EPIDEMIOLOGÍA DE LA SARNA SARCÓPTICA...

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


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


_____________________________________________________________________________Índice

Results ................................................................................................. 78

Discussion ........................................................................................... 78


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


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


Discussion ......................................................................................... 140

Conclusions ....................................................................................... 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


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


References ........................................................................................ 200

DISCUSIÓN GENERAL ........................................................................... 205

D.1-Síntesis de los hallazgos más relevantes ..................................... 207


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


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)


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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

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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.


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


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


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.


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


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 -


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.


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.


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

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________________________________________________________________________ Capítulo 1

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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


32 _____________________________________________________________________________

________________________________________________________________________ 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.


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.


36 _____________________________________________________________________________

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.


38 _____________________________________________________________________________

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).


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.


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.



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.


46 _____________________________________________________________________________

________________________________________________________________________ Capítulo 1

_____________________________________________________________________________ 47

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.


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

_____________________________________________________________________________ 49

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).


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).


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.


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).

________________________________________________________________________ Capítulo 1

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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


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).


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.

________________________________________________________________________ Capítulo 1

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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.


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

________________________________________________________________________ Capítulo 1

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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


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,

________________________________________________________________________ Capítulo 1

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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


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

________________________________________________________________________ Capítulo 1

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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


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.


66 _____________________________________________________________________________

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________________________________________________________________________ Capítulo 2

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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


72 _____________________________________________________________________________

________________________________________________________________________ Capítulo 2

_____________________________________________________________________________ 73

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


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

________________________________________________________________________ Capítulo 2

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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 pyrenaica) 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.


76 _____________________________________________________________________________

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.


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

________________________________________________________________________ Capítulo 2

_____________________________________________________________________________ 77

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


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

________________________________________________________________________ Capítulo 2

_____________________________________________________________________________ 79

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).


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


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

_____________________________________________________________________________ 83

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


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.


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


100 _____________________________________________________________________________

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


102 _____________________________________________________________________________

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.


104 _____________________________________________________________________________

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.


106 _____________________________________________________________________________

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.


110 _____________________________________________________________________________

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).


112 _____________________________________________________________________________

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,

________________________________________________________________________ Capítulo 3

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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


114 _____________________________________________________________________________

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


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


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


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

________________________________________________________________________ Capítulo 3

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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


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


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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N.W., Wunder, B.A., Lavelle, M.J., 2007. Elk use of wallows and potential chronic wasting disease transmission. J. Wildl. Dis. 43, 784–788.

Wall, R., Shearer, D., 1997. Veterinary Entomology. Chapman and Hall,

London. Walton, S.F., Dougall, A., Pizzutto, S., Holt, D., Taplin, D., Arlian, L.G.,

Morgan, M., Currie, B.J., Kemp, D.J., 2004. Genetic epidemiology of Sarcoptes scabiei (Acari: Sarcoptidae) in northern Australia. Int. J. Parasitol. 34, 839–849.

Wobeser, G., 1992. Traumatic, degenerative, and developmental lesions

in wolves and coyotes from Saskatchewan. J. Wildl. Dis. 28, 268–275. Yager, J.A., Scott, D.W., 1993. The skin and appendages. Mites. In:

Jubb, K.V.F., Kennedy, P.C., Palmer, N. (Eds.), Pathology of Domestic Animals, vol. 1, pp. 681–683.


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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).


132 _____________________________________________________________________________

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_____________________________________________________________________________ 133

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.


134 _____________________________________________________________________________

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.


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


136 _____________________________________________________________________________

(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


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


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

________________________________________________________________________ Capítulo 3

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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


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


144 _____________________________________________________________________________

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

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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.


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


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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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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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.


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.


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


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


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.


160 _____________________________________________________________________________

REFERENCES

Alasaad, S., Oleaga, A., Casais, R., Rossi, L., Min, A.M., Soringer, 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. Parasits. Vectors 4, 151.

Arlian, L.G., 1996. Immunology of scabies. In: Wikel SK (ed) The

immunology of host ectoparasitic arthropod relationships. Wallingford, United Kingdom, pp 232–258.

Bates, P., 2003. Sarcoptic mange (Sarcoptic scabiei var. vulpes) in a red fox

(Vulpes vulpes) population in north-west Surrey. Vet. Rec. 152, 112–114. Casais, R., Prieto, J.M., Balseiro, A., Solano, P., Parra, F., Martin Alonso, J.M.,

2007. Identification and heterologous expression of a Sarcoptes scabiei cDNA encoding a structural antigen with immunodiagnostic potential. Vet. Res. 38, 435–450.

Fernández-Morán, J., Gómez, S., Ballesteros, F., González-Quirós, P.,

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.

Gortázar, C., Villafuerte, R., Blanco, J.C., Fernández-De-Luco, D., 1998.

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.

_______________________________________________________ _ Capítulo 5

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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


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.



164 _____________________________________________________________________________

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_____________________________________________________________________________ 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).


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


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


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.


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.


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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_____________________________________________________________________________ 175

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.


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.

_______________________________________________________ _ Capítulo 5

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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



184 _____________________________________________________________________________

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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”.


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


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.


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


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),

_______________________________________________________ _ Capítulo 5

_____________________________________________________________________________ 191

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).


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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_____________________________________________________________________________ 193

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.


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.

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_____________________________________________________________________________ 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


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.


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

_______________________________________________________ _ Capítulo 5

_____________________________________________________________________________ 199

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


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


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


206 _____________________________________________________________________________

_______________________________________________________ _ Discusión

_____________________________________________________________________________ 207

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


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

_______________________________________________________ _ Discusión

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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)


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


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


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


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.


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

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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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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


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.

_______________________________________________________ _ Discusión

_____________________________________________________________________________ 223

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


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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.

_______________________________________________________ _ Discusión

_____________________________________________________________________________ 225

- 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])


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


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


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.

_______________________________________________________ _ Discusión

_____________________________________________________________________________ 231

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


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]).

_______________________________________________________ _ Discusión

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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,


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.

_______________________________________________________ _ Discusión

_____________________________________________________________________________ 235

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