.
- Las características genéticas propias de las poblaciones hospedadoras dentro
de una misma especie, y las diferencias existentes entre las mismas, pueden jugar
también un papel importante en la competencia del sistema inmunitario para hacer
frente a S. scabiei. Esta es una materia sólo someramente explorada para el caso de
la sarna sarcóptica, de indudable interés atendiendo a números reducidos de
individuos “fundadores” en algunas de las poblaciones afectadas (ciervo - Pérez et
al., 1998), o al “cuello de botella” experimentado por otras en un pasado reciente
(rebeco – Fernández-Moran et al., 1997; González-Quirós et al., 2002).
D.2.d-) Aspectos epidemiológicos del proceso pendientes de investigación:
- A pesar de verse afectadas con una frecuencia notablemente inferior a la de
los machos de su especie, el papel que las hembras de ciervo desempeñan en el
mantenimiento y circulación de la sarna sarcóptica permanece sin esclarecer. La
detección en 2008 y 2009 de ácaros S. scabiei en las extremidades de 2 de 8
ciervas sin lesiones macroscópicas (aparentemente sanas, y abatidas en cacería
ordinaria) muestreadas al azar y sometidas al protocolo de búsqueda del parásito
pone de manifiesto la posible relevancia de las hembras a nivel epidemiológico. Su
aparente menor morbilidad puede hacerlas un hospedador más “prolongado en el
tiempo” y un vehículo más eficaz del parásito en esta especie.
- Una de las cuestiones planteadas en el primer capítulo y que no pudo ser
esclarecida es la llamativa concentración a partir de 2002 de ciervos afectados por
sarna en una superficie llamativamente pequeña del Valle del Nalón. Para poder
descartar el posible efecto de diferentes grados o esfuerzos de prospección en
distintas áreas de la zona de estudio resulta necesario establecer protocolos de
búsqueda y seguimiento de animales sanos y/o enfermos que aseguren un esfuerzo
de búsqueda uniforme y con ello la comparabilidad de los datos obtenidos en las
diferentes áreas y a lo largo del tiempo.
- Un mejor conocimiento a nivel biológico, ecológico y etológico tanto de rebeco
como de ciervo en Asturias pueden ayudar en la identificación de posibles zonas y
_______________________________________________________ _ Discusión
_____________________________________________________________________________ 227
momentos propicios o “puntos críticos” para el contacto entre ambas especies y la
transmisión interespecífica del ácaro señalada en la presente tesis.
- Al igual que con otros agentes patógenos (moquillo, parvovirus…), un
seguimiento preciso de las tasas de fertilidad, natalidad, reclutamiento y
supervivencia de cachorros en lobo parece la única forma de evaluar correctamente
el verdadero efecto a nivel de mortalidad que la enfermedad puede tener, y con ello,
su relevancia real a nivel de conservación en esta especie. El uso de técnicas de
radioseguimiento de los grupos se antoja una herramienta clave a este nivel. La
localización mediante teleseguimiento de camadas de lobo permitiría no sólo un
mayor muestreo de cachorros y un análisis de sus seroprevalencias, sino
especialmente la detección de patrones temporales de exposición (por ejemplo
frente al virus del moquillo canino) a nivel de grupo y población, como se ha
comprobado y utilizado en la interpretación de resultados en otros trabajos con lobo
(Almberg et al., 2009). Esta localización de camadas permitiría, asimismo, la toma de
muestras “no invasivas” como excrementos en fresco, con indudable interés en el
estudio de otros procesos como CPV.
- Resulta necesario continuar con los muestreos serológicos y su estudio a lo
largo del tiempo para determinar la evolución de exposición a diferentes agentes
(incluyendo S. scabiei) e identificar tendencias a largo plazo en las especies
estudiadas.
- A diferencia de lo observado en otras especies (bóvidos y cérvidos silvestres,
zorro…), las características del tipo de respuesta inmune descrita y del proceso
clínico apreciado en lobo sugieren las grandes pérdidas de calor y energía
provocadas por la ausencia de pelo como una de las consecuencias más
importantes de la sarna sarcóptica en este cánido. El posible efecto de la sarna en el
comportamiento de este carnívoro social, motivado por una mayor demanda de
energía para suplir esa pérdida extra de calorías, puede tener relevancia a nivel
epidemiológico (por ejemplo con mayores o más frecuentes desplazamientos en
búsqueda de alimento que incrementarían el riesgo de contacto y contagio a otros
animales). Tanto estas posibles alteraciones de comportamiento como la evolución y
desenlace del proceso en diferentes animales afectados por S. scabiei son
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
228 _____________________________________________________________________________
susceptibles de ser investigados mediante teleseguimiento (vía radio-tracking,
GPS…).
D.2.e-) Desarrollo de infecciones experimentales:
- La evaluación de parámetros bioquímicos en sangre y sus variaciones
durante la evolución del proceso resulta abordable exclusivamente mediante la
ejecución de infecciones experimentales y su estricto seguimiento. La determinación
de estos parámetros en las diferentes especies ayudaría a comprender mejor la
patogenia y efectos fisiológicos de la enfermedad y las posibles diferencias en la
evolución clínica entre las mismas.
- Una infección experimental en ciervos permitiría describir y entender mejor la
enfermedad y sus fases en esta especie poco estudiada, así como interpretar mejor
y sacar mayor partido de las pruebas serológicas, al permitir caracterizar las
diferentes fases de seroconversión y sus características y efecto sobre el ácaro
(Casais et al., 2014). Este tipo de trabajo permitiría asimismo determinar la
capacidad de ciervos (tanto macho como hembras) de sobrevivir a la enfermedad, y
en caso de confirmarse, cuáles son los factores que determinan esa supervivencia.
- Del mismo modo, un mayor conocimiento de las pautas y plazos de
seroconversión y mantenimiento de anticuerpos en lobo permitiría una mejor
interpretación de los resultados obtenidos con el ELISA empleado (este punto es
totalmente aplicable a las otras 4 especies estudiadas, con infecciones
experimentales o capturas-recapturas y estudio serológico continuado de animales
afectados como única vía de información útil al respecto).
D.2.f-) Aspectos clínicos y patogénicos del proceso pendientes de
investigación:
- El estudio histológico pormenorizado de los principales órganos en todos los
individuos afectados por sarna sarcóptica permitiría una mejor comprensión y
caracterización del proceso en las especies afectadas, así como una mejor
determinación de las diferencias existentes en la evolución clínica del proceso entre
las mismas. Este estudio anatomo-histo-patológico exhaustivo permitiría también
indagar en la patogenia última de la enfermedad y los mecanismos o alteraciones
_______________________________________________________ _ Discusión
_____________________________________________________________________________ 229
fisiológicas a través de los cuales acaba desencadenando la muerte en ciertas
especies (y no en otras).
- Las diferencias registradas tanto a nivel lesional (macro y microscópicamente)
como en el aparente pronóstico de la sarna sarcóptica que presentan dos especies
tan próximas filogenéticamente como el zorro y el lobo en Asturias sugieren un
estudio y comparación detallados entre ambas. El estudio del tipo de respuesta
inmune desplegada y su eficacia ofrecería una oportunidad muy interesante de
mejorar nuestro conocimiento y comprensión de la patogenia de S. scabiei y el modo
en que el organismo es capaz de defenderse de él de forma efectiva.
- La inmunohistoquímica puede proporcionar valiosos datos acerca del tipo y
eficacia de la respuesta inmune celular desarrollada por los diferentes
individuos/especies.
- Determinar las diferencias o razones por las que no todos los lobos
confirmados con sarna provocaron la “inmunotinción” de glóbulos blancos permitiría
optimizar el uso de la inmunohistoquímica en el diagnóstico de esta enfermedad en
lobo y otras especies con una respuesta inmune similar o un escaso número de
ácaros vivos en piel.
D.2.g-) Estudio y control del papel desempeñado por animales domésticos en
contacto con fauna silvestre:
- El seguimiento estricto del ganado presente en el monte, sus datos de
pertenencia y la detección de posibles síntomas de enfermedad son medidas
imprescindibles para tratar de evitar la transmisión de enfermedades a fauna
silvestre tal como ocurrió con la devastadora epizootia de sarna en rebeco en 1993.
Aprovechar el trabajo desarrollado en censos de fauna silvestre (por ejemplo
itinerarios de censo de rebeco) para tratar de incluir datos sobre presencia de
ganado en el campo y su abundancia permitiría detectar tendencias en el tiempo
acerca de su presencia y por tanto de su probabilidad de contacto (y riesgo de
transmisión de enfermedad) con fauna silvestre.
- En relación al punto anterior, parecen recomendables un correcto seguimiento
de rebeco y cabra doméstica y el empleo de las técnicas adecuadas para determinar
las tasas de contacto entre fauna silvestre y doméstica, así como para identificar
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
230 _____________________________________________________________________________
lugares y momentos en que estos contactos se producen con mayor frecuencia. Esta
información permitiría comprender mejor la epidemiología de estas y otras
enfermedades compartidas por ambos bóvidos, así como plantear medidas
encaminadas a minimizar el riesgo de transmisión de las mismas.
- Parece posible efectuar un buen seguimiento serológico de sarna en cabras
del Núcleo Occidental aprovechando los muestreos de las campañas de
erradicación de enfermedades transmisibles (tuberculosis y brucelosis) impuestos
por la Unión Europea. Estos estudios serológicos permitirían detectar aumentos
reseñables de seroprevalencia en cabras que pudiesen alertar de un posible riesgo
para la población simpátrica de rebecos. Dichas campañas de saneamiento
obligatorio podrían asimismo evaluar la presencia de lesiones compatibles con sarna
en ganado durante su muestreo.
- Extremar los controles en el movimiento del ganado e implantar tratamientos
preventivos frente a sarna sarcóptica previos a la salida de los animales a pasto
pueden ser actuaciones profilácticas recomendables en aquellas áreas en que la
sarna no ha sido aún detectada en fauna silvestre.
- Determinar si los perros de las zonas rurales sirven como “chivo expiatorio” de
incrementos en la morbilidad en cánidos silvestres (Clayton, 2003), o por el contrario
actúan como fuente de infección para los mismos, reviste indudable interés y parece
posible mediante un adecuado seguimiento de la sarna sarcóptica en perros de las
zonas estudiadas y su comparación con los datos obtenidos en carnívoros silvestres
de las mismas.
- Muestreos serológicos continuados en el tiempo y en paralelo de perros,
lobos y zorros de las misma áreas nos ayudarían a comparar y comprender mejor la
epidemiología y tendencias temporales de los distintos procesos que comparten.
- La vacunación de los perros rurales frente a diferentes agentes patógenos
compartidos con cánidos silvestres (moquillo canino, parvovirosis…) ha sido
propuesto como una medida necesaria en poblaciones de lobo más amenazadas
(Müller et al., 2011), y puede mejorar el estado sanitario de las poblaciones
asturianas de lobo a tenor de los contactos confirmados entre ambas especies.
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D.2.h-) Empleo de la biología molecular en el estudio de la sarna sarcóptica:
- El capítulo 5 puede ser considerado una “primera aproximación” al estudio
epidemiológico de la sarna sarcóptica en Asturias a través de la biología molecular
de S. scabiei. El muestreo y análisis de un mayor número de individuos (y taxones,
como mustélidos o jabalí) permitirá una mejor comprensión de las relaciones
epidemiológicas entre las diferentes especies.
- El muestreo y seguimiento a lo largo del tiempo de las características
genéticas de los ácaros aislados en cada especie y sus variaciones pueden
asimismo aportar información muy valiosa desde un punto de vista clínico y
epidemiológico. Una de las interesantes cuestiones planteadas a este nivel es si los
ácaros aislados en ciervo pueden acabar presentando con el paso de los años
variaciones con respecto a sus características descritas en este trabajo. Su aparente
transición de hospedador “accidental” o “alternativo” a hospedador capaz de
mantener y transmitir el ácaro con independencia del rebeco puede suponer la
aparición de “adaptaciones” del ácaro que faciliten su ciclo sobre cérvidos.
- Del mismo modo parece interesante continuar con el muestreo y evaluación
de las características moleculares de ácaros procedentes de lobo. Esto permitiría
evaluar si con el paso del tiempo llegan a registrarse variaciones o tendencias en las
mismas que nos permitan sospechar la evolución y/o aparición de una variedad
“adaptada” o “propia de lobo”. Este tipo de variaciones podrían relacionarse con
cambios a nivel de patogenia, morbilidad o epidemiología del proceso en este gran
carnívoro y sus poblaciones.
- La comparación de los aspectos clínicos e histopatológicos registrados con
los datos moleculares procedentes de los ácaros presentes en un mayor número de
lobos estudiados permitiría determinar las posibles diferencias clínicas (caso de
existir) derivadas del tipo de ácaro (carnívoro/herbívoro) presente en cada uno de los
cánidos estudiados. Si bien en los intentos efectuados en el capítulo 5 (con un
número discreto de animales analizados a este nivel) no llegó a ser detectada, es
posible que exista una diferente capacidad patogénica para el lobo en función del
tipo de hospedador (herbívoros o zorro) sobre el que el ácaro haya “evolucionado”.
- Uno de los trabajos pendientes más interesantes es la inclusión en los análisis
moleculares efectuados de ácaros procedentes de animales domésticos. A pesar de
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
232 _____________________________________________________________________________
que fue planteado como uno de los puntos a estudiar en la presente tesis, la
imposibilidad de obtener ácaros S. scabiei procedentes de cabra o perro impidió su
inclusión en los trabajos presentados. La caracterización genética de esos ácaros, y
su comparación con los aislados a partir de fauna silvestre simpátrica, ofrecería
valiosa información sobre la circulación y posible transmisión del parásito entre
ambos grupos, permitiendo incluso en algún caso la confirmación o desestimación
laboratorial de hipótesis empíricamente aceptadas (como el origen en cabras del
brote de sarna sarcóptica en rebecos en 1993 [Fernández-Morán et al., 1997; Lavín
et al., 2000]).
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D.3- CONCLUSIONES
1. La sarna sarcóptica está ampliamente distribuida y afecta a
diferentes especies de fauna silvestre en Asturias (incluyendo rebeco, ciervo,
corzo, zorro y lobo) con diferencias en los grados de morbilidad y mortalidad
registrados.
2. Tras el brote epizoótico inicial (1993), la enfermedad continúa
presente de forma endémica en el rebeco cantábrico en su núcleo oriental de
distribución en el Principado, siendo actualmente detectada de forma continua
todos los años pero con baja prevalencia.
3. Atendiendo a los datos serológicos, la cabra doméstica continúa
representando una amenaza sanitaria para el núcleo occidental de distribución
del rebeco cantábrico en el Principado de Asturias, libre de sarna sarcóptica
hasta la fecha. Estos resultados ponen de manifiesto la necesidad de
complementar los programas de vigilancia y control de fauna silvestre con el
seguimiento y control sanitarios de animales domésticos.
4. La sarna sarcóptica en ciervos puede ser considerada endémica
en buena parte del oriente del Principado de Asturias, cursando con baja
morbilidad y elevada mortalidad. La evolución espacio-temporal del proceso
en este cérvido es independiente de la que continúa experimentando el rebeco
cantábrico simpátrico, y no parece afectar a la dinámica de las poblaciones de
ciervo en Asturias.
5. Las características clínicas del proceso en lobo, junto con los
datos de campo recabados, sugieren que esta parasitación no constituye
actualmente una amenaza para el mantenimiento de las poblaciones asturianas
de lobo. Sin embargo una exposición combinada a otros patógenos, como el
virus del moquillo canino, podría potenciar los efectos adversos de los
agentes infecciosos, aspecto que ha de ser elucidado.
6. Las características clínicas de la sarna sarcóptica presentan
notables similitudes en las tres especies de ungulados estudiados (ciervo,
Tesis doctoral ___________________________________________________________ Álvaro Oleaga
234 _____________________________________________________________________________
rebeco y corzo) con predominio de una respuesta de hipersensibilidad Tipo I
(forma paraqueratótica), frente a una respuesta de hipersensibilidad Tipo IV
(forma alópecica) en el lobo que presentó una evolución y características
histopatológicas macro y microscópicas diferentes. En el caso del zorro se
detectaron ambos tipos de respuesta inmune y cuadros clínicos similares
tanto a los registrados en ungulados como en lobo. La inmunohistoquímica se
mostró como una herramienta interesante en el estudio y diagnóstico de la
sarna sarcóptica, especialmente valiosa en especies o individuos que albergan
un número reducido de ácaros.
7. Resulta necesario considerar diferentes especies simpátricas
susceptibles a S. scabiei en los programas de seguimiento sanitario de fauna
silvestre para un mejor entendimiento de la epidemiología de la sarna
sarcóptica, y continuar con los programas y técnicas de monitorización
poblacional y sanitaria empleados hasta la fecha.
8. Los resultados moleculares obtenidos de los ácaros aislados
permiten confirmar la sarna sarcóptica en rebeco como origen de los casos
registrados en cérvidos asturianos, y descartar la sarna en zorros como una
amenaza para las poblaciones de rebeco aún libres de sarna en el Principado.
El lobo presenta ácaros relacionados tanto con zorro como con ungulados, no
habiéndose detectado la presencia de una variedad genética específica de este
cánido.
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_____________________________________________________________________________ 235
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