Susceptibility to acaricides and genetic diversity of · Spécialité : Pathologie et recherche...

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N°: 2009 ENAM XXXX présentée et soutenue publiquement par Fang FANG Le 15 Avril 2016 Susceptibility to acaricides and genetic diversity of Sarcoptes scabiei from animals UNIVERSITÉ PARIS-EST T H È S E Pour obtenir le grade de docteur délivré par Ecole doctorale Sciences de la Vie et de la Santé Spécialité : Pathologie et recherche clinique Directeur de thèse : Pr Jacques GUILLOT Unité de Parasitologie, Mycologie, Dermatologie, Ecole nationale vétérinaire d'Alfort, Maisons-Alfort, France EA 7380 Dynamyc, Faculté de Médecine, Créteil, France Jury M. Pascal DELAUNAY, MCU-PH, Faculté de Médecine de Nice, France Rapporteur M. Michel FRANC, Professeur, Parasitologie, Ecole nationale vétérinaire de Toulouse, France Rapporteur Mme Weiyi HUANG, Professeur, Faculté vétérinaire, Université du Guangxi, Chine Examinateur Mme Françoise BOTTEREL, Professeur, Equipe Dynamyc, Paris-Est Créteil, France Examinateur Mme Lénaïg HALOS, Docteur vétérinaire, Merial, Lyon, France Examinateur M. Olivier CHOSIDOW, Professeur, Dermatologie, Hôpital Henri Mondor, Créteil, France Examinateur M. Rémy DURAND, MCU-PH, Parasitologie, Hôpital Avicenne, Bobigny, France Examinateur

Transcript of Susceptibility to acaricides and genetic diversity of · Spécialité : Pathologie et recherche...

N°:    2009  ENAM  XXXX    

 

                     

présentée et soutenue publiquement par

Fang FANG

Le 15 Avril 2016

Susceptibility to acaricides and genetic diversity of Sarcoptes scabiei from animals

 

UNIVERSITÉ PARIS-EST

T H È S E Pour obtenir le grade de docteur délivré par

Ecole doctorale Sciences de la Vie et de la Santé Spécialité : Pathologie et recherche clinique

Directeur de thèse : Pr Jacques GUILLOT

Unité de Parasitologie, Mycologie, Dermatologie, Ecole nationale vétérinaire d'Alfort, Maisons-Alfort, France EA 7380 Dynamyc, Faculté de Médecine, Créteil, France

Jury M. Pascal DELAUNAY, MCU-PH, Faculté de Médecine de Nice, France Rapporteur M. Michel FRANC, Professeur, Parasitologie, Ecole nationale vétérinaire de Toulouse, France Rapporteur Mme Weiyi HUANG, Professeur, Faculté vétérinaire, Université du Guangxi, Chine Examinateur Mme Françoise BOTTEREL, Professeur, Equipe Dynamyc, Paris-Est Créteil, France Examinateur Mme Lénaïg HALOS, Docteur vétérinaire, Merial, Lyon, France Examinateur M. Olivier CHOSIDOW, Professeur, Dermatologie, Hôpital Henri Mondor, Créteil, France Examinateur M. Rémy DURAND, MCU-PH, Parasitologie, Hôpital Avicenne, Bobigny, France Examinateur

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Acknowledgements  

 

On   the  occasion  of   the  completion  of  my  dissertation  and  subsequent  PhD,   I  would  

like  to  appreciate,  first  and  foremost,  my  director  Professor  Jacques  Guillot.  It  has  been  an  

honor  to  be  his  PhD  student.  Jacques  is  someone  who  is  nice  and  cheerful,  who  is  always  

optimistic  and  work  productively.  I  have  learned  a  lot  from  him  under  the  influence  of  his  

good   characters   during   the   whole   period   of   my   PhD   study.   I   really   appreciate   all   his  

contributions  of  time,  ideas,  and  funding  for  my  PhD.    

I  am  grateful  to  the  China  Scholarship  Council,  which  provided  a  PhD  grant  for  me  and  

gave  me  the  opportunity  to  study  in  France.  

I  am  particularly  thankful  to  the  jury  members  of  my  thesis:  Dr  Pascal  Delaunay  and  Pr  

Michel   Franc   who   spent   time   to   review   my   thesis,   and   Pr   Weiyi   Huang,   Pr   Françoise  

Botterel,  Dr  Lénaïg  Halos,  Pr  Olivier  Chosidow  and  Dr  Rémy  Durand  who  kindly  accepted  

to  be  members  of  the  PhD  jury.  

Special   thanks   to  Dr   Sarah  Bonnet   from  BIPAR,  who  participated   to  my   “Comité  de  

pilotage”  and  gave  good  suggestions  on  my  PhD  project.    

I  would   like   to   thank  every  members  of   the   research   team  Dynamyc:   Elise  Melloul,  

Charlotte  Bernigaud,  Stéphanie  Luigi,  Françoise  Botterel,  Françoise  Foulet,  Veronica  Risco,  

Pascal  Arné,  René  Chermette.  I  would  like  to  express  my  deeply  gratitude  to  Charlotte  and  

Elise,  two  other  PhD  students,  who  helped  me  a   lot.  We  worked  and  travelled  together,  

had  lots  of  fun.  Thanks  to  them,  my  PhD  life  has  been  cheerful  and  colorful.  

I  would   like  to  thank  the  teachers  of  Parasitology  group  in  EnvA.  To  Jacques  Guillot,  

Bruno   Polack,   René   Chermette   and   Radu   Blaga,   for   their   excellent   classes   in   veterinary  

Parasitology.  To  Odile  Crosaz  who  is  always  nice  and  ready  to  answer  my  questions  with  

patience.  To  Radia  Guechi  who  helped  me  in  experiment  preparation.  

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Thanks   to   the   members   of   the   Parasitology   department   of   Avicenne   Hospital:   Dr  

Arezki   Izri  who  provided  some  essential  oils  and  products,  Candy  Kerdalidec  who  helped  

me  with  in  vitro  tests,  Rémy  Durand  and  Valérie  Andriantsoanirina  who  were  in  charge  of  

the  molecular  analysis.  

Thanks   to   Thomas   Lilin   and   Francis   Moreau   from   the   Centre   de   Recherche  

Biomédicale.    

I  really  appreciate  my  families  and  friends.  Words  cannot  express  how  grateful  I  am  to  

my  mom  and  dad   for  all   their   love  and  support  on  me.  Million   thanks   to  all  my   friends,  

without  them,  my  life  won’t  have  been  so  happy.  My  appreciation  especially  goes  to  my  

dear  boyfriend,  who  is  ready  to  encourage  me  no  matter  day  or  night.  His  unconditional  

love  and  support  has  enlightened  me  not  only  through  PhD,  but  also  through  life.    

Last   but   not   the   least,   I   would   like   to   express  my   deepest   gratitude   to   the   French  

people,   who   have   always   attached   great   importance   to   protecting   their   heritages   and  

cultures  as  well  as  those  around  the  world.  Thanks  to  their  effort  and  persistent  love  for  

art,   I  was  able   to  admire   the   fabulous  museums,   the   splendid   castles  and  all  wonderful  

arts  around  the  world.  Here  I  would  like  to  quote  the  words  of  Hemingway  to  express  my  

affection  of  the  life  in  Paris:  If  you  are  lucky  enough  to  have  lived  in  Paris  as  a  young  man,  

then  wherever  you  go  for  the  rest  of  your   life,   it  stays  with  you,   for  Paris   is  a  moveable  

feast.  

 

 

 

     

 

 

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 TABLE  OF  CONTENTS  

 

Acknowledgements........................................................................................................ 1  

Table  of  contents ........................................................................................................... 3  

Abstract ......................................................................................................................... 5  

Résumé.......................................................................................................................... 6    

I.  Background  and  outline  of  the  thesis   ........................................................................ 7  1.  Sarcoptes  scabiei ........................................................................................................... 8  1.1.  Classification........................................................................................................... 8  

        1.2.  Morphology............................................................................................................ 9  1.3.  Life  cycle............................................................................................................... 11  1.4.  Survival  capacities  and  modes  of  transmission   .................................................. 13  1.5.  Variability  and  host  specificity ............................................................................. 14  

1.5.1  Morphological  variability.............................................................................. 14  1.5.2.  Population  genetics  of  Sarcoptes  scabiei .................................................... 15  1.5.3.Host  specificity  and  cross-­‐infectivity ............................................................ 23  

2.  Infection  by  Sarcoptes  scabiei  in  animals.................................................................... 26  2.1.  Distribution .......................................................................................................... 29  2.2.  Clinical  features.................................................................................................... 30  2.3.  Diagnosis  in  animals ............................................................................................. 37  2.4.  Animal  models...................................................................................................... 39  

3.  Infection  by  Sarcoptes  scabiei  in  humans ................................................................... 41  4.  Control......................................................................................................................... 47  4.1.  Acaricides ............................................................................................................. 47  4.2.  Current  treatments  in  animals ............................................................................. 52  4.3.  Current  treatments  in  humans............................................................................. 53  4.4.  Drug  resistance   ................................................................................................... 55  

5.  Outline  of  the  thesis .................................................................................................... 56    

II.  Evaluation  of  afoxolaner  for  the  treatment  of  Sarcoptes  scabiei  infection  in  pigs ......57  1.  Introduction ................................................................................................................ 58  2.  Materials  and  Methods ............................................................................................... 59  2.1.  Experimental  pig  model ....................................................................................... 59  2.2.  Study  design ......................................................................................................... 60  2.3.  Clinical  monitoring ............................................................................................... 61  2.4.  Afoxolaner  and  ivermectin  pharmacokinetics...................................................... 63  2.5.  Statistical  Analysis   .............................................................................................. 64  

     

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3.  Results   ....................................................................................................................... 65  3.1.  Experimental  pig  model ....................................................................................... 65  3.2.  Clinical  outcomes   ............................................................................................... 66  

4.  Discussion   .................................................................................................................. 71    

III.   In   vitro   evaluation   of   acaricides,   repellents   and   essential   oils   for   the   control   of  Sarcoptes  scabiei ................................................................................................................ 75  1.  Introduction ................................................................................................................ 76  2.  Materials  and  Methods ............................................................................................... 78  2.1  Sarcoptes  mites ..................................................................................................... 78  2.2  Solutions  preparation  and  bioassays  of  ivermectin  and  moxidectin .................... 78  2.3  Products  and  bioassays  for  environmental  control............................................... 78  2.4  Essential  oils  and  bioassays ................................................................................... 80  2.5  Statistical  analyses ................................................................................................ 81  

3.  Results   ....................................................................................................................... 81  3.1  In  vitro  evaluation  of  ivermectin  and  moxidectin  efficacy.................................. 81  3.2  Evaluation  of  products  for  environmental  control  of  S.  scabiei.......................... 82  3.3  In  vitro  evaluation  of  essential  oils ...................................................................... 84  

4.  Discussion   .................................................................................................................. 86    IV.  Characterization  of  the  genetic  diversity  of  Sarcoptes  scabiei  from  animals ............. 91  1.  Introduction ................................................................................................................ 92  2.  Materials  and  Methods ............................................................................................... 93  2.1  Collection  of  S.  scabiei  mites   ............................................................................... 93  2.2  DNA  extraction  and  gene  amplification   .............................................................. 96  2.3  Sequence  and  phylogenetic  analyses   .................................................................. 96  

3.  Results   ....................................................................................................................... 97  4.  Discussion   ................................................................................................................ 100  

V.  Conclusion  and  perspectives ....................................................................................... 103  

 

References ........................................................................................................................ 108  

 

Annexes ............................................................................................................................ 124  

 

 

 

 

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Abstract  

Sarcoptes  scabiei   is  an  ectoparasite  responsible  for  the  emerging/re-­‐emerging  disease  called  

scabies   in   humans   or   mange   in   animals.   It   was   reported   in   104   species   across   27   families   of  

domestic   and  wild   animals.   Current   treatments   for   scabies/mange  are   limited   and   there   are   no  

efficient  products  for  the  environment  control  of  S.  scabiei.  Moreover,  the  taxonomic  status  of  S.  

scabiei   is   still   under   controversy   and   the   question   remains   that   whether   it   represents   a   single  

species  or  several  taxa.    

The   objectives   of   the   thesis  were   to   assess   the   susceptibility   to   acaricides   and   analyse   the  

genetic   diversity  of  S.   scabiei   from  animals.   In   the   first   part   of   the   thesis,   an   animal  model  was  

used   to   evaluate   the   efficacy   of   afoxolaner,   a   new   acaricide   from   the   isoaxazoline   family.   The  

primary  outcome  of  efficacy  was  based  on  the  reduction   in  the  number  of   live  mites  counted   in  

skin   scrapings   following   treatment.   At   day   8,   four   afoxolaner-­‐treated   pigs   (out   of   four)   were  

mite-­‐free,  while  mites  were  still  found  in  three  (out  of  three)  ivermectin-­‐treated  pigs.  All  treated  

pigs   were   cured   at   the   end   of   the   study   (day   35)   and   all   pigs   in   the   control   group   remained  

infected.   Secondary   outcomes   included  measures   on   the   reduction   of   skin   lesions   and   pruritus.  

The  clinical  lesions  of  scabies  infection  were  allowed  to  disappear  completely  for  all  the  pigs  in  the  

afoxolaner  group  but  not   in  the   ivermectin  group  at  14  days  after   the  treatment.  An   increase  of  

the  pruritus  was  observed  right  after   treatment,   followed  by  a  decrease  of   the  pruritus  score   in  

both   treated   groups.   The   second   part   of   the   thesis   was   to   evaluate   the   scabicidal   effect   of  

molecules   or   products   using   an   in   vitro   test.   A   gradient   of   concentrations   of   ivermectin   and  

moxidectin  as  well  as  11  essential  oils  have  been  evaluated  in  vitro  against  S.  scabiei.  After  24h  of  

exposure  to  ivermectin  and  moxidectin,  the  median  lethal  concentrations  were  150.2±31.4  µg/mL  

and  608.3±88.0  µg/mL,  respectively.  Doses  of  ivermectin  under  1  ng/mL  and  moxidectin  under  10  

ng/mL   showed   no   scabicidal   effect.   Fumigation   and   contact   bioassays   were   used   for   the  

assessment   of   essential   oils   efficacy.   Among   Lavandula   augustifolia,   Melaleuca   altenifolia,  

Pelargonium  asperum,  Eucalyptus  radiate,  Leptospermum  scoparium,  Cryptomeria  japonica,  Citrus  

aurantium   ssp   amara   and   3   other   unknown   oils   (BOB4,   BOB5,   BOB9)   tested   with   the   contact  

bioassay,  the  essential  oil  identified  as  BOB4  demonstrated  the  best  scabicidal  effect  (1%  solution  

killed  all  the  mites   in  20  min).  Among  the  10  essential  oils   listed  before  plus  Juniperus  oxycedrus  

with  the  fumigation  bioassay,  the  oil  Melaleuca  altenifolia  demonstrated  the  best  scabicidal  effect  

(all  the  mites  died  in  only  4  min).  For  environmental  control  of  S.  scabiei,  the  efficacy  of  biocides  

or  repellents  was  assessed.  The  median  survival  time  was  calculated  for  permethrin  (4%  and  0.6%),  

esdepallethrin   and   bioresmethrin,   bifenthrin,   cypermethrin   and   imiprothrin,   cyfluthrin,  

tetramethrin  and  sumithrin,  DEET  (25%  and  50%),  icaridin  and  IR3535.  The  third  part  of  the  thesis  

included  the  study  of  the  genetic  diversity  of  populations  of  S.  scabiei  from  animals.  A  part  of  cox1  

was  used  for  phylogenetic  analyses.  The  results  showed  that  Sarcoptes  mites  from  dogs  seem  to  

derive  from  humans.  

 

Key  words:  Sarcoptes  scabiei,  acaricides,  animal  model,  in  vitro  test,  genetic  diversity.  

 

 

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Résumé  

Sarcoptes  scabiei  est  un  acarien  ectoparasite  obligatoire.  Sa  présence  dans  la  couche  cornée  

de   l’épiderme  est  à   l’origine  d’une  gale  dite  sarcoptique.  Cette  ectoparasitose  a  été  décrite  chez  

104  espèces  de  mammifères  représentant  27  familles  distinctes.  Les  traitements  actuels  de  la  gale  

sarcoptique   ne   sont   pas   toujours   satisfaisants   et   il   n’existe   pas   de   produits   qui   permettent  

d’éliminer  S.   scabiei  dans   l’environnement.  Par  ailleurs,   la  diversité  génétique  de  S.   scabiei  n’est  

pas  clairement  définie  et  l’unicité  de  l’espèce  fait  toujours  l’objet  de  controverses.    

L’objectif   de   cette   thèse   a   été   d’évaluer   l’efficacité   d’acaricides   vis-­‐à-­‐vis   de   S.   scabiei   en  

utilisant  un  modèle  animal  ou  par  le  biais  de  tests  in  vitro.  La  diversité  génétique  d’isolats  d’origine  

animale   a   également   été   étudiée.   La   première   partie   du   travail   de   thèse   a   concerné   un   essai  

thérapeutique     L’efficacité   d’une   administration   orale   unique   d’afoxolaner,   une   molécule   du  

groupe   des   isoaxazolines,   a   été   évaluée   sur   des   porcs   expérimentalement   infestés.   Le   critère  

principal  d’évaluation  a  été  la  réduction  du  nombre  de  sarcoptes  mis  en  évidence  dans  les  raclages  

cutanés.  Huit  jours  après  le  traitement,  aucun  sarcopte  n’a  été  détecté  sur  les  4  porcs  ayant  reçu  

l’afoxolaner   alors   que   des   sarcoptes   étaient   toujours   présents   sur   les   3   porcs   ayant   reçu   de  

l’ivermectine.  Tous  les  porcs  traités  étaient  guéris  à  la  fin  de  l’essai  (J35)  alors  que  les  animaux  non  

traités   sont   demeurés   infestés.   Les   autres   critères   d’évaluation   étaient   l’évolution   du   score  

clinique   et   de   prurit.   Les   lésions   cutanées   ont   rapidement   régressé   dans   le   groupe   traité   par  

l’afoxolaner  alors  qu’elles  étaient  encore  présentes  à  J14  dans  le  groupe  traité  avec  l’ivermectine.  

La  deuxième  partie  du  travail  de  thèse  a  porté  sur  l’évaluation  in  vitro  de  différentes  molécules  ou  

produits  acaricides.  Plusieurs  concentrations  d’une  solution  d’ivermectin  ou  de  moxidectine  ainsi  

11  huiles  essentielles  ont  été  testées.  Après  24h  de  contact  avec  l’ivermectine  et  la  moxidectine,  la  

dose   létale   50%   étaient   de   150,2±31,4   µg/mL   et   608,3±88,0   µg/mL,   respectivement.   Une  

concentration   inférieure  à  1  ng/mL  (pour   l’ivermectine)  ou  à  10  ng/mL  (pour   la  moxidectine)  n’a  

aucune  activité  acaricide.  Pour   les  huiles  essentielles,  des   tests  par   fumigation  et  par   immersion  

ont   été   réalisés.   Parmi   Lavandula   augustifolia,   Melaleuca   altenifolia,   Pelargonium   asperum,  

Eucalyptus  radiate,  Leptospermum  scoparium,  Cryptomeria  japonica,  Citrus  aurantium  ssp  amara  

et   3   l’huile   essentielle   identifiée   (BOB4,   BOB5,   BOB9)   testés   par   immersion,   l’huile   essentielle  

identifiée  BOB4  s’est  révélée  la  plus  efficace  (une  solution  à  1%  tue  tous  les  acariens  en  20  min).  

Parmi  les  10  huiles  essentielles  énumérées  avant,  plus  Juniperus  oxycedrus  testés  par  immersion,  

l’huile   essentielle   de  Melaleuca   altenifolia   s’est   révélée   la   plus   efficace   (tous   les   acariens   sont  

morts   en   4   min).   Pour   le   contrôle   de   S.   scabiei   dans   l’environnement,   différents   biocides   ou  

répulsifs   ont   été   examinés.   La   durée   moyenne   de   survie   a   été   calculée   pour   les   produits  

comportant  de  la  perméthrine,  de  l’esdépallethrine  et  de  la  bioresmethrine,  de  la  bifenthrine,  de  

la  cyperméthrine  et  de  l’imiprothrine,  de  la  cyfluthrine,  de  la  tétramethrine  et  de  la  sumithrine,  du  

DEET,   de   l’icaridine   et   le   produit   IR3535.   La   deuxième   partie   du   travail   de   thèse   a   porté   sur   la  

diversité   génétique   d’isolats   de   S.   scabiei   provenant   d’animaux.   Une   partie   du   gène   cox1   a   été  

amplifiée.   L’analyse   des   séquences   ainsi   obtenues   semble   montrer   que   les   sarcoptes   circulant  

chez  le  Chien  sont  issus  de  population  de  sarcoptes  d’origine  humaine.  

 

Mots  clés  :  Sarcoptes  scabiei,  acaricides,  modèle  animal,  tests  in  vitro,  diversité  génétique.    

I.  Background  

 7  

       

 I.  Background  and  outline  of  the  thesis  

 

 

 

 

 

I.  Background  

 8  

1.  Sarcoptes  scabiei    

1.1 Classification  

    Sarcoptes   scabiei   is   an   arthropod,   subphylum   Chelicerata,   class   Arachnida,   order  

Acarina,  suborder  Astigmata  (Sarcoptiformes)  and  family  Sarcoptidae  (figure  1).    

    The  word  arthropod  comes   from  the  Greek  words  arthro   that  means   joint  and  podos  

that  means  foot.  Arthropods  are  characterized  by  their  jointed  limbs  and  cuticle  made  of  

chitin,   often  mineralized  with   calcium   carbonate.   The   Phylum   Arthropoda   includes   the  

insects,  myriapods,  crustaceans,  chelicerates  and  trilobites.  There  are  around  1.3  million  

different  kinds  arthropods  that  have  been  found,  which  is  the  most  numerous  phylum  of  

all   living   organisms   (Averof   and  Akam,   1995;  Mangowi,   2014).   Arachnida   are   a   class   of  

arthropods  with  8   legs.  The  order   Acarina   (or  Acari),   including  mites  and  ticks,  contains  

numerous   economically   and  medically   important   species   that   are   parasitic   for   humans,  

domestic  or  wild  animals,  and  crops,  food,  etc.  The  sub-­‐order  Astigmata   is  a  large  group  

of   relatively   slow   moving,   similar   mites   with   thinly   sclerotized   integument   and   no  

detectable   spiracles   or   tracheal   system.   The   families   Sarcoptidae,   Psoroptidae   and  

Cnemidocoptidae   are   of  major   veterinary   importance.   Sarcoptidae   are   characterized   by  

short   legs   and   short   capitulum.   Psoroptidae   are   characterized   by   long   legs   and   long  

capitulum;   the   size   of   these   parasites   is   relatively   bigger   than   that   of   Sarcoptidae.  

Cnemidocoptidae   (or   Knemidocoptidae)   are   parasites   of   birds.   The   family   Sarcoptidae  

includes   three  genera:  Sarcoptes,  Notoedres  and  Trixacarus.  All  of   them  are  parasites   in  

mammals  (Mehlhorn  and  Armstrong,  2001;  Taylor  et  al.,  2007).  

 

I.  Background  

 9  

 

Figure  1.  Simplified  classification  of  mites  of  veterinary  importance  

1.2  Morphology  

    Sarcoptes   scabiei   has   a   characteristic   oval,   ventrally   flattened   and   dorsally   convex,  

tortoise-­‐like   body.   The  most   striking   parts   of   the   ventral   surface   are   the   chitinous   bars  

(called  epimeres),  which  strengthen  the  places  where  forelegs  and  hindlegs  are  inserted  in  

the  body.  On  the  dorsal  surface  of  the  mite,  there  are  transversely  arranged  thorns  and  10  

pairs   of   spines   arranged   on   two   sides,   3   pairs   on   the   anterior   part   and   7   pairs   on   the  

posterior   part   of   the   dorsal   surface.   The   female   is  300   to   500  µm   long   by   230-­‐420  µm  

wide,  and  the  male  is  210  to  285  µm  long  by  160-­‐210  µm  wide,  around  two-­‐thirds  the  size  

of   the   female.   Larvae   have   six   legs,   nymphs   and   adults   have   eight   legs,   with   suckers  

present  on   legs  1  and  2   in  both  sexes  and   leg  4  only   in  male  (figures  2  &  3).  The  anus   is  

terminal  in  both  sexes.  The  eggs  are  oval,  whitish  and  glossy,  with  slightly  tapering  at  the  

pole   lying   anteriorly   in   the   female   mite,   and   this   pole   is   attached   to   the   floor   of   the  

burrow  by  means  of  sticky  substance,  which  may  fasten  the  egg  to  the  burrow  securely.  

The   dimensions   of   the   eggs   are   167-­‐175  µm   by   88-­‐97  µm,   and   increase   during  

development  (figure  4)  (Heilesen,  1946).  

I.  Background  

 10  

 

Figure  2.  Male  and  female  of  Sarcoptes  scabiei  (Parasitology,  EnvA)  

 

Figure  3.  Microscopic  pictures  of  Sarcoptes  scabiei  var.  suis    

(A:  capitulum.  B:  suckers.  C:  thorns  and  spines  on  the  upper  part  of  dorsal  surface.  D:  spines)    

(Parasitology,  EnvA)  

I.  Background  

 13  

1.4  Survival  capacities  and  modes  of  transmission  

    Arlian   et   al.   (1984)   demonstrated   that   S.   scabiei   could   survive   for   24-­‐36h   at   room  

conditions   (21°C   and   40-­‐80%   relative   humidity),   have   the   capability   of   penetration   and  

remain   infective.   Females   and   nymphs   survive   longer   than   larvae   and   males   in  

comparable   conditions.   Low   temperatures   (10-­‐15°C)   and   high   relative   humidity   favored  

survival,  with  nymphs  surviving  up  to  21  days  at  10°C  and  97%  relative  humidity  (Arlian  et  

al.,  1989).  It  was  inferred  that  mites  remain  infective  for  at  least  one  half  to  two  thirds  of  

their  survival  time  when  dislodged  from  the  host.  It  should  be  noted  that  at  temperatures  

below  20°C,  S.  scabiei  mites  are  virtually  immobile,  while  the  activity  is  greatly  increased  

at  35°C.  

    The  transmission  of  S.  scabiei  can  be  reached  by  direct  contact  between  individuals,  or  

indirectly   by   fomites   (Burkhart  et   al.,   2000).   Studies   in   pigs   and   foxes   showed   that   the  

transmission   of   S.  scabiei   occurred   when   uninfected   animals   were   exposed   to   fomites  

(Samuel  et  al.,  2001;  Smith,  1986).  In  humans,  it  was  shown  that  fomites  play  a  little  role  

in   transmission   in   the   case  of  ordinary   scabies   (with   an  average  burden  of   less   than  20  

mites)  (Mellanby,  1941).  Studies  about  life  cycle  demonstrated  that  all  life  stages  of  mites  

leave  the  burrow  frequently,  wander  on  the  skin  and  may  fall  from  the  host  (L.  G.  Arlian  et  

al.,   1984a).   A   survey   in   homes   and   nursing   homes   environment   with   scabies   patients  

confirmed  the  presence  of  mites   in   fomites   (Arlian  et  al.,  1988a).  These   factors  coupled  

with   the   survival   and   infectivity   of   mites   suggest   that   fomites   could   be   a   source   of  

infection.  Especially  in  cases  of  crusted  scabies  which  is  characterized  by  the  presence  of  

thousands  of  mites  (CDC,  2011;  Chosidow,  2000;  Walton  et  al.,  1999b).  Kim  et  al.  (1990)  

reported  a  case  of  medical  staffs  who  were  infected  by  a  crusted  scabies  patient  by  means  

of  contaminated  medical  instruments.  

 

 

 

I.  Background  

 14  

1.5.  Variability  and  host  specificity  

1.5.1  Morphological  variability    

    The   mites   from   different   hosts   or   different   geographic   areas   tend   to   exhibit   some  

variable  morphologic  characteristics  including  the  size,  the  dorsal  field  of  spines,  and  the  

ventrolateral   spines.   Therefore,   it   remains   unclear   whether   different   isolates   represent  

different  species  or  simply  different  varieties  of  one  species.  Fain   (Fain,  1978,  1968)  did  

not  consider   that   the  variation  between  strains   from  different  hosts  have   taxonomically  

significance  and  proposed   that   the  genus  Sarcoptes   contains  only  one  valid  but  variable  

species  with  numerous  varieties.  He  summarized  the  bare  area  in  the  dorsal  field  of  scales  

into  4  different   types  and  divided   the   strains  of  S.   scabiei   into  3  main  groups  of   strains  

(figure  7):  1)  Strains  with  a  bare  area  in  most  or  in  all  the  specimens.  This  group  contains  

strains   completely   devoid   of   ventrolateral   scales   (strains   from   humans,   camels,  

dromedaries,  peccaries,  gibbons,  wild  sheep,  cabiais)  and  strains  having  ventral  scales   in  

all  specimens  (strains  from  domestic  and  wild  pigs)  or  in  some  specimens  (strains  from  a  

tapir   from   the   Vienna   zoo,   a   chimpanzee,   a   goat   from   South   Africa,   some   African  

antelopes,   horses   from   USA   and   South   Africa).   2)   Strains   with   most   of   the   specimens  

devoid   of   a   bare   area.   This   group   contains   strains   completely   devoid   of   ventrolateral  

scales   (strains   from   cattle   in  Holland   and  Belgium)   and   strains  with   ventrolateral   scales  

present  in  all  the  specimens  (strains  from  dogs,  ferrets,  polecats,  foxes,  llamas,  sheep  and  

goats   from  Austria,   chamois,   red   deers,  mountain   dogs)   or   in   almost   all   the   specimens  

(strains   from   horses   from   Mayaguez   and   from   Holland,   wombats,   chimpanzees).  

3)  Intermediate   forms.   This   group   contains   strains  with   intermediate   characteristics,   for  

both  the  bare  area  and  the  ventrolateral  scales,  which  prevents  Fain  from  putting  them  in  

either  of  the  2  preceding  groups.  They  are  probably  unstable  strains  still  in  the  process  of  

adaptation  to  a  new  host.    

 

I.  Background  

 15  

 

Figure  7.  Dorsum  features  of  females  Sarcoptes  scabiei  

Left:  absence  of  bare  area;  Right:  presence  of  bare  area  (from  Fain,  1978)  

 

1.5.2.  Population  genetics  of  Sarcoptes  scabiei  

    Molecular   biotechnology   is   an   important   tool   in   population   systematic   analysis   and  

DNA   sequencing  methods   have   advantages   over  morphology   or   protein  methodologies  

for   population   studies   in  mites   (Shelley   F.  Walton   et   al.,   2004a).   In   order   to   clarify   the  

taxonomic   status,  population  dynamics  and  epidemiology  of  S.   scabiei   infection,   several  

molecular   markers   have   been   used   since   the   late   1990s.   These   markers   include:   (1)  

microsatellite   DNA;   (2)   12S   rRNA,   16S   rRNA   and   COX1   gene   of   mitonchondrial   DNA  

(mtDNA);  (3)  the  second  internal  transcribed  spacer  (ITS2)  of  the  rRNA  gene  (Table  1).  

 

 

 

I.  Background  

 16  

Table  1.  Genetic  studies  about  Sarcoptes  scabiei  with  information  of  markers,  origin  of  the  mites  and  conclusions.  

Markers   Origin  of  the  mites  (host/country)   Conclusions   Reference  

Sarms  1,  15,  20  712  scabies  mites  from  humans  and  dogs  /  Ohio,  

Panama  and  Australia  

S.  scabiei  from  dogs  and  humans  clustered  by  host  species  rather  than  by  

geographic  location  

(Walton  et  al.,  

1999a)  

Sarms  33-­‐38,  40,  41,  44  and  

45  

Chamois  and  red  foxes  /  Italy   Gene  flow  between  mite  varieties  on  sympatric  Alpine  chamois  and  red  foxes  

was  absent  or  extremely  rare  

(Soglia  et  al.,  2007)  

Sarms  33-­‐38,  40,  41,  44  and  

45  

15  wild  mammals  from  10  species  /Italy,  France  and  

Spain  

There  was  a  lack  of  gene  flow  or  recent  admixture  between  carnivore-­‐,  

herbivore,  and  omnivore-­‐derived  Sarcoptes  populations  

(Rasero  et  al.,  2010)  

Sarms  34-­‐37,  40,  41,  44  and  

45  

Herbivores  (Thomson's  gazelle  and  wildebeest),  

carnivores  (lion  and  cheetah)  /  Masai  Mara,  Kenya  

Sarcoptes  infection  in  wild  animals  was  prey-­‐to-­‐predator-­‐wise   (Gakuya  et  al.,  

2011)  

Sarms  33-­‐38,  40,  41,  44   Pyrenean  chamois,  red  deers,  roe  deers  and  red  

foxes  /  Asturias,  Spain  

Little  change  in  the  genetic  diversity  with  the  mites  collected  from  animals  

between  an  11-­‐year  interval  period  

(Alasaad  et  al.,  

2011)  

microsatellite  

DNA  

Sarms  33–38  except  35,  40,  

41,  44  and  45  

Raccoons,  red  foxes,  chamois,  wild  boars  /  Germany,  

Italy  and  Switzerland  

The  raccoon-­‐derived  mites  clustered  together  with  the  foxes  samples  and  

were  clearly  differentiated  from  those  of  the  wild  boar  and  chamois  samples,  

which  suggests  a  fox  origin  for  the  raccoon  mange  infection  

(Rentería-­‐Solís  et  

al.,  2014)  

16S  rRNA   Chamois  and  foxes  /  Italy  and  Spain   Mite  populations  from  distinct  geographic  origins  were  genetically  separated,  

while  the  two  sympatric  populations  of  mites  collected  on  different  hosts  

from  north-­‐eastern  Italy  did  not  show  significant  levels  of  genetic  variation  

(Berrilli  et  al.,  2002)  

16S  rRNA  and  COX1   Humans,  dogs,  chimpanzees,  wallabies  and  wombats  

/  Panama,  Australia,  USA,  Sweden  

There  was  substantial  divergence  between  human-­‐associated  mite  

populations  and  other  animal-­‐associated  mite  populations  and  they  may  not  

have  shared  a  common  mitochondrial  ancestor  since  2-­‐4  million  years  ago  

(S.  F.  Walton  et  al.,  

2004)  

16S  rRNA  and  COX1   buffaloes,  cattle,  sheep,  rabbits  /  Egypt   COX1  and  16S  rRNA  indicated  the  presence  of  both  host-­‐adapted  and  

geographically  segregated  mites  from  different  hosts  

(Alasaad  et  al.,  

2014)  

mtDNA  

16S  rRNA  and  COX1   dogs,  humans  /  China   COX1  was  suitable  DNA  barcode  for  phylogenetic  study  of  Sarcoptes  mites  but   (Zhao  et  al.,  2015)  

I.  Background  

 17  

not  16S  rRNA  

COX1   pigs,  rabbits,  foxes,  jackals  and  hedgehogs  /Israel   COX1  analysis  showed  genetic  linkage  to  geographic  location,  but  not  to  the  

host.  

(Erster  et  al.,  2015)  

12S  rRNA   Wombats,  dogs  and  humans  /  Australia   Wombats  may  was  introduced  to  Australia  with  people  and/or  their  dogs   (Skerratt   et   al.,  

2002)  

 

12S  rRNA   Humans  and  dogs  /  France   Mange  in  wombats  is  due  to  the  introduction  of  S.  scabiei  into  Australia  by  

immigrating  individuals  and/or  their  companion  animals  

(Andriantsoanirina  

et  al.,  2015b)  

ITS2   dogs,  pigs,  cattle,  foxes,  lynxes,  wombats,  

dromedaris  and  chamois  /  Germany  

Unable  to  see  any  association  between  mite  haplotype  and  host  species   (Zahler  et  al.,  1999)  

ITS2   Chamois  and  foxes  /  Italy  and  Spain   The  ITS-­‐2  nucleotide  sequences  were  genetically  polymorphic.  The  variable  

sites  were  randomly  distributed  in  the  individuals  from  different  hosts  and  

localities  

(Berrilli  et  al.,  2002)  

ITS2   9  wild  animal  species  /  Switzerland,  Italy,  France,  

Spain  

ITS2  did  not  appear  to  be  suitable  for  examining  genetic  diversity  among  mite  

populations  

(Alasaad   et   al.,  

2009)  

ITS2   Buffaloes,  cattle,  sheep,  rabbits  /  Egypt   ITS2  showed  no  host  segregation  or  geographical  isolation   (Alasaad   et   al.,  

2014)  

rRNA  gene  

ITS2   Rabbits  and  pigs  /China   The  results  did  not  suggest  any  genetic  separation   (Gu  and  Yang,  2008)  

I.  Background  

 18  

Microsatellite  DNA          

    Microsatellites  or  simple  sequence  repeats  (SSRs)  are  tandemly  repeated  motifs  of  1–6  

bases   found   in   all   prokaryotic   and   eukaryotic   genomes   analysed   to   date.   They   are  

characterized  by   a   high  degree  of   length  polymorphism  and  have  been  used   as   genetic  

markers   in   relationship   studies   within   and   between   populations,   as   well   as   for   linkage  

analysis  and  genetic  mapping  (Zane  et  al.,  2002).  The  microsatellites  as  genetic  marker  for  

S.  scabiei  were  first  described  by  Walton  (1997)  who  isolated  18  microsatellites  and  chose  

three  hyper   variable  microsatellites   as  useful  markers   (namely   Sam1,  15  and  20).   Then,  

Walton  used   these   three  microsatellites  markers   to  analyze   scabies  mites   from  humans  

and  dogs  in  three  different  places  in  Australia,  showing  that  genotypes  of  dog-­‐derived  and  

human-­‐derived  mites   cluster   by   host   rather   than  by   geographic   location   (Walton  et   al.,  

1999a).   Later,   (S.   F.   Walton   et   al.,   2004))   identified   10   more   highly   polymorphic  

dinucleotide   repeats   (Sarms   23,   33,   34,   35,   36,   37,   40,   41,   44,   45)   and   two   slightly  

polymorphic  microsatellite  loci  (Sarms  31  and  38).  

    There   are   two   main   phenomena   have   been   described   by   applying   microsatellite  

markers   in   the  molecular  epidemiology   study  of  S.   scabiei   infection   in  animals:   (i)   three  

separate   clusters   (namely   herbivore-­‐,   carnivore-­‐   and   omnivore-­‐derived   Sarcoptes  

populations)  are  present  in  European  wild  animals  (Rasero  et  al.,  2010)  and  (ii)  there  is  a  

prey-­‐to-­‐predator  Sarcoptes  gene  flow  in  the  Masai  Mara  (Kenya)  ecosystem  (Gakuya  et  al.,  

2011).   Additional   studies   also   demonstrated   a   gene   flow   between   Sarcoptes   mite  

populations  in  sympatric  humans  and  dogs  (S.  F.  Walton  et  al.,  2004;  Walton  et  al.,  1999a),  

sympatric  Alpine  chamois  and  red  foxes  (Soglia  et  al.,  2007),  sympatric  Pyrenean  chamois,  

red   deers,   red   foxes   and   Iberian   wolfes   (Oleaga   et   al.,   2013)   and   raccoons   and   foxes  

(Rentería-­‐Solís  et  al.,  2014).    

I.  Background  

 19  

 

 

Figure  9.  Multilocus  microsatellite  clustering  analysis  of  individual  Sarcoptes  scabiei  using  a  

similarity  matrix  based  on  the  proportion  of  shared  alleles.  (A)  analysis  assumes  single  alleles  are  

homozygous.  (B)  Analysis  assumes  single  alleles  are  heterozygous-­‐nulls  (from  Walton  et  al.  2004)  

I.  Background  

 20  

12S  rRNA,  16S  rRNA  and  COX1  gene  of  mtDNA  

    Mitochondrial  DNA,  which  has  higher  rate  of  base  substitution  than  most  nuclear  genes,  

has   been   proven   to   be   a   useful   phylogenetic   tool   in   mites   and   ticks   to   investigate  

relationships  between  closely   related  species  and  at   the   intraspecific   level   (Cruickshank,  

2002;   Curole   and   Kocher,   1999).   By   analyzing   12S   rRNA   gene,   it   was   showed   that  

Sarcoptes  mites   from  wombats,   dogs   and   humans   could   not   be   separated   according   to  

host  or  geographical  origin  (Andriantsoanirina  et  al.,  2015b;  Skerratt  et  al.,  2002).  (Berrilli  

et   al.,   2002)   used   a   460bp   portion   of   16S   rRNA   to   investigate   the   phylogenetic  

relationships  of  S.  scabies  and  found  geographic  but  no  host  isolation  between  red  foxes  

and   chamois   from   different   regions.   16S   rRNA   and   COX1   sequences   indicated   the  

presence  of   both  host-­‐adapted   and   geographically   segregated  populations   of  S.   scabiei,  

however,  16S  rRNA  seems  to  have  less  variable  nucleotide  positions  (Amer  et  al.,  2014;  S.  

F.  Walton  et  al.,  2004;  Zhao  et  al.,  2015).    

 

Figure  10.  Un-­‐rooted  neighbour-­‐joining  tree  of  Sarcoptes  mites  based  on  COX1  sequences    

(Amer  et  al.,  2014)  

I.  Background  

 21  

 

 

Figure  11.  Un-­‐rooted  neighbour-­‐joining  tree  of  Sarcoptes  mites  based  on  mitochondrial  16S  rRNA  

sequences  (Amer  et  al.,  2014)  

 

The  second  internal  transcribed  spacer  (ITS2)  of  the  rRNA  gene  

  ITS2  sequence  analysis  revealed  very  little  variation  in  S.  scabiei  collected  from  different  

hosts   and   geographic   locations   (Alasaad   et   al.,   2009;   Berrilli   et   al.,   2002;   Gu   and   Yang,  

2008;  Zahler  et  al.,  1999).  Alasaad  et  al.  concluded  that  ITS2  rDNA  may  not  be  suitable  for  

examining  genetic  diversity  among  Sarcoptes  mite  populations  (table  2,  figure  12).  

 

 

I.  Background  

 22  

Table   2.   Countries,   geographical   locations   and   host   species,   together   with   the   number   of   host  

animals  and  Sarcoptes  mite  samples,  and  GenBankTM  accession  numbers  for  ITS-­‐2  sequences.    

Codes  Countries  and  

Codes  

Geographical  locations  and  codes   Host  species  and  codes  

No.  of  

  animals  

No.  of  

mites  

GenBankTM  

accession  number  

SwVv   Switzerland   Sw   Different  locations  

  Vulpes  vulpes   Vv   13   15   AM980676–AM980690  ItNERr   Italy   It   Northeast   NE   Rupicapra  

rupicapra  Rr   11   33   AM980691–AM980723  

ItNECe   Italy   It   Northeast   NE   Cervus  elaphus   Ce   1   2   AM980724–AM980725  

ItNESs   Italy   It   Northeast   NE   Sus  scrofa   Ss   2   6   AM980726–AM980731  

ItNEOam   Italy   It   Northeast   NE   Ovis  aries  musimon  

Oam   2   6   AM980732–AM980737  

ItNECi   Italy   It   Northeast   NE   Capra  ibex   Ci   2   5   AM980738–AM980742  

ItNEVv   Italy   It   Northeast   NE   Vulpes  vulpes   Vv   5   14   AM980743–AM980756  

ItNWVv   Italy   It   Northwest   NW   Vulpes  vulpes   Vv   10   26   AM980757–AM980782  

ItNWMf   Italy   It   Northwest   NW   Martes  foina   Mf   1   3   AM980783–AM980785  

FrNESs   France   Fr   Northeast   NE   Sus  scrofa   Ss   3   4   AM980786–AM980789  

SpNEVv   Spain   Sp   Northeast   NE   Vulpes  vulpes   Vv   1   4   AM980790–AM980793  

SpNWRp   Spain   Sp   Northwest   NW   Rupicapra  pyrenaica  

Rp   3   9   AM980794–AM980802  

SpSCp   Spain   Sp   South   S   Capra  pyrenaica   Cp   21   21   AM980803–AM980823  

 

 

Figure  12.  UPGMA  tree  showing  clustering  of  the  148  Sarcoptes  mites  from  13  wild  animal  

populations  belonging  to  nine  species  in  four  European  countries,  based  on  ITS-­‐2  ribosomal  DNA  

sequences,  using  Notoedres  cati  (AF251801)  as  the  out-­‐group.  Codes  in  this  figure  represent  the  

sample  codes  in  table  2  (from  Alasaad  et  al.,  2009)  

I.  Background  

 23  

1.5.3.Host  specificity  and  cross-­‐infectivity  

    Sarcoptes  scabiei   infects  humans  and  mammals,  which  has  the  largest  variety  of  hosts  

among   all   the   permanent   parasitic   mites   (Currier   et   al.,   2011).   It   was   proposed   that  

Sarcoptes   mites   originated   from   humans,   and   then   were   transmitted   to   animals   (Fain,  

1978).   Natural   transmission   of   animal  mange   between   different   host   species   has   been  

reported  and  animal-­‐derived  mites  are  also   responsible   for  outbreaks   in  humans   (figure  

13,  table  3).  

    (Neveu-­‐Lemaire  and  others,  1938)  described  in  detail  the  ability  of  cross  transmission  of  

mite   variants   between   different   hosts   including   human   beings.   Experimental   infections  

showed   that  mites   from   goats   can   infected   sheep   and   camels   (Abu-­‐Samra  et   al.,   1984;  

Nayel   and   Abu-­‐Samra,   1986);   mites   from   goats   can   infect   chamois   (Lavín  et  al.,  2000);  

mites  from  dogs  can  infect  rabbits  permanently,  and  can  infect  goats,  calves,  cats  and  pigs  

ranging  from  a  period  of  4  to  13  weeks  (Arlian  et  al.,  1988b);  mites  from  dogs  and  foxes  

are   readily   interchanged   and   seem  morphologically   identical   (Samuel   1981;   Bornestein  

1991;   (Soulsbury  et  al.,  2007).  Nonhuman  Sarcoptes   strains  which   infect  humans  usually  

come   from   dogs   (Aydıngöz   and  Mansur,   2011;   Beck,   1965;   Charlesworth   and   Johnson,  

1974;  Emde,  1961;  Smith  and  Claypoole,  1967),  but  strains  from  the  camels,  horses,  pigs,  

goats,   sheep,   chamois,   ferrets,   foxes,   wombats,   lions   and   the   llamas   have   also   been  

reported  as  zoonotic  on  various  occasions  (Mitra  et  al.,  1992;  Neveu-­‐Lemaire  and  others,  

1938;  Salifou  et  al.,  2013).  However,  none  of   these  human   infections  have  been  proved  

permanent  except  a  case  of  a  14-­‐year-­‐old  girl  with  crusted  scabies  due  to  S.  scabiei  var.  

canis.   The  patient   lived  with   three   severely   infected  dogs,   and   several  members  of   her  

family   developed   self-­‐limiting   rashes   after   sleeping  with   her.   Additionally   a   normal   dog  

was   successfully   infected  with  mites   from   the   girl   but   the   investigators  were   unable   to  

initiate  an  infection  on  rabbits  or  nude  mice  (Ruiz-­‐Maldonado  R  et  al.,  1977).  A  study  in  an  

experimentally   infected   human  with   S.   scabiei   var.   canis   showed   that   canine  mites   can  

burrow,  feed  and  lay  eggs  in  human  skin  (Estes  et  al.,  1983).  

I.  Background  

 24  

    The   mechanisms   for   the   host   specificity   of   S.  scabiei   are   largely   unknown.  

Host-­‐specificity  may   be   attributed   to  many   factors   and   interactions   between   hosts   and  

parasite,  such  as  physiological  differences  in  the  requirements  of  mite  strains;  differences  

in  dietary  and  non-­‐dietary  properties  of  the  host  skin  environment;  ability  of  the  host  to  

mount  an   immune  response;  antigenicity  of   the  parasite;  and  resistance  of   the  mites   to  

the  host  immune  response  (Arlian,  1989).  

 

 

Figure  13.  Cross  infections  of  Sarcoptes  scabiei  between  different  hosts  

 

 

 

I.  Background  

 25  

Table  3.  References  of  cross  infections  of  Sarcoptes  scabiei  between  hosts  

  To  humans   To  dogs   To  pigs   To  sheep   To  goats   To  horses  

From  

humans             A  

From    

dogs    A,  B,  C,  D,  E,  F     G     G   A  

From    

foxes    A   H,  I,  J         A  

From    

Pigs    A            

From    

sheep  A   A       A,  K    

From    

goats    A,  L     A   A     A  

From    

horses    A            

From    

llamas  A       A     A  

References:    

A.  Neveu-­‐Lemaire  et  al.  1938  

B.  Emde  1961  

C.  Beck  1965  

D.  Smith  and  Claypoole  1967  

E.  Charlesworth  and  Johnson  1974  

F.  Aydıngöz  and  Mansur  2011  

G.  Arlian  et  al.  1988b  

H.  Samuel  1981  

I.  Bornstein  1991    

J.  Soulsbury  et  al.  2007  

K.  Abu-­‐Samra  et  al.  1984  

L.  Salifou  et  al.  2013  

I.  Background  

 26  

2.  Infection  by  Sarcoptes  scabiei  in  animals    

 

    Sarcoptic  mange,  the  disease  caused  by  S.  scabiei   in  animals,  has  been  reported  from  

10  orders,   27   families,   and  104   species   of   domestic,   free-­‐ranging   and  wild  mammals.  A  

complete   list   of   these   hosts   has   been   published   and   recent   findings   have   been   added  

(table  4)  (Samuel  et  al.,  2001).  

Table  4.  List  of  reported  animal  species  infected  with  Sarcoptes  scabiei  (Samuel  et  al.,  2001).  

 

Order/Family   Species   Scientific  Name   Locality   Selected  References  PRIMATES          Cercopithecidae   Java-­‐macaca   Macaca  fascicularis   Denmarka   Leerhøy  and  Jensen  1967  Hominidae   Man   Homo  sapiens   Global   Fain  1978  Pongidae   Chimpanzee   Pan  troglodytes   Africa   Zumpt  and  Ledger  1973     Pygmy  

chimpanzee   Pan  paniscus   Africa   Zumpt  and  Ledger  1973  

  Orangutang   Pongo  pygmaeus   The  Netherlandsa   Fain  1968  

  Gibbon   Hylobates  leuciscus   USAa   Fain  1968  CARNIVORA          Canidae   Arctic  fox   Alopex  lagopus   Europe   Mörner  et  al.  1988     Dog   Canis  familiaris   Globala   Muller  et  al.  1989,(Xhaxhiu  et  

al.,  2009),(Chen  et  al.,  2014)     Dingo   Canis  familiaris  dingo   Australia   Gray  1937,  McCarthy  1960  

  Coyote   Canis  latrans   America  Samuel  1981,  Todd  et  al.  1981,  Pence  and  Windberg  1994  

  Gray  wolf   Canis  lupus   North  America   Todd  et  al.  1981,  Mörner,  1992     Jackal   Canis  mesomelas   Africa   Zumpt  and  Ledger  1973     Red  wolf   Canis  rufus   North  America   Pence  et  al.  1981     Crab-­‐eating  fox   Cerdocyon  thous   South  America   Fain  1968     Wild  dog   Lycaon  pictus   Africa   Mwanzia  et  al.  1995  

  Racoon  dog   Nyctereutes  procynoides   Europe   Henriksson  1972  

  Gray  fox   Urocyon  cinereoargenteus   North  America   Stone  et  al.  1982  

   

Red  fox   Vulpes  vulpes   Australia,  Holarctic  

Gray  1937,  Trainer  and  Hale  1969,  Mörner  1981  

Felidae   Cheetah   Acinonyx  jubatus   Africa   Mwanzia  et  al.  1995,(Gakuya  et  al.,  2012)  

  Cat   Felis  catus   Globala   Kershaw  1989     Cougar   Felis  concolor   USAa   Blair  1922     Serval   Felis  serval   Africa   Zumpt  and  Ledger  1973  

  Lynx   Lynx  lynx   Europe  Holt  and  Berg  1990,  Mörner  1992,  (Ryser-­‐Degiorgis  et  al.,  2002)  

  Lion   Panthera  leo   Africa   Young  1975,(Gakuya  et  al.,  2012)  

  Jaguar   Panthera  onca   USAa   Blair  1922     Leopard   Panthera  pardus   Germanya,  USAa   Blair  1922     Tiger   Panthera  tigris   Vietnama   Houdemer  1938  

  Snow  leopard   Uncia  uncia   The  Netherlandsa   Peters  and  Zwart  1973  

 

I.  Background  

 27  

Table  4  (continued)  

Order/Family   Species   Scientific  Name   Locality   Selected  References  Mustelidae   Stone  marten   Martes  foina   Europe   Wetzel  and  Rieck  1962  

  Pine  marten   Martes  martes   Europe   Holt  and  Berg  1990,  Mörner  1992  

  Fisher   Martes  pennanti   North  America   O’Meara  et  al.  1960     Badger   Mele  meles   Europe   Holt  and  Berg  1990     Siberian  polecat   Mustela  putorius   Europe   Wetzel  and  Rieck  1962     Stoat   Mustela  putorius  furo   Globala   Ryland  and  Gorham  1978  Procyonidae   Red  panda   Ailurus  fulgens   Swedena   Bornstein  1992     Coati   Nasua  nasua   Englanda   Fain  1968  Protelidae   Aardwolf   Proteles  cirstatus   Africa   Zumpt  and  Ledger  1973  Ursidae   Polar  bear   Thalarctos  maritimus   Czech  Republica   Jedlicka  and  Hojocova  1972     Black  bear   Ursus  americanus   North  America   Schmitt  et  al.  1987     Brown  bear   Ursus  arctos   Czech  Republica   Jedlicka  and  Hojocova  1972  ARTIODACTYLA          Bovidae   Impala   Aepyceros  melampus   Africa   Zumpt  and  Ledger  1973     Hartebeest   Alcelaphus  buselaphus   Africa   Zumpt  and  Ledger  1973     Barbary  sheep   Ammontragus  lervia   Israela   Yeruham  et  al.  1996     Springbok   Antidorcas  marsupialis   Africa   Zumpt  and  Ledger  1973     Pronghorn   Antilope  cervicapra   Czech  Republica   Frolka  and  Rostinska  1984  

  Cattle   Bos  taurus   Globala   Fain  1968,  Chakrabarti  and  Chaudhury  1984  

  Water  buffalo   Bubalus  bubalis   Asiaa   Chakrabarti  et  al.  1981  

  Goat   Capra  hircus   Europea,  Africaa,  Asiaa  

Fain  1968,  Garg  1973,  Ibrahim  and  Abu-­‐Samra  1985  

  Ibex   Capra  ibex   Europe   Rossi  et  al.  1995     Nubian  ibex   Capra  rubiana   Israela   Yeruham  et  al.  1996  

  Iberian  ibex   Capra  pyrenaica   Europe   Palomares  and  Ruíz-­‐Martìnez  1993  

  Siberian  ibex   Capra  sibirica   Asia   Jakunin  1958,  Vyrypaev  1985  

  Oryx   Connochaetes  taurinus   Africa   Zumpt  and  Ledger  1973     Mountain  gazelle   Gazella  gazella   Israela   Yeruham  et  al.  1996     Grants  gazelle   Gazella  granti   Africa   Wetzel  1984  

  Thomson’s  gazelle   Gazellea  thomsoni   Africa   Sachs  and  Sachs  1968,(Gakuya  et  al.,  2012)  

  Sable  antelope   Hippotragus  niger   Africa   Young  1975     Waterbuck   Kobus  ellipsiprymnus   Czech  Republica   Frolka  and  Rostinska  1984     Arabian  oryx   Oryx  leucoryx   Israela   Yeruham  et  al.  1996  

  Sheep   Ovis  aries   Europea,  Africaa,  Asiaa  

Fain  1968,  Okoh  and  Gadzama  1982,  Chakrabarti  and  Chaudhury  1984,(Rahbari  et  al.,  2009)  

  Mouflon   Ovis  musimon   Europe   Kerschlagl  1938,  Kutzer  1970     Steenbok   Raphicerus  campestris   Africa   Zumpt  and  Ledger  1973  

  Chamois   Rupicapra  rupicapra   Europe   Onderscheka  et  al.  1968,  Rossi  et  al.  1995  

  African  buffalo   Syncerus  caffer   Africa   Zumpt  and  Ledger  1973     Eland  antelope   Taurotragus  oryx   Israela   Yeruham  et  al.  1996  

  Kudu   Tragelaphus  strepsiceros   Africa   Zumpt  and  Ledger  1973  

Camelidae   Bactrian  camel   Camelus  bactrianus   Englanda   Fain  1968  

  Dromedary   Camelus  dromedarius   Asiaa,  Arabiaa,  Africaa  

Lodha  1966,  Higgins  et  al.1984,  Nayel  and  Abu-­‐Samra  1986  

  Lama   Lama  glama   -­‐   Kutzer  1970       Guanaco   Lama  guanicoe   -­‐   Kutzer  1970  

  Alpaca   Vicugna  pacos   Europe   Kutzer  1970,(Twomey  et  al.,  2009)  

  Vicuna   Vicugna  vicugna   -­‐   Kutzer  1970  

 

I.  Background  

 28  

Table  4  (continued)  

Order/Family   Species   Scientific  Name   Locality   Selected  References  Cervidae   Moose   Alces  alces   Germanya   Ullrich  1938     Roe  deer   Capreolus  capreolus   -­‐   Kutzer  1970     Red  deer   Cervus  elaphus   Europe   Kutzer  1970     Sambar   Cervus  unicolor   Africa   Fain  1968     Reindeer   Rangifer  tarandus   Russiaa   Lange  and  Sokolova  1992  

Giraffidae  Giraffe   Giraffa  camolopardalis   Francea   Mégnin  1877  

  Warthog   Phacochoerus  aethiopicus   Africa   Fain  1968  

  Wild  boar   Sus  scrofa   Europe,  North  America  

Wetzer  and  Rieck  1962,  Smith  et  al.  1982  

  Swine   Sus  scrofa  domestica   Globala  Chakrabarti  1990,  Davis  and  Moon  1990b,(Damriyasa  et  al.,  2004)  

Tayassuidae   White-­‐lipped  peccary   Tayassu  pecari   America   Fain  1968  

  Collared  peccary   Tayassu  tajacu   USAa   Meierhenry  and  Clausen  1977  PINNIPEDIA          Phocidae   Harbour  seal   Phoca  vitulina   Europe   Jacobsen  1966  HYRACOIDEA          Procaviidae   Gray  hyrax   Heterohyrax  syriacus   Africa   Wetzel  1984     Rock  dassie   Procavia  johnstoni   Africa   Wetzel  1984  PERISSODACTYLA        Equidae   Donkey   Equus  asinus   Arabiaa   Abu  Yaman  1978  

  Horse   Equus  caballus   Globala  Fain  1968,  Abu  Yaman1978,  Chakrabarti  and  Chaudhury  1984  

Tapiridae   Tapir   Tapirus  terrestris   Europea,  USAa   Kutzer  and  Grünberg  1967,  Fain  1968,  Frolka  and  Rostinska  1984  

RODENTIA          Caviidae   Guinea  pig   Cavia  porcellus   Francea   Fain  1968     Capybara   Hydrochaeris  

hydrochaeris   Europea   Fiebeger  1913,  Fain  1968  Erethizontidae   Porcupine   Erethizon  dorsatum   North  America   Payne  and  O’Meara  1958  Muridae   African  giant  

pouched  rat   Cricetomys  gambianus   Africa   Fain  1968  

  House  mouse   Mus  musculus   USAa   Meierhenry  and  Clausen  1977  Sciuridae   Fox  squirrel   Sciurus  niger   North  America   Allen  1942  LAGOMORPHA        Leporidae   Brown  hare   Lepus  europaeus   Europe   Restani  et  al.  1985     Mountain  hare   Lepus  timidus   Europe   Bornstein  1985     Rabbit   Oryctolagus  cuniculus   Europe,  USAa,   Fain  1968,  Arlian  et  al.  1984a     Marsh  rabbit   Sylvilagus  palustris   USA   Stringer  et  al.  1969  MARSUPIALIA        Phascolarctidae   Koala   Phascolarctos  cinereus   Australia   Barker  1974,  Brown  et  al.  1981  Vombatidae   Wombat   Lasiorhinus  latifrons   Australia   Fain  1968,  Wells  1971     Wombat   Vombatus  ursinus   Australia   Gray  1937  INSECTIVORA        Erinaceidae   African  hedgehog   Atelerix  albiventris   Africa   Okaeme  and  Osakawe  1985     Hedgehog   Erinaceus  europaeus   Israel,  

Germany   Kuttin  et  al.  1977,  Saupe  1988  

  Long-­‐eared  hedgehog   Hemiechinus  auritus   Israela   Yeruham  et  al.  1996  

a  Indicates  that  the  infection  has  occurred  in  mammals  in  captivity  

 

 

I.  Background  

 29  

2.1.  Distribution    

  Mange   has   been   reported   from   many   wild   mammals   worldwide   and   could   reach  

epizootic   proportions   in   certain   wildlife   populations.   These   include   (i)   coyotes   (Canis  

latrans),  red  foxes  (Vulpes  vulpes)  and  grey  wolves  (Canis  lupus)  in  North  America  (Little  et  

al.,  1998;  Pence  et  al.,  1983;  Pence  and  Windberg,  1994;  Todd  et  al.,  1981);  (ii)  arctic  foxes  

(Alopex  lagopus),  red  foxes,  grey  wolves,  wild  boars  (Sus  scrofa),  lynx  (Lynx  lynx),  chamois  

(Rupicapra   rupicapra),   ibex   (Capra   ibex),   Iberian   ibex   (Capra   pyrenaica)   and   red   deers  

(Cervus  elaphus)  in  Europe  (Fernández-­‐Morán  et  al.,  1997;  Gortázar  et  al.,  1998;  Lindström  

et   al.,   1994;   Mörner,   1992;   Pence   et   al.,   1983;   Pence   and   Ueckermann,   2002;  

Ryser-­‐Degiorgis   et   al.,   2002);   (iii)   red   foxes,   dingoes   (Canis   familiaris   dingo),   wombats  

(Vombatus  ursinus),  koalas  (Phascolarctos  cinereus)  in  Australia  (Brown  et  al.,  1982;  Gray,  

1937;  Martin  et  al.,  1998;  Potkay,  1977;  Skerratt  et  al.,  1998);  and  (iv)  mountain  gorillas  

(Gorilla   gorilla   berengei),   lions   (Panthera   leo),   cheetahs   (Acinonyx   jubatus),   impalas  

(Aepyceros   melampus),   hartebeests   (Alcelaphus   buselaphus),   springboks   (Antidorcas  

marsupialis),   wildebeests   (Connochaetes   taurinus),   buffaloes   (Syncerus   caffer),   elands  

(Taurotragus   oryx),   kudus   (Tragelaphus   strepsiceros),   Grant’s   and   Thompson’s   gazelles  

(Gazella   gazelle   and   G.   thompsoni)   and   sable   antelopes   (Hippotragus   niger)   in   Africa  

(Gakuya  et  al.,  2012;  Kalema  et  al.,  1998;  Pence  and  Ueckermann,  2002;  Sachs  and  Sachs,  

1968;  Samuel  et  al.,  2001;  Young,  1975).    

    Sarcoptic  mange  is  an  important  and  common  disease  in  companion  animals.  From  the  

reported  data  worldwide,  the  prevalence  of  S.  scabiei  in  dogs  is  5.6%  in  Iran  (Mosallanejad  

et  al.,  2012),  4.4%  in  Albania  (Xhaxhiu  et  al.,  2009),  4.7%  in  Kenya  (Gakuya  et  al.,  2012),  

0.6-­‐1.4%  in  Southern  China  (Chen  et  al.,  2014),  and  2-­‐3.4%  in  Nigeria  (Ugbomoiko  et  al.,  

2008).  

  Sarcoptes   scabiei   is   also   considered   as   a   common   parasite   among   economically  

important  livestock  such  as  pigs,  camels,  sheep,  buffaloes,  and  cattle.  Sarcoptic  mange  has  

been  documented  from  livestock  in  many  countries  including  Australia,  Belgium,  Denmark,  

Germany,  Greece,  Netherlands,  Spain,  Belgium,  Italy,  Switzerland,  France,  United  Kingdom,  

I.  Background  

 30  

Jordan,  India  and  the  United  States  of  America  (Al-­‐Rawashdeh  et  al.,  2000;  Davies  et  al.,  

1996;  de  Vega  et  al.,  1998;  Fanneau  de  la  Horie,  1990;  Fthenakis  et  al.,  2001;  Gakuya  et  al.,  

2012;  Rehbein  et  al.,  2003;  Tikaram  and  Ruprah,  1986).  The  prevalence  of  S.  scabiei  in  pig  

herds  varied  from  8.3%  to  86.6%  from  the  reports  of  European  countries  (Colebrook  and  

Wall,  2004).  It  has  been  estimated  that  between  50  and  95%  of  pig  herds  worldwide  have  

sarcoptic  mange  problem  (Cargill  et  al.,  1997).  

    The  economic  loss  due  to  S.  scabiei  infection  in  pig  industry  was  estimated  at  least  €100  

per  sow  per  year,  not  including  losses  such  as  lower  daily  weight  gain  and  depressed  feed  

conversion   in   fatteners   (Arends   et   al.,   1990;   Damriyasa   et   al.,   2004).   Other   economic  

losses  due  to  sarcoptic  mange  in  farms  related  to  the  decrease  of  milk  production,  weight  

loss,   and   leather   alterations.   Furthermore   significant   costs   are   associated   with   the  

continuous  use  of  acaricides  in  infected  herds.    

   

2.2.  Clinical  features  

    In   many   animal   species,   acute   sarcoptic   mange   is   characterized   by   intense   pruritus  

accompanied  by  erythematous  eruptions,  papules,  seborrhea,  and  alopecia.  These  clinical  

signs   are   not   always   observed   because   thick   fur   covers   the   lesions   in  many   animals.   In  

chronic  cases,  crusting,  hyperkeratosis,  lichenification,  and  thickening  of  the  skin  are  seen,  

and   animals   develop   a   foul   aromatic   odor.   In   severe   cases,   lymphadenopathy   occurs.  

Subcutaneous   edema  may  be  present,   often   seen  on   the   face   as   squinting   eyes   shortly  

before  death  (Samuel  et  al.,  2001).  

 

 

 

 

I.  Background  

 32  

In  foxes  

    In  foxes,  clinical  signs  usually  begin  around  the  haunches  and  the  base  of  the  tail  where  

alopecia   is   localized   (figure   15).   Presumably   this   is   related   to   the   area   being   heavily  

scented  and  a  site  of  social  communication  among  foxes.  Signs  are  often  quick  to  manifest  

on  the  head  while  the  fox  is  grooming  the  affected  area. As  the  infection  spreads,  the  hair  

loss  increases  along  with  areas  of  raw  skin  (damaged  during  scratching  and  grooming)  and  

the   fox,   unable   to   maintain   its   body   temperature   without   fur   and   less   able   to   hunt  

because   of   the   constant   itching,   begins   losing   condition.   As   the   animal’s   condition  

deteriorates,   it   becomes   susceptible   to   secondary   bacterial   infections,   caused   by  

opportunistic  microorganisms   (e.g.   Streptococcus  and   Staphylococcus   bacteria)   living   on  

the   skin.   Lloyd   et   al.   (Lloyd   and   others,   1980)   noted   the   affected   animals   develop   a  

“mangy”   odour,   which  may   be   due   to   a   secondary   bacterial   infection. Conjunctivitis   is  

also  common  in  the  late  stages  of  the  disease,  giving  a  swollen-­‐eyed  crusty  appearance  to  

the   face. At   the   end,   the   bacterial   infection,   starvation   and   hypothermia,   if   untreated,  

prove  fatal  (Bornstein  et  al.,  1994;  Mörner  and  Christensson,  1984).  

 

 

Figure  15.  A  fox  with  sarcoptic  mange,  alopecia  and  crusts  on  the  haunch  and  flank    

(Parasitology,  EnvA)  

 

 

 

I.  Background  

 33  

In  cattle  

    In   cattle,   clinical   signs   begin   from   the   head   and   the   neck.   The   disease   is   also   called  

“neck  and  tail  mange”  because  mites  have  partial  site  preferences.  Mild  infections  merely  

show  scaly  skin  with  little  hair  loss,  but,  in  severe  cases,  the  skin  becomes  thickened,  there  

is  marked  loss  of  hair  and  crusts  form  on  the  less  well  haired  parts  of  the  body  (figure  16).  

The  edema  and  inflammation  cause  the  formation  of  characteristic  skin  folds.  The  disease  

extends  rapidly  to  the  entire  body.  Scratching  is  continual  and  is  responsible  for  extensive  

mechanical  lesions  (Pouplard  et  al.,  1990).  

 

Figure  16.  Characteristic  lesions  of  sarcoptic  mange  in  a  cattle    

(from  Faculté  de  médecine  vétérinaire  de  St-­‐Hyacinthe,  Canada)  

 

In  sheep  and  goats  

    Sarcoptic  mange  is  a  common  condition  in  sheep.  In  breeds  kept  for  wool  production,  

lesions  are  observed  on  the  regions  without  wool,  such  as  head  and  legs.  Affected  areas  

are  at  first  erythematous  and  scurfy.  Because  of  intense  pruritus,  the  sheep  continuously  

scratch  and  rub  so  as  to  unable  to  graze  and  then  emaciate  progressively.  Thick  crusts  are  

observed.  In  haired  sheep,  the  entire  body  may  be  affected.  Sarcoptic  mange  in  goats  is  a  

chronic   condition,   which   may   have   been   present   simply   as   “skin   disease”   for   many  

months   before   definitive   diagnosis.   Like   other   sarcoptic   infections,   the   lesions   include  

irritation   with   crusts,   loss   of   hair   and   excoriation   from   rubbing   and   scratching.   In  

long-­‐standing   cases,   the   skin   becomes   thickened   and   nodules  may   develop   on   the   less  

I.  Background  

 34  

well  haired  parts  of   the   skin,   including   the  muzzle,   around   the  eyes  and   inside   the  ears  

(Lefevre  et  al.,  2010).  

     

Figure  17.  Sarcoptic  mange  in  a  sheep  (left,  from  Parasitology,  EnvA)    

and  a  goat  (right,  from  Salifou  et  al.,  2013)  

 

In  rabbits  

    In  rabbits,  cutaneous  lesions  appear  first  on  the  paws,  lips  and  nose,  later  around  the  

head,   neck,   and   sometimes   around   the   genitalia.   The   mites   can   lead   to   heavy  

scratching  by  the  rabbit,  which  will  lick  the  affected  areas.  Alopecia  is  often  observed.  

Crusts   are   usually   seen   on   these   locations.   One   can   also   observe   the   secretion   of   a  

watery   stuff   that   forms   crusts   upon   drying   (figure   18).   Self-­‐mutilation   will   lead   to  

wounds   and   secondary   bacterial   infection.   Severe   infection   leads   to   anemia   and  

leucopenia.  The  rabbit  becomes  lethargic  and  can  die  within  a  few  weeks  (Casais  et  al.,  

2014a).  

I.  Background  

 36  

 

Figure  19.  Crusted  scabies  in  a  pig  (from  MCP)  

 

 

In  llamas  

    In   llamas,  Sarcoptes   scabiei  distribute  mainly  over   the   legs,   the  ventral  abdomen,   the  

face  and  ears  (figure  20)  (Bornstein  and  de-­‐Verdier,  2010;  Twomey  et  al.,  2009).  The  early  

acute  manifestations  include  mild  to  severe  pruritus  with  erythema,  papules  and  pustules.  

In   chronic   infections,   the   skin  becomes   thickened  and  covered  with  hard  dry   scabs   that  

flake  off  and  remove  a  considerable  quantity  of  wool.  In  very  severe  infections  the  disease  

may  result  in  death  (Leguía,  1991).  

 

I.  Background  

 37  

 

Figure  20.  Sarcoptic  mange  in  a  llama.  Skin  lesions  include  hyperkeratosis,  alopecia,  thick  scabs,  

squamosis  and  ulceration  on  the  face,  shoulders,  flanks  and  legs    

(from  Yannick  Caron,  Faculté  vétérinaire  de  Liège,  Belgique)  

 

2.3.  Diagnosis  in  animals  

    In   dogs,   the  pinnal-­‐pedal   reflex   test   can  be  used   to   aid   in   a   diagnosis.   The   tip  of   the  

dog’s  one  earflap  is  rubbed  and  the  test  is  considered  positive  if  the  dog’s  hind  leg  made  a  

scratching  movement.  Between  75%  and  90%  of  dogs  with  mange  and  ear  lesions  have  a  

positive   pinnal-­‐pedal   reflex.   The   test   may   be   negative   if   no   ear   lesions   are   present  

(Mueller  et  al.,  2001).    

    Deep  skin   scrapings  are  made  with  a  scalpel  or  similar  blade-­‐   like  tool  to  the  point  of  

oozing  blood.  Direct  microscopic  examination  of   scrapings   is  often  not  worth   the  effort.  

However,  if  the  scrapings  are  heated  gently,  mites  become  active  and  more  observable.  If  

the  host  is  dead,  pieces  of  mangy  skin  are  removed  and  placed  in  a  Petri  dish.  Heat  from  

the  light  source  of  a  stereomicroscope  stimulates  mites  to  migrate  from  the  skin.  Failing  

I.  Background  

 38  

this,   scrapings  are  put   in  10%–20%  aqueous  potassium  hydroxide   (KOH)   solution   (figure  

21)  (Samuel  et  al.,  2001).  

 

Figure  21.  Microscopically  examination  of  skin  scrapings  from  a  fox,  showing  a  female  and  3  eggs  

(from  Parasitology,  ENVA).    

An   enzyme-­‐linked   immunosorbent   assay   (ELISA)   test   exists   for   the   diagnosis   of  

sarcoptic  mange  in  animals  including  pigs,  dogs,  chamois,  deers  and  red  foxes(Bornstein  et  

al.,   2006)   (Bornstein   et   al.,   2006;   Casais   et   al.,   2007;   Kessler   et   al.,   2003;   Lower   et   al.,  

2001).   In  Europe,  ELISA   for   the  detection  of   IgG  antibodies   to  S.   scabiei   is   commercially  

available   in  dogs   (Sarcoptes-­‐ELISA  2001®  Dog)  and  pigs   (Sarcoptes-­‐ELISA  2001®  Pig).  The  

sensitivity   and   the   specificity   of   the   test   for   dogs   were   92.1%   and   94.6%,   respectively,  

after  3  weeks  infection.  In  pigs,  the  sensitivity  and  the  specificity  were  95%  and  97%,  and  

the  test  is  positive  after  5  weeks  infection.        

    It   is   frequently   difficult   to   find   the   mites.   As   a   consequence,   if   sarcoptic   mange   is  

suspected,  treatment  should  begin  immediately.  If  the  clinical  signs  resolve  following  the  

specific  treatment  for  sarcoptic  mange,  then  the  diagnosis  is  confirmed.  

 

 

I.  Background  

 39  

2.4.  Animal  models  

    Scabies  and  sarcoptic  mange  have  historically  been  difficult  diseases  to  study.  A  major  

problem   is   the   lack  of   stable  and   large  amount  of  mite   sources.  At  present,   there   is  no  

method  available  for  culturing  and  propagating  Sarcoptes  mites  in  vitro.  Therefore,  animal  

models  are  still  required  in  order  to  widen  and  deepen  our  knowledge  of  Sarcoptes  scabiei  

and  the  corresponding  diseases.  

    To  date,  there  are  three  animal  models  that  have  been  successfully  established:  a  rabbit  

model  developed  by  Arlian  et  al.  in  the  USA  in  1984;  a  pig  model  developed  by  Mounsey  

and  Fischer  in  Australia  in  2010;  and  a  rabbit  model  developed  by  Casais  et  al.  in  Spain  in  

2014.  Using   these  models,   features  of   scabies  biology,   immunology  and  pathology  have  

been  revealed  (Arlian  et  al.,  1984b;  Casais  et  al.,  2014a;  Harumal  et  al.,  2003;  Mounsey  et  

al.,  2010).  

    Arlian   et   al.   (1984)   established   the   first   animal   model   in   1984.   They   experimentally  

transfered  S.  scabiei  var.  canis  to  rabbits.  Heavily  infected  crusts  from  a  dog  were  placed  

on  the  shaved  back  of  a  rabbit  for  12  to  22  h.  A  plastic  Petri  dish  covered  the  crusts  and  

fastened   by   tape.   Among   23   experimentally   infected   rabbits,   6   animals   established  

permanent  infection  with  a  single  inoculation,  10  and  7  rabbits  with  a  temporary  infection  

of   1   to   5   weeks   and   5   to   8   weeks,   respectively.   Some   of   these   rabbits   developed   a  

permanent   infection   when   re-­‐exposed,   but   5   rabbits   did   not   develop   a   permanent  

infection  even  after  secondary  exposure.  Twenty-­‐four  hours  after  exposure  in  all  exposed  

rabbits,  red  papules  and  general  erythema  were  observed  in  the  primary  area  of  infection.  

Within   2   weeks,   skin   scaling   also   became   evident   on   the   rabbits   that   would   develop  

permanent   infections.  Heavy  crusts  formed  during  the  subsequent  4  to  8  weeks  and  the  

lesions  and  crusts  gradually  spread  from  the   inoculation  area  over  the  entire  body,  with  

heavy  involvement  of  ears,  nose,  face,  feet,  and  back  (Arlian  et  al.,  1984b).    

    A   pig  model  was   established   by   an  Australian   research   team   (Mounsey  et   al.,   2010).  

Pigs  have  the  advantages  of  similar  epidermal,  morphological  and  immunological  changes  

to   humans  when   infected  with   S.   scabiei,   as   well   as   the   complement   system   in   pigs   is  

I.  Background  

 40  

comparable   to   humans   (Salvesen   and   Mollnes,   2009;   Van   Neste   and   Staquet,   1986)  

(annex  1).  Over   five  years  of  experiments,  a   sustainable  pig  model  with   regularly  use  of  

glucocorticoids  was  developed,  the   infection  can   last  for  at   least  12  months.  Crusts  with  

large  amount  of  mites  were  harvested  from  infected  pigs  and  dissected  into  small  pieces.  

Crusts  were  inserted  deep  into  both  ear  canals  of  2  to  3  week-­‐old  piglets.  Mounsey  et  al.  

observed  that  naturally  infected  pigs  self  cured  over  time.  In  order  to  maintain  infection,  a  

synthetic  glucocorticoid  dexamethasone  of  0.2  mg/kg  was  given  to  piglets  one  week  prior  

to   infection  and   for   the  entire  study.  Side  effects   like  growth  retardation  and  change  of  

body  shape  were  found  with  the  corticosteroids  treatment.  

    Recently,   Casais   et   al.   developed   an   animal  model   using   New   Zealand  White   rabbits  

experimentally   infected   with   S.   scabiei   isolated   from   a   European   wild   rabbit.   Three  

month-­‐old   New   Zealand   White   rabbits   were   contaminated   by   direct   contact   for   24h  

period  with  an  infected  rabbit  or  inoculation  of  crusts  on  each  shaved  hind  limb  and  worn  

for  24h,  without  use  of  any  immunosuppressant.  In  direct  contact  rabbits,  crusts  appeared  

in  10  out  of  10  rabbits  2  weeks  post  infection.  The  crusts  started  at  the  root  of  the  claw  

and   then   gradually   up   the   paw,   later   on   in   the   ears   and   nostrils.   In   rabbits   infected   by  

means  of  inoculation,  lesions  became  visible  at  2  weeks  in  2  out  of  10  rabbits  and  spread  

mainly  down  the  claw.  

 

 

 

I.  Background  

 41  

3.  Infection  by  Sarcoptes  scabiei  in  humans  

    The  disease  caused  by  S.  scabiei  is  called  scabies  in  humans.  

 

3.1  Epidemiology  

    Scabies   is   an   emerging   or   re-­‐emerging   disease   all   around   the   world.   The   cases   of  

scabies   in   humans   have   been   estimated   to   be   100  million   (Vos   et   al.,   2012)   up   to   300  

million   (Chosidow,   2006a)   per   year   and   the   occurrence   rates   vary   from   0.2%   to   46%  

(Fuller,  2013).  Overwhelmingly,  scabies  is  endemic  in  tropical  and  subtropical  areas,  such  

as   Africa   (Emodi   et   al.,   2010;   Odueko   et   al.,   2000;   Schmeller   and   Dzikus,   2001)  

(Abdel-­‐Hafez  et  al.,   2003),   South  America   (Feldmeier  et  al.,   2009),  northern  and  central  

Australia  (Walton  et  al.,  2004a),  India  (Sharma  et  al.,  1984),  and  Southeast  Asia  (Mahmud,  

2010;   Pruksachatkunakorn  et   al.,   2003).   In   temperate   zones,   the   incidence  of   scabies   is  

reported  higher  in  cooler  seasons  which  might  be  related  to  increased  personal  contact  as  

well   as   increased   survival   of   the   mites   (Hay   et   al.,   2012;   Mimouni   et   al.,   2003).   The  

prevalence  of  scabies   is   related  to  poverty,  overcrowding  and  demographic   forces   (wars  

and  migration  influences)  (Currier  et  al.,  2011).  In  developed  countries,  however,  scabies  

also   causes   significant   public   health   issues   in   schools,   hospitals,   and   other   institutions  

(Bouvresse  and  Chosidow,  2010;  Buehlmann  et  al.,  2009;  Capobussi  et  al.,  2014;  Ejidokun  

et   al.,   2007),   especially   among   vulnerable   individuals,   such   as   elderly   and  

immunocompromised   patients   like   HIV-­‐infected   individuals,   organ   transplant   recipients,  

and  patients  on  biologics  or  other   immunosuppressive   therapies   (Brites  et  al.,   2002).   In  

England,  a  national  study  has  reported  an  estimated  incidence  of  233  to  470  per  100,000  

person-­‐years  from  1994  to  2003  (Pannell  et  al.,  2005).  

3.2  Clinical  features  

    Although   scabies   can   present   clinically   in   many   different   forms,   the   two   most  

commonly  described  manifestations  are  ordinary  scabies  (also  known  as  classical  scabies)  

and  crusted  scabies  (also  known  as  Norwegian  scabies)  (Mounsey  et  al.,  2013).  Moreover,  

human  infection  of  animal  derived  mites  could  manifest  differently.  

I.  Background  

 42  

    Ordinary   scabies   presents   as  papular  or   vesicular   lesions   at   the   site  of  burrowing,   as  

well  as  a  generalized  allergic   rash  accompanied  by   itching   that   is  more   intense  at  night.  

The  lesions  localize  classically  in  the  webs  of  the  fingers,  the  flexor  surfaces  of  the  wrists,  

the  extensor  surfaces  of  the  elbows,  the  periumbilical  skin,  the  buttocks,  the  ankles,  the  

penis   in  males,   and   the   periareolar   region   in   females   (figures   22   and   23).   This   form   of  

scabies  is  commonly  seen  in  healthy  adults  in  whom  sexual  transmission  plays  a  significant  

role,   with   subsequent   intrafamilial   contact   transmission,   especially   to   children.   It   is  

reported   that   the   number   of  mites   per   patient   is   low   (approximately   12   female  mites),  

and   could   be   less  with   repeat   infections,   suggesting   acquisition   of   protective   immunity  

against  mite  reproduction  (Mellanby,  1944).    

    Crusted  scabies  was  first  described  among  leprosy  patients  in  Norway  in  1848  and  thus  

is   also   known   as  Norwegian   scabies.   It   is   a   severe   debilitating   disease   characterized   by  

millions   of   mites   and   the   development   of   large   hyperkeratotic   crusts   (figure   24).   This  

condition   is   often   seen   in   the   elderly   or   immunocompromised   persons   such   as   in  

transplant  recipients  (Gregorini  et  al.,  2012),  corticosteroid  use,  or  in  association  with  HIV  

or  HTLV-­‐1   infection  (Brites  et  al.,  2002).  Crusting  can  be   localized  or  extensive,  and  may  

occur  outside  normal  predilection  sites  of  mites,   including  the  face,  ears,  and  scalp.  The  

presence   of   pruritus   is   variable,   patients   with   extremely   poor   condition   may   not   itch  

(Fujimoto  et  al.,  2014).  The  crusted  scabies  patient  has  very   large  mite  burdens   in   their  

living   environment,   clothing   and   bedding,   posing   great   risk   to   families   and   caretakers  

(Carslaw  et  al.,  1975).  

      There  are  a  number  of   reports  on   transmission  of  Sarcoptes  scabiei   from  animals   to  

humans.  Human  infection  with  S.  scabiei  var.  canis  is  one  of  the  most  frequent  zoonoses  

due   to   close   contact   of   people   with   companion   animals   (Aydıngöz  and  Mansur,  2011).  

Compared  with   scabies   induced  by  S.   scabiei  var.  hominis,   the  disease  differs  a   little  bit  

clinically.   First,   the   incubation   period   is   usually   shorter   between   24-­‐96h   after   exposure  

which   takes  4-­‐6  weeks   in  human  scabies  of   the  primary   infection   (Walton  and  Oprescu,  

2013).   Secondly,   the   distribution   of   the   lesions   is   atypical.   Patients   develop   pruritic  

I.  Background  

 43  

papular  or  vesicular  erythemic  lesions,  primarily  on  the  trunk,  forearms,  axillae  and  thighs  

(figure   25)   (Beck,   1965;   Charlesworth   and   Johnson,   1974;   Tannenbaum,   1965).   No  

burrows  have  ever  been  seen  and  the  skin  scrapings  are  rarely  positive  in  man  (Smith  and  

Claypoole,  1967).  Thirdly,  the  severity  and  course  tend  to  be  milder  and  transient  which  is  

usually  described  as  self-­‐limited  with  the  infection  lasts  for  four  to  five  weeks  (Faust  et  al.,  

1962).  

 Figure  22.  Lesions  of  ordinary  scabies.  A.  Typical  lesions  in  the  finger  webs.  B.  Involvement  of  the  

male  genitalia  in  a  patient  with  excoriated  and  papular  scabies.  C.  The  breast  of  a  woman  with  

papular  scabies  lesions  on  the  nipple  and  areolar  area.  D.  A  typical,  specific,  scabious,  linear  

burrow  with  a  tiny  vesicle  at  the  distal  end.  E.  The  chronic  pruritus  of  scabies  rapidly  leads  to  

scratching  and  explains  why  eczema  is  frequently  observed  (from  Chosidow,  2006)  

 Figure  23.  Areas  where  scabies  rash  symptoms  often  appear  (modified  from  

http://www.cdc.gov/dpdx/scabies/)  

I.  Background  

 45  

programs   directed   at   scabies   without   antibiotic   intervention   have   shown   a   significant  

impact  on  the  prevalence  and  severity  of  pyoderma  (Carapetis  et  al.,  1997;  Taplin  et  al.,  

1991).  More  significant   is   the  streptococcal   infections  may  result   in   the  development  of  

glomerulonephritis   and   acute   rheumatic   fever.   Rates   of   end-­‐stage   renal   failure   in   the  

Aboriginal   people   of   scabies   epidemic   regions   are   21   times   that   seen   in   the   general  

Australian   population   and   have   been   shown   to   be   associated   with   post-­‐streptococcal  

glomerulonephritis  in  childhood  (White  et  al.,  2001).  

 

3.3  Diagnosis  in  humans  

To   efficiently   and   accurately   diagnose   human   scabies   is   still   a   challenge.   Detecting  

visible   lesions  can  be  difficult,  as   they  are  often  obscured  by  eczema  or   impetigo  or  are  

atypical.  Presumptive  diagnosis  can  be  made  on  the  basis  of  a  typical  history  of  pruritus,  

the  distribution  of  the  inflammatory  papules,  and  a  history  of  contact  with  other  sarcoptic  

infection  cases.  

Microscopy   is   a   conventional   diagnosis   for   scabies.   It   involves   the   use   of   skin  

scrapings  which  are   then  digested  with  one  or   two  drops  of  10%  KOH.  The  presence  of  

adult   mites,   nymphs,   or   eggs   can   be   confirmed.   While   this   method   has   an   excellent  

specificity  and  a  short  turnaround  time,  experience  has  shown  that  the  sensitivity  of  the  

test  is  less  than  50%,  which  further  varies  according  to  the  quality  and  quantity  of  the  skin  

scrapings  received  (Hong  et  al.,  2010).    

Epiluminescence  microscopy  and  high-­‐resolution  videodermatoscopy  are  noninvasive  

techniques   that  allow  detailed   inspection  of   the   skin   from  the   surface   to   the   superficial  

papillary   dermis.   (Dupuy   et   al.,   2007)   reported   91%   sensitivity   and   86%   specificity   for  

dermoscopy   by   experienced   users.   The   wrists   and   finger   webs   are   the   best   sites   for  

diagnostic   test   and   the   image  of   triangle  or   “delta  wing   jet”   sign   in   the   skin   represents  

mite  and  its  burrow  (figure  26).    

I.  Background  

 46  

 

Figure  26.  Dermoscopic  image  of  triangle  or  “delta  wing  jet”  sign  of  Sarcoptes  scabiei  infection  of  

human  skin  (Fox,  2009)  

 

    The  burrow  ink  test  is  using  ink  to  mark  the  suspicious  papules  and  then  wiped  off  with  

an  alcohol  pad  to  remove  the  surface   ink   from  the   lesion.  A  positive  BIT  test   (figure  27)  

occurs  when  the  characteristic  zigzagged  line  appears  as  the  result  of  the  ink  tracks  down  

the  mite  burrow.  This   test   is  useful  when  other  diagnostic  methods  are  unavailable   like  

microscope  or  dermatoscope.    

 

Figure  27.  Burrow  demonstrating  a  positive  BIT  result  (Golant  and  Levitt,  2012)  

I.  Background  

 47  

4.  Control  

    Since   scabies   and   mange   are   contagious   diseases   and   the   survival   of   mites   in   the  

environment,  the  treatment  requires  threefold,  that   is  treatment  of  the   infected  patient  

or  animal,   the   treatment  of  clothing  and   the  environment,  and  subjects  with  whom  the  

patient  or  animal  had  close  contacts  (Buffet  and  Dupin,  2003).  

4.1.  Acaricides  

Macrocyclic  Lactones      

    Macrocyclic   lactones   (MLs)   are   a   large   family   of   broad-­‐spectrum   antiparasitic   drugs  

widely  used  for   the  treatment  of  arthropods  and  nematodes  parasites   in  veterinary  and  

human   medicine   (Hennessy   and   Alvinerie,   2002).   Macrocyclic   Lactones   includes   two  

distinct   chemical   families.   These   are   milbemycins   (moxidectin,   milbemycin   oxime)   and  

avermectins  (ivermectin,  abamectin,  doramectin,  eprinomectin,  selamectin).  Milbemycins  

were   first   described   in   1974,   and   avermectins   were   first   described   in   1975.   They   are  

produced   by   fermentation   of   the   actinocycete   Streptomyces   spp.  Moxidectin   is   derived  

from   chemical   modification   of   nemadectin,   a   fermentation   product   of   Streptomyces  

cyanogriseus.   Ivermectin   is   a   semi-­‐synthetic   derivative   of   the   natural   avermectins,  

produced  during   fermentation  of  Streptomyces  avermitilis.  Mutation  of  S.   avermitilis   by  

chemical   modification   results   in   production   of   doramectin,   structurally   closer   to  

abamectin  than  ivermectin  (figure  28)  (McKellar  and  Benchaoui,  1996;  Shoop  et  al.,  1995).  

The   mechanism   of   action   of   MLs   in   parasites   is   based   on   their   high   affinity   for  

glutamate-­‐gated   chloride   channels   (GluCls)   that   is   confined   to   invertebrates  

(Kiki-­‐Mvouaka  et  al.,  2010;  Wolstenholme  and  Rogers,  2005).  GluCls  have  a  wide  range  of  

functions   in   invertebrate   nervous   systems   including   the   control   and   modulation   of  

locomotion,   the   regulation   of   feeding,   and   the   mediation   of   sensory   inputs  

(Wolstenholme,  2012).  GABA  receptors  appear  as  a  secondary  target  of  MLs  resulting   in  

somatic  muscle  paralysis  of  nematodes  (Beech  et  al.,  2010;  Brown  et  al.,  2012).  

I.  Background  

 48  

 

Figure  28.  Macrocyclic  lactones  family  (Prichard  et  al.,  2012)  

   

Pyrethroids  

    Pyrethroids  are  synthetic   insecticides  and  acaricides  that  are  derived  structurally  from  

the   natural   pyrethrins.   They   are   widely   used   throughout   the   world   and   generally  

considered  to  be  the  safest  class  of  insecticides/acaricides  available.  Many  pyrethroids  are  

registered   for   the   control   of   arthropods   in   agriculture   and   in   veterinary   medicine  

(Soderlund  et  al.,  2002).  Pyrethroids  are  known  to  work  on  insect  nerves  by  modifying  the  

kinetics   of   voltage-­‐gated   sodium   channels,  which  mediate   the   transient   increase   in   the  

sodium   permeability   of   the   nerve   membrane,   causing   prolonged   depolarization   of  

nerve-­‐cell   membranes   and   disrupting   neurotransmission   (Soderlund   and   Bloomquist,  

1989).  

Formamidines  

    The   formamidines   are   a   class   of   compounds   used   in   agriculture   and   in   veterinary  

medicine   mainly   as   acaricides.   This   class   includes   chlordimeform   and   amitraz  

(Hollingworth,  1976).  In  invertebrates,  these  compounds  exert  their  toxicity  by  activating  

an  octopamine-­‐dependent  adenylate  cyclase  (Nathanson,  1985).    

I.  Background  

 49  

Organophosphates    

    The   organophosphates   are   a   group   of   compounds   used   as   pesticides,   insecticides,  

acaricides   (Bajgar,   2004;   Senthilkumaran,   2015).   The  mechanism   of   action   is   known   to  

inactivate  acetylcholinesterase  (AChE),  which  is  essential  to  nerve  function  in  insects  and  

many  other  animals  (Fukuto,  1990).  Organophosphates  degrade  rapidly  by  hydrolysis  on  

exposure  to  sunlight,  air,  and  soil,  but  they  also  pose  risks  to  people  who  may  be  exposed  

to  large  amounts  (Costa,  2006).  

Phenylpyrazoles  

    Phenylpyrazoles   are   a  chemical   class   of   pesticides,   insecticides   and   acaricides  

introduced  in  the  early  1990's  both  for  agricultural  and  veterinary  use.  This  class  includes  

fipronil  and  pyriprole,  which  are  effective  against  a  number  of  parasites  such  as  fleas,  flies,  

ticks,   lice   and   mites.   Phenylpyrazoles   are   known   to   inhibit   both   GABA-­‐   and   L-­‐  

glutamate-­‐gated   chloride   channels   (Bloomquist,   2003;   Narahashi   et   al.,   2010).  

Phenylpyrazoles   are   quite   lipophilic   and   when   applied   topically   to   animals   they   are  

deposited  in  the  sebaceous  glands  of  the  skin  from  where  they  are  slowly  released.  This  

allows  a  rather  long  residual  effect  against  several  external  parasites  like  fleas.  

Isoxazolines    

    Isoxazolines   are   insecticides   and  acaricides  of   a  new  chemical   class   introduced   in   the  

2000s,   including   afoxolaner,   fluralaner   and   sarolaner   (figure   29).   They   have   a   broad  

spectrum   of   insecticidal   and   acaricidal   activity   and   are   effective   against   a   number   of  

ectoparasites   such   as   fleas,   ticks   and   lice.   So   far,   the   available   products   have   been  

introduced   only   for   oral   administration   in   dogs   (Dumont   et   al.,   2014;   Letendre   et   al.,  

2014a).    

    The   isoxazoline   compounds   have   been   proven   to   act   on   specific   GABA/glutamate  

receptor   inhibiting   the  chloride   ion  channels  of  arthropods   (García-­‐Reynaga  et  al.,  2013;  

Gassel  et  al.,  2014;  Shoop  et  al.,  2014).  In  comparison  with  fipronil,  which  also  showed  to  

inhibit  the  chloride  ion  channels,   isoxazoline  derivatives  exhibit  markedly  higher  potency  

I.  Background  

 50  

toward  GABA  chloride  channels.  Furthermore,  with  their  distinct  target  site,   it   is  unlikely  

to   have   cross-­‐resistance   to   isoxazolines   on   arthropods   that   exhibit   resistance   to  

commonly   used   insecticides   or   acaricides   (Shoop   et   al.,   2014;  Weber   and   Selzer,   2016;  

Zhao  and  Casida,  2014a)  (figure  30).  

    Isoxazolines   also   have   advantage   in   terms   of   pharmacokinetic   properties.   Fluralaner  

could   be   quantified   in   plasma   (>   10   ng/mL)   for   up   to   112   days   after   single   oral   and  

intravenous  treatment.  The  half-­‐life  of  both  fluralaner  and  afoxolaner  are  as  long  as  about  

15  days  (Kilp  et  al.,  2014;  Letendre  et  al.,  2014a).      

    With  an   increased   interest   in   the  development  of   isoxazoline-­‐derived  treatments,   this  

new   chemical   class   could   be   a   promising   treatment   for   parasites   including   S.  scabiei   in  

humans  and  animals.  

 

 afoxolaner                                   fluralaner                                         sarolaner          

Figure  29.  Molecule  structure  of  isoxazoline-­‐derived  compounds  

 

 

Figure  30.  Schematic  representation  of  cysteine-­‐loop  ligand-­‐gated  chloride  channels  in  a  sectional  

view  with  only  three  units  of  the  pentameric  transmembrane  region  depicted  for  clarity.  

Positioning  of  isoxazolines  is  putative  (Weber  and  Selzer,  2016).  

I.  Background  

 51  

Botanical  extracts  

    In  recent  years,  as  the  use  of  synthetic  products  is  becoming  increasingly  problematic,  

increasing  studies  have  focused  on  botanical  products  as  alternative  acaricides.  Recently,  

a  topical  treatment  of  5%  tea  tree  oil  combined  with  25%  benzyl  benzoate  has  been  used  

in  Australia  for  scabies.  This  therapy  proved  not  only  efficient,  but  the  addition  of  tea  tree  

oil  helped  to  reduce  the  significant  irritation  experienced  with  benzyl  benzoate  (Currie  et  

al.,  2004).    

    Besides   tea   tree   oil,   treatments   with   many   other   botanical   extracts,   that   are   of  

promising   scabicidal   properties,   have   also   been   described.   Tabassam   et   al.   (2008)  

reported   that   20%   crude   aqueous-­‐methanol   of   neem   (Azadirachta   indica)   seed   kernel  

completely   treated  mange   in   sheep  on  alternate  days   for  14  days;  Seddiek  et  al.   (2013)  

found  that  rabbits  were  cured  after  treated  with  25%  aqueous  neem  leaf  extract  every  3  

days   for   3   consecutive  weeks.  A  neem   seed  based  product  was  used   to   treat  mange   in  

dogs  and  80%  of  dogs  were  cured  after  14  days  consecutive  treatment  (Abdel-­‐Ghaffar  et  

al.,  2008).  Magi  et  al.  2006  showed  that  1%  oil  of  citronella  (Cymbopogon  nardus)  and  tea  

tree,  10%  ethanol  extract  of  hogweed  (Heracleum  sosnowskyi),  tansy  (Tanacetum  vulgare)  

and  wormwood  (Artemisia  absinthium)  reduced   in  vivo  mite   infection   levels  to   less  than  

10%   of   the   pretreatment   level   of   mange   in   pigs.   Jatropha   curcas   showed   a   better  

scabicidal  effect   then  benzyl  benzoate   for   the  comparison  treatment  of  mange   in  sheep  

(Dimri   and   Sharma,   2004).   Nong   et   al.   (2013)   found   that   1.0,   0.5g/mL   (w/v)   ethanol  

extract  of  Eupatorium  adenophorum   reached  100%  clinical   therapeutic   efficacy   after   14  

days  after  treated  twice  on  days  0  and  7  on  mange  in  rabbits,  which  was  as  effective  as  

ivermectin.   For   the   treatment   of   human   scabies,   two   essential   oils   Lippia   oil   (Lippia  

multiflora)   and   Camphor   oil   (Eucalyptus   globulus)   demonstrated   a   better   therapeutic  

effect  than  benzyl  benzoate  (Oladimeji  et  al.  2000;  Morsy  et  al.  2003).  

    So   far,   there   are   8   botanical   extracts   have   been   tested   in   vitro  on   S.  scabiei.   Among  

them,   neem   (Azadirachta   indica)   oil   and   tea   tree   (Melaleuca   alternifolia)   oil   have   been  

intensely  studied.  Others  include  Clove  oil  (Eugenia  caryophyllata),  Nugmeg  oil  (Myristica  

fragrans)   and   YlangYlang   oil   (Cananga   odorata),   as   well   as   plant   extract   include  

I.  Background  

 52  

Eupatorium  adenophorum,  Ailanthus  altissima  and  Ligularia  virgaurea  were  found  to  be  at  

least,  if  not  more  effective  against  S.  scabiei  than  their  positive  controls  such  as  ivermectin  

(Emtenan  M.H  NAM  et  al.,  2010;  Liao  et  al.,  2014;  Seddiek  et  al.,  2013;  Shelley  F.  Walton  et  

al.,  2004b)(Luo  et  al.,  2015),  benzyl  benzoate  (Oladimeji  et  al.,  2000;  Pasay  et  al.,  2010),  

amitraz   (Nong  et   al.,   2012)   and  permethrin   (Deng  et   al.,   2012;   Shelley   F.  Walton  et   al.,  

2004b).  An  overview  of  the  literature  reporting  studies  in  vitro  on  the  scabicidal  effect  of  

botanical  extracts  and  their  components  is  presented  in  annex  2.  

4.2.  Current  treatments  in  animals  

    In  animals,  different  drugs  and  formulation  can  be  used  for  the  treatment  of  sarcoptic  

mange   (table   5).   Currently   available   formulations   include   subcutaneous   injection,   oral  

products  and  topical  products  (pour-­‐on,  spot  on,  spray  or  dip).  The  choice  of  the  product  

usually  depends  on  the  animal  species,  the  age  of  animal,  or  the  number  of  animals.  For  

example,  amitraz  should  not  be  used  in  pregnant  or  nursing  bitches  or  puppies  less  than  3  

months.  Ivermectin  should  be  avoided  in  collies  and  sheep  dogs  or  their  crosses  as  it  can  

affect   central   nervous   system   causing   ataxia,   tremors,   mydriasis,   salivation,   depression  

and  even  coma  and  death  (Curtis,  2004).  

Table  5.  List  of  drugs  registered  for  the  treatment  of  sarcoptic  mange  in  France  (January  2016)  

Family   Molecule   Brand  name   Formulation  /  dosage   Animal  species  

ivermectin   Ivomec®   SC,  pour  on   Cattle,  sheep,  pigs  

selamectin   Stronghold®   Spot-­‐on,6-­‐10  mg/kg   Dogs  

Dectomax®     SC   Cattle,  sheep,  pigs  doramectin  

Doramec®   SC   Cattle,  sheep,  pigs  

eprinomectin   Eprinex®   Pour-­‐on,  0.1  mL/kg   Cattle  

Cydectin®     Pour-­‐on,  0.5  mg/kg;  SC,  0.2  mg/kg   Cattle,  sheep  moxidectin  

Advocate®   Spot-­‐on,  0.1  mL/kg   Dogs  

Macrocyclic  

lactones  

milbemycin  oxime   Interceptor®   Oral,  1.0-­‐1.5  mg/kg   Dogs  

fenvalerate   Acadrex®  60   Solution   Cattle  Pyrethroids  

deltamethrin   Butox®   Solution  5%   Cattle,  sheep  

Formamidine   amitraz   Taktic®   Solution   Cattle,  sheep,  goats,  pigs  

Organophosph. phoxim   Sebacil®   Solution   Pigs  

I.  Background  

 53  

4.3.  Current  treatments  in  humans  

    There  are  a  variety  of  treatments  available  on  the  market  for  human  scabies.  They  can  

be  divided   into  topical  agents  and  oral  agents.  The  choice   is   largely  based  on  the  age  of  

the  patient,  the  extent  of  eczematisation,  the  potential  toxicity  of  the  drug,  the  cost  and  

availability.  

Permethrin  

    Permethrin  is  a  synthetic  pyrethroid,  which  is  recommended  by  the  Centers  for  Disease  

Control  and  Prevention   (CDC)  as   first-­‐line  topical   therapy   for  scabies   (Chosidow,  2006a).  

The  creams  are  applied  overnight  once  a  week  for   two  weeks  to  the  entire  body  with  a  

contact  period  of  8  hours.  It  has  an  excellent  record  for  safety  and  low  toxicity.  Treatment  

trials   reported  more   than   90%   cure   rate   in   14   or   28   days   (Goldust   et   al.,   2013,   2012;  

Sharma  et  al.,  2011).  Previous  studies  of   in  vitro   tests  against  S.  scabiei  showed  that  the  

lethal  time  of  5  %  permethrin  was  480  min  (Walton  et  al.,  2000).  

Lindane  

    Lindane,  also  know  as  gamma  benzene  hexachloride,  is  an  organochloride  compound.  It  

was   first   used   to   treat   scabies   in   1948   (Wooldridge,   1948).   Lindane   used   to   be   the  

treatment   mainstay   with   a   single   6-­‐12h   application,   which   is   effective   against   mite.  

Nevertheless,  the  potential  neurotoxicity  of  lindane,  especially  with  repeated  applications,  

has   limited   its   use.   A   review   of   lindane   showed   that   43%   of   serious   adverse   reactions  

occurred  when  the  drug  was  used  as  labelled  (Nolan  et  al.,  2012).  The  product  is  no  longer  

available  (Chosidow,  2006a).    

Benzyl  benzoate  

    Benzyl   benzoate   is   used   at   a   concentration   of   25%   emulsion.   Its   advantages   include  

high  efficacy,  absence  of  resistance  and  low  cost.  It  is  therefore  a  very  popular  treatment  

in  Africa  and  parts  of  Europe,  although  it  is  not  available  in  the  United  States.  A  limitation  

is   that   the   drug   is   prone   to   cause   burning   and   stinging   and   possible   neurological  

I.  Background  

 54  

complications  with  misuse.  Therefore  in  children,  the  dosage  is  reduced  to  10%  or  12.5%  

(Mounsey  and  McCarthy,  2013).    

Crotamiton  

    Crotamiton   is   used   as   10%   cream   or   lotion.   It   is   often   used   on   scabies   nodules   in  

children.  The  clinical  efficacy   is  variable  between  50%  and  70%,  so  multiple  applications  

are  advised.  Previous  studies  demonstrated  that  ivermectin  and  permethrin  are  superior  

to   crotamiton   cream   10%   at   four   weeks   treatment   (Goldust   et   al.,   2014;   Taplin   et   al.,  

1990).  

Sulphur  

    Sulphur   is   used   as   an   ointment   (2%–10%).   The   technique   is   very   simple:   after   a  

preliminary   bath,   the   sulphur   ointment   is   applied   and   thoroughly   rubbed   onto   the   skin  

over  the  whole  body  for  two  or  three  consecutive  nights  (Lin  et  al.,  1988).  Patients  should  

apply   the   ointment   personally,   as   it   ensures   that   their   hands  will   be  well   impregnated.  

Due  to  its  messy  application  and  malodor,  topical  sulphur  ointment  is  not  a  popular  choice.  

It  is  still  used  in  some  areas  because  of  its  low  price  and  is  wide  margin  of  safety  in  infants,  

children,   and   pregnant   women.   So   it   should   be   used   only   in   situations   where   adults  

cannot  tolerate  lindane,  permethrin,  or  ivermectin  (Karthikeyan,  2005).  

Ivermectin  

    Ivermectin  has  been  used   in  humans   since   the  mid  1980s.  As  a   treatment  of   scabies,  

ivermectin   can   be   used   topically   and   orally   (Mounsey   and   McCarthy,   2013).   Oral  

ivermectin  is  most  commonly  administered  at  a  weight-­‐based  dose  of  200  µg/kg.  Since  it  

is   more   practical   than   topical   application,   oral   ivermectin   is   increasingly   used   for   the  

treatment   of   scabies   all   over   the   World,   especially   in   institutional   outbreaks   and   for  

crusted  scabies.  Despise  its  recorded  side  effects  such  as  headache,  pruritus,  pain  in  joints  

and  muscles,  maculopapular  rash  and  lymphadenopathy,  it  is  a  relatively  safe  drug.  

 

I.  Background  

 55  

4.4.  Drug  resistance  

  Treatment   failures  can  be  attributed  to  various  reasons   including   incorrect  application,  

reinfection  and  drug  resistance.  Resistance  of  S.  scabiei  has  been  reported  with  drugs  such  

as   lindane,   crotamiton,   permethrin   and   ivermectin   (Roth,   1991;   Thomas   et   al.,   2015).  

Permethrin   resistance   is  widespread   in  many   ectoparasites   (Heukelbach   and   Feldmeier,  

2004).   Evidence   of   increasing   resistance   to   permethrin   leading   to   scabies   treatment  

failures   has   been   reported   (Pasay   et   al.,   2006).   In   vitro   sensitivity   data   indicated   that  

S.  scabiei  mites  are  becoming   increasingly  tolerant  to  permethrin   in  Australia  (Walton  et  

al.,   2000).   In   the   last   decade,   there   have   been   reports   of   clinical   treatment   failure   of  

ivermectin  in  human  and  animals  (Currie  et  al.,  2004;  Terada  et  al.,  2010).  A  10-­‐year  study  

of   in   vitro   sensitivity   tests  demonstrated   that  S.  scabiei  mites  are  becoming   increasingly  

tolerant   to   ivermectin   in   remote   Aboriginal   communities   across   northern   Australia  

(Mounsey  et  al.,  2009a).    

    The  mechanism  of  resistance  in  S.  scabiei  is  complex.  Laboratory  studies  demonstrated  

that   acaricide   resistance   is   likely   mediated   by   P-­‐glycoprotein   mediated   efflux   (for  

ivermectin)  (Mounsey  et  al.,  2010),  sodium  channel  mutations  (for  permethrin)  (Pasay  et  

al.,   2008),   and   increased   activity   of  metabolic   enzymes,   such   as   esterases,   cytochrome  

P450   and   glutathion   S-­‐tranferases   (for   permethrin   and   ivermectin)   (Pasay   et   al.,   2009,  

2008).  Of  note,  it  has  been  shown  that  the  addition  of  a  synergistic  enzyme  to  permethrin  

can  reverse  resistance  in  vitro,  (Pasay  et  al.,  2009).  

 

 

 

 

 

I.  Background  

 56  

5.  Outline  of  the  thesis  

     

    Current   treatments   for  S.   scabiei   infection   are   limited,   especially   in   human  medicine.  

Moreover,   adverse   effects   and   resistance   of   S.   scabiei   to   conventional   acaricides   are  

reported.  For  the  environment  control  of  S.  scabiei,  there  are  no  efficient  products  so  far.  

In  addition,  the  taxonomic  status  of  S.  scabiei  is  still  under  controversy.  A  widely  accepted  

hypothesis   suggests   that  humans  were   the   initial   source  of  mites   for   animals,  however,  

without  factual  data.  

    The   objectives   of   the   thesis   were   to   assess   the   susceptibility   to   acaricides   and   to  

better  characterize  the  genetic  diversity  of  S.  scabiei  from  animals.  

    The   first   part   of   the   experimental   study   evaluated   the   efficacy   of   a   new   acaricide  

(afoxolaner)  in  an  animal  model.  

    The   second   part  assessed   the   scabicidal   effect   of  molecules,   commercial   products   or  

essential  oils  using  in  vitro  test.    

    The   third   part   of   the   study   characterized   the   genetic   diversity   S.   scabiei   mites   from  

animals.  

 

II.  In  vivo  evaluation  of  afoxolaner  

 57  

         

II.  Evaluation  of  afoxolaner  for  the  treatment  of  Sarcoptes  scabiei  infection  in  pigs  

   

 

 

 

 

 

II.  In  vivo  evaluation  of  afoxolaner  

 58  

1.  Introduction    

 

For   S.   scabiei   infection,   the  most   common   treatments   are   topical   acaricides   and/or  

systemic  treatment  with  a  macrocyclic  lactone.  These  treatments  are  limited  and  may  be  

not   sufficient   to   control   the   disease   burden.   In   addition,   the   problem   of   resistance   to  

current   acaricides   is   becoming   increasingly   severe   (Mounsey  et  al.,   2008).   Ivermectin,   a  

member  of  the  macrocyclic  lactones,  is  the  only  oral  drug  available  for  scabies.  The  drug  

must  be  given   twice  at   the  dosage  of  0.2  mg/kg   (Currie  and  McCarthy,  2010)  due   to   its  

limited   ovicidal   effect   and   a   short   half-­‐life   (Mounsey   et   al.,   2015).   Additionally,   the  

situation   is   complicated   by   the   low   research   and   lack   of   interest   from   pharmaceutical  

companies.  Alternative  approaches  such  as  vaccination  (Liu  et  al.,  2014)  seem  to  have  a  

long  way  to  go.  Therefore,  the  development  of  potent  acaricides  is  essential  and  urgent.    

    On   the   other   hand,   extensive   research   has   been   performed   in   the   veterinary   field.  

Afoxolaner,  a  member  of   isoxazolines,  has  been  demonstrated   to  be  a   safe  drug,  highly  

effective  against  fleas  and  ticks  (Beugnet  et  al.,  2014;  Dumont  et  al.,  2014;  Letendre  et  al.,  

2014b).  Moreover,  afoxolaner  has  shown  to  have  advantage   in  pharmacokinetics  profile  

with   long-­‐lasting   insecticidal   and   acaricidal   activity   (Letendre  et   al.,   2014b).   As   a   result,  

afoxolaner   might   also   be   effective   against   Sarcoptes  mites,   and   might   be   a   promising  

alternative  treatment  for  mange  and  scabies.  

In  this  context,  using  the  experimental  pig  model  described  by  Mounsey  et  al.  in  2010,  

we   conducted   a   preclinical   study   for   comparing   the   efficacy   of   a   single   dose   of   orally  

administrated  afoxolaner  with  two  doses  of  oral  ivermectin  against  S.  scabiei  var  suis.  

 

 

 

 

II.  In  vivo  evaluation  of  afoxolaner  

 59  

2.  Materials  and  methods  

 

2.1.  Experimental  pig  model  

  Twelve   three-­‐week   old   Sus   scrofa   domesticus   «  Large   white  »   breed,   female,   siblings  

from   the   same   pig   farm   (Christian   Lebeau,   Gambais,   France)   were   housed   at   the  

experimental   facility  CRBM  (Centre  de  Recherche  Biomédicale)   in   the  Veterinary  college  

of  Alfort,   France.  The  mean  weight   (±  SD)  at   the  arrival  was  7.15  kg   (±  0.63).  Pigs  were  

initially  free  of  sarcoptic  mange  and  they  had  never  received  any  antiparasitic  treatments.  

At   their   arrival,   drawing   lots   randomly   assigned   pigs   into   three   groups   of   four   pigs.   To  

reduce  stress  and  to  acclimate,  the  pigs  were  housed  two  weeks  before  starting  the  study  

in   small   groups   of   the   same   gender.   Pigs   where   placed   in   similar   experimental  

climate-­‐controlled  units  by  group  (temperature  of  21°C  ±  2°,  humidity  50%  ±  10%,  surface  

of   12   m2).   Environmental   enrichment   included   wood   shavings   on   concrete   floors   that  

were   cleaned   once   daily.   Feed   was   given   once   a   day   and   tap   water   was   continuously  

provided.  A  12/12h  light/dark  cycle  was  maintained  (on  at  7am  and  off  at  7pm).  A  physical  

examination   of   each   animal   by   a   veterinarian   twice   a   week   ascertained   management  

according  to  animal  welfare  standards.  Care  was  taken  to  reduce  stress  or  pain  of  the  pigs.  

Invasive   procedures   (e.g.   blood   samples,   skin   biopsies)   were   kept   to   a   minimum   and  

performed  under  a  short-­‐term  mild  sedation,  using  a  mixture  of  0.2  mL/kg  chlorhydrate  of  

ketamine  (Ketamine  1000®,  Virbac,  Carros,  France)  and  0.02  mL/kg  of  xylazine  (Rompun®  

2%,  Bayer  Healthcare,  Loos,  France)  given  by  a  single  intra-­‐muscular  injection.  

        The  infection  of  the  pigs  was  in  accordance  with  the  method  described  by  Mounsey  

et   al.   (2010).   The   synthetic   glucocorticoid   immune-­‐suppressant   dexamethasone   (Fagron  

SAS,  Thiais,  France)  was  used  to  promote  initial  infection,  increase  intensity  and  duration  

of   infection.   A   daily   oral   dosage   of   0.2   mg/kg   was   administered.   Dexamethasone  

treatment   was   initiated   in   pigs   one   week   prior   to   infection   and   continued   during   the  

entire   study   period   (figure   31).   The   infection   was   accomplished   by   directly   introducing  

mite-­‐infected   skin   crusts   deep   into   the   ear   canals   of   the   pigs.   Sarcoptes   mites   were  

initially  obtained  from  a  cohort  of  naturally  infected  pigs  in  a  farm  in  Brittany  (Dominique  

II.  In  vivo  evaluation  of  afoxolaner  

 61  

 

Figure  31.  Study  design.  Pictorial  representation  showing  the  three  different  experimental  phases:  housing  and  

acclimation  phase,  experimental  phase  1  and  phase  2.  DXM:  dexamethasone,  PK:  pharmacokinetics.  

 

2.3.  Clinical  monitoring  

    The  primary  outcome  was  based  on  the  reduction  in  the  number  of  live  mites  counted  

in  skin  scrapings  after  treatment.  The  end  point  was  the  complete  absence  of  live  mites  at  

day  14  post-­‐treatment.  Mites  were  collected  and  counted  in  skin  scrapings,  taken  on  day  0  

(just  before  treatment)  and  subsequently  on  days  2,  4,  8,  10,  14,  21,  28,  35  and  45  after  

treatment  in  order  to  estimate  the  percentage  efficacy  of  treatment  and  the  percentage  

of   mite   count   reduction.   Skin   scrapings   were   obtained   from   each   pig,   around   0.2   g   of  

crusts  were   scraped  using   a   scalpel   blade   from   the   ears   or   other   skin   areas   until   blood  

seeped   from   the   abrasion.   Samples  were   examined   in   a   Petri   dish  within   2   hours   after  

collection.  Under  a   light  heat  source,  mites  were  encouraged  to  crawl  out  of   the  crusts.  

The  mites  were  examined  under  a  stereomicroscope  (Nikon©,  SMZ645).  Only  alive  mites  

were   counted   and   the   number   of   life   stages   (adult   or   immature   stages)   was   noted.  

II.  In  vivo  evaluation  of  afoxolaner  

 62  

Immature  stages  include  larvaes  and  nymphs.    

    A  clinical   score   (figure  32)  was  designed  based  on  the  skin  surface  involved  by  scabies  

lesions  (scale  from  0  to  6),  the  intensity  of  the  erythema  of  the  skin  (from  0  to  4)  and  the  

intensity  of  the  encrustment  (from  0  to  4).  The  score  was  calculated  for  five  anatomic  sites  

(ears,  legs,  tail,  back  and  head)  and  added  up.  Clinical  examination  and  scoring  of  animals  

were   carried   out   weekly   after   infection   and   on   day   0   (just   before   treatment)   and  

subsequently  on  days  2,  4,  8,  10,  14,  21,  28,  35  and  45  after  treatment.  All  animals  were  

individually  examined.  Photographs  were  taken  from  each  pig.  

    Pigs  were  observed  weekly  for  recording  pruritus  within  15  min.  Movements  of  rubbing  

and  scratching  such  as   flapping  of   the  ears,   rubbing  on  a  surface,  scratching  ears  with  a  

posterior  leg  were  recorded.  Scoring  of  pruritus  was  carried  out  after  infection  and  on  day  

0  (just  before  treatment)  and  subsequently  on  days  2,  4,  8,  10,  14,  21,  28,  35  and  45  after  

treatment.  All  animals  were  individually  examined.  

To  estimate  the  hatchability  of  the  eggs,  eggs  were  collected  from  the  skin  scrapings  

taken   at   day   0   (just   before   treatment)   and   subsequently   on   days   2,   4,   8   and   14   after  

treatments.  Each   time,  10  eggs  were  collected   from  each  group   in  a  plastic   sterile  Petri  

dish.  The  eggs  were  placed  in  an  incubator  at  35°C  and  90%  humidity.    

 

 Figure  32.  Table  of  clinical  score  

 

 

II.  In  vivo  evaluation  of  afoxolaner  

 63  

2.4.  Afoxolaner  and  ivermectin  pharmacokinetics  

Blood   samples   were   collected   by   jugular   vein   puncture   on   heparinized   tubes   (BD  

Vacutainer®,   BD-­‐Plymouth,   UK)   on   day   0   (just   before   treatment)   and   subsequently   on  

hours  2,  4,  6,  24  and  days  2,  4,  5,  8  (4  hours  after  the  second  administration  of  drug),  10,  

14,  21,  28,  35,  45  and  50  after  treatment.  Plasma  was  prepared  by  centrifugation  of  blood  

samples   at   2   000   g   for   10   minutes.   Skin   biopsies   were   made   by   using   a   standard  

5-­‐mm-­‐diameter  punch  biopsy   tool   (KAI   Europe®,  GmbH,  Germany)   to  extract   a  piece  of  

epidermis  and  dermis   from  the  neck  region  of  the  pigs  on  day  0  (just  before  treatment)  

and   subsequently   on   days   1,   2,   4,   5,   8,   10,   14,   21,   28,   35,   45   and   50   after   treatment.  

Plasma  and   tissue   samples  were   stored  at   -­‐20°C  until  drug  analysis.   IVM  concentrations  

were  measured   in   plasma   and   skin   by   high   performance   liquid   chromatography   (HPLC)  

with  fluorescence  detection  using  a  procedure  previously  described  and  validated  (figure  

33)  (Lespine  et  al.,  2005;  Lifschitz  et  al.,  1999).  The  procedure  was  performed  in  Toxalim  

laboratory,   INRA,  Toulouse,  France.  AFX  concentrations  will  be  measured   in  plasma  and  

skin   by   liquid   chromatography-­‐mass   spectrometry   (LC-­‐MS)   in   Merial   laboratories   in  

Missouri,  USA.  The  extracted  analytes  were  chromatographed  by  reverse-­‐phase  HPLC  and  

quantified   by   a   triple   quadrupole   mass   spectrometer   system   using   the   electrospray  

interface(Letendre  et  al.,  2014b).  For  IVM  concentrations,  the  linearity  was  similar  in  the  

plasma  and  in  the  skin  (r  =  0.99  over  a  0.1–100  ng/mL  concentration  range)  and  the  limit  

of  quantitation   (LOQ)  were  0.05  ng/mL   in   the  plasma  and  0.1  ng/g   in   the   skin.   For  AFX  

concentrations,  the  lower  LOQ  was  1  ng/mL  in  plasma.  The  pharmacokinetics  parameters  

were   determined   using   a   non-­‐compartmental   analysis   (Kinetica   computer   program  

version   4.2,   InnaPhase®,   Philadelphia,   PA   for   IVM   and  WinNonlin®   software,   Pharsight  

Corp,  version  5.0.1  for  AFX).  The  area  under  the  concentration–time  curve  (AUC)  and  the  

mean  residence  time  (MRT)  were  calculated  from  the  time  of  administration  to  the  time  

of  the  last  measurable  concentration  (tlast),  using  the  arithmetic  trapezoidal  rule.  The  peak  

plasma  concentration  (Cmax)  and  time  of  peak  plasma  concentration  (Tmax)  were  read  from  

the  plotted  concentration  versus  time  for  each  pig.  

II.  In  vivo  evaluation  of  afoxolaner  

 64  

Figure  33.  High  performance  liquid  chromatography  (HPLC)  

HPLC  system  used  in  Toxalim  laboratory,  INRA,  Toulouse  for  the  dosage  of  IVM  in  skin  and  plasma.  

 

 

2.5.  Statistical  Analysis  

    A   Kruskal-­‐Wallis   H   test   was   used   in   order   to   compare   the   groups   at   baseline.   The  

primary  outcome  was  based  on  the  reduction  in  the  number  of  live  mites  counted  in  skin  

scrapings  following  treatment.  The  percentage  of  efficacy  was  calculated  according  to  the  

following   formula:   Efficacy   (%)   =   [(C   –   T)/C]   x   100   where   C   was   the   arithmetic   mean  

number  of  live  mites  for  the  control  group  and  T  was  the  arithmetic  mean  number  of  live  

mites   for   the   treated   group   for   each   time   point.   The   percentage   reduction   of   the  mite  

count  was  calculated  according  to  the  formula:  Reduction  (%)=  [(Mpre  –  Mpost)  /  Mpre]  x  

100  where  Mpre  was  the  arithmetic  mean  number  of   live  mites  at  baseline  (day  0),  and  

Mpost  the  arithmetic  mean  number  of  live  mites  post-­‐treatment  (days  2,  4,  8,  10,  14,  21,  

28,  35  and  45).  The  decrease  over   time   in  mite  count  and   in  clinical  and  pruritus  scores  

within  each  group  of  pigs  was  tested  for  significance  (p  <  0.05)  by  repeated  measures  in  a  

mixed  model  with  a  robust  variance  estimate  using  STATA  version12®  software.  We  use  a  

negative  binomial  regression  model  to  assess  the  relationship  between  parasites  (variable  

II.  In  vivo  evaluation  of  afoxolaner  

 65  

to  explain),   treatments  and   time(Hilbe,  2011).  Pharmacokinetics  parameters  obtained   in  

the   different   groups   were   compared   by   a   non-­‐parametric   Mann-­‐Whitney   test   at   a  

significance  level  of  p  <  0.05.  

 

3.  Results  

 

3.1.  Experimental  pig  model  

    Twelve   pigs   were   enrolled   into   the   study   in   January   2015   and   were   infected   with  

S.  scabiei  var.  suis  the  30th  of  January  2015.  They  were  treated  nine  weeks  after  infection.  

One  pig   (in   the   IVM-­‐treated  group)  died  during   the  study.  A  congenital  malformation  of  

the  digestive  tract  was  revealed  during  the  autopsy  (Annexe  3).  No  clinical  signs  of  drug  

intolerance  were  noticed  during  the  50-­‐day  period  of  observation  after  administration  of  

the  two  drugs.  The  side  effects  of  steroid  observed  in  the  study  were  mild  (augmentation  

of  the  appetite  and  hairiness).  

    First   cutaneous   lesions   were   visible   two   weeks   after   infection   and   encrustment  

occurred   after   four   weeks.   The   ear   was   the   first   localization   to   develop   lesions   then  

lesions   spread   to   the   entire   body.   Clinical   score   slowly   increased   after   infection   (Fig  4).  

Pruritus  appeared  two  weeks  after  infection  and  pruritus  scores  increased  rapidly  with  a  

peak  at  week  5  after  infection  (Fig  5).    

    On  week  9  post-­‐infection  (day  0),  pigs  from  all  the  three  groups  showed  clinical  signs  of  

successful  mite   infection   associated  with   intensive   itching.   At   baseline,   the   two   treated  

groups   and   the   control   group   were   comparable   in   terms   of   mite   count   in   the   skin  

scrapings  (p>0.05),  clinical  scores  (p>0.05)  and  pruritus  scores  (p>0.05).        

 

 

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 66  

3.2.  Clinical  outcomes  

Primary  outcomes  

    For   the  end  point,  on  day  14,   four  out  of   four  AFX-­‐treated  pigs  and  one  out  of   three  

IVM-­‐treated   pigs   were   mite-­‐free.   The   percentage   efficacy   of   the   treatment   and   the  

percentage  reduction  in  the  number  of  live  mites  in  skin  scrapings  in  the  AFX,  IVM-­‐treated  

groups   and   in   the   control   group   over   time   are   presented   in   figure   34   and   table   6.   On  

day  14,   the   drug   efficacy   was   100%   in   AFX-­‐treated   pigs   compared   to   95.4%  

(range  87.7−98.6%)  in  IVM-­‐treated  pigs.  The  percentage  reduction  of  the  mite  count  was  

100%   in  AFX-­‐treated  pigs  whereas   it  was  94.7%   (range  82.7−100%)   in   IVM-­‐treated  pigs.  

From  day  8  onwards,  no  more  mites  were  observed  in  the  scrapings  from  the  AFX-­‐treated  

pigs  at  all  further  points.  In  the  IVM-­‐treated  pigs,  one  pig  was  still  infected  with  live  mites  

at  the  end  of  the  study.  All  pigs  from  the  untreated  control  group  had  mites  throughout  

the   study   and   the   mite   count   remained   stable   for   all   animals.   After   treatment,   the  

decrease  over  time  in  mite  count  in  both  treated  groups  was  statistically  different  (p<0.05)  

and   statistically   different   from   the   count   of   the   control   group   (p<0.05).   The   figure   34  

presents  the  gerometric  mean  Immanuel  and  adult  stages  of  mite  counts  (immature  and  

adults).      

 

Fig  34.  Geometric  mean  immature  and  adult  stages  of  mite  counts  before  and  after  treatments  

with  ivermectin  or  afoxolaner  

 

II.  In  vivo  evaluation  of  afoxolaner  

 67  

Table   6.  Mite   count   values   and   efficacies   for   all   groups   over   time.   The   table   presents   the  mite  

counts  values  (number  of  mites,  mean  and  SD)  in  the  AFX,  IVM-­‐treated  and  control  groups  of  pigs  

on  the  various  assessments  days.   It  also  presents  the  percentages  of  reduction   in  S.  scabiei  mite  

count  and  efficacies  of  AFX  and  IVM  treatments  over  time.    Day  of  Study  

Afoxolaner  Group  (n=4)  No.  of  mites  (mean  mite  counts  ±  SD)  Count  range  (n)  Reduction  %  (range)  Efficacy  %  (range)  

Ivermectin  Group  (n=4)  No.  of  mites  (mean  mite  counts  ±  SD)  Count  range  (n)  Reduction  %  (range)  Efficacy  %  (range)  

Control  Group  (n=4)  No.  of  mites  (mean  mite  counts  ±  SD)  Count  range  (n)  Reduction  %  (range)  Efficacy  %  (range)  

Day  0   277  (69.3  ±  61.9)  4−127  

252  (63  ±  38.8)  13−98  

226  (56.5  ±  40.7)  15−106  

Day  2   78  (19.5  ±  12.9)  7−33  71.8%  (65.5−94%)  63%  (37.4−86.7%)  

256  (64  ±  33.8)  21−103  NA  NA  

211  (52.8  ±  42.7)  11−93  NA  NA  

Day  4   19  (4.8  ±  8.2)  0−17  93.1%  (75−100%)    93.5%  (76.9−100%)  

95  (31.7  ±  22.7)    13−57  49.7%  (35.9−75%)  56.9%  (22.4−82.3%)  

294  (73.5  ±  52.3)  8−124  NA  NA  

Day  8   0  0−0  100%  100%  

31  (10.3  ±  15.4)  0−28  83.6%  (53.6−100%)  81.3%  (66−100%)  

221  (55.3  ±  43.7)  9−105  NA  NA  

Day  10   0  0−0  100%  100%  

9.3  (11.8  ±  4.5)  5−14  85.2%  (61.5−89.9%)  87.6%  (49.3−88%)  

301  (75.3  ±  79.2)  1−151  NA  NA  

Day  14   0  0−0  100%  100%  

10  (3.3  ±  4.9)  0−9  94.7%  (82.7−100%)  95.4%  (87.7−98.6%)  

293  (73.3  ±  43.8)  22−124  NA  NA  

Day  21   0  0−0  100%  100%  

1  (0.33  ±  0.6)  0−1  99.5%  (98.1−100%)  99.5%  (98.4−100%)  

250  (62.5  ±  59.3)  18−144  NA  NA  

Day  28   0  0−0  100%  100%  

6  (2  ±  3.5)  0−6  96.8%  (88.5−100%)  96.3%  (89−100%)  

219  (54.8  ±  50.1)  5−117  NA  NA  

Day  35   0  0−0  100%  100%  

1  (0.3  ±  0.6)  0−1  99.5%  (98.1−100%)  99.4%  (98.2−100%)  

227  (56.8  ±  74.5)  3−167  NA  NA  

Day  45   0  0−0  100%  100%  

2  (0.7  ±  1.2)  0−2  98.9%  (96.2−100%)  98.9%  (96.7−100%)  

244  (61  ±  61.1)  7−148  NA  NA  

a  Standard  deviation    b  Not  applicable  

 

 

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 68  

Secondary  outcomes  

Clinical  scores  of  infection  

The  mean  clinical  scores  over  time  in  the  three  groups  are  presented  in  figure  35.  The  

clinical   lesions   of   sarcoptic   mange   disappeared   completely   for   all   the   AFX-­‐treated   pigs  

whereas  two  out  of  three  IVM-­‐treated  group  still  had  few  lesions  at  the  end  of  the  study.  

After   treatment,   the   mean   clinical   scores   of   both   treated   groups   were   statistically  

different  (p<0.05)  and  statistically  different  from  those  of  the  control  group.    

 

 

Figure  35.  Clinical  scores  in  the  three  groups  of  pigs.  

Mean  clinical  scores  (±  SD)  in  the  AFX,  IVM-­‐treated  and  control  groups  of  pigs  over  time,  after  

infection  and  after  treatment.  Clinical  scores  were  based  on  the  skin  surface  involved  by  scabies  

lesions  (from  0  to  6),  the  erythema  of  the  skin  (from  0  to  4)  and  the  encrustment  (from  0  to  4)  on  

5  different  anatomic  sites.  

   

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 69  

Pruritus    

Two   days   after   treatments,   an   increase   of   pruritus   was   observed   in   both   treated  

groups,   followed   by   a   decrease   of   the   pruritus   score   (figure   36).   Another   peak   was  

observed  just  after  the  second  dose  of  IVM  in  IVM-­‐treated  group  at  day  8.  After  treatment,  

the  mean  pruritus  scores  of  both  treated  groups  were  not  statistically  different  (p>0.05)  

but  were  statistically  different  from  those  of  the  control  group  (p<0.05).  

 

 

   

Figure  36.  Pruritus  scores  over  time  in  the  three  groups.  

Mean  pruritus  scores  (±  SD)  over  time  after  infection  and  after  treatments  in  the  AFX,  IVM-­‐treated  

and  control  groups  of  pigs.  

 

 

 

 

 

 

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 70  

 

Hatchability  of  the  eggs      

Before  treatment  (D0),  almost  all  eggs  collected  from  the  three  groups  were  able  to  

hatch  in  the  incubator.  At  day  2  (D2)  and  day  4  (D4)  after  treatment,  all  eggs  except  one  

from  IVM-­‐treated  at  D2  were  able  to  hatch.  At  days  8  (D8)  and  14  (D14)  after  treatments,  

no  eggs   from  the  AFX-­‐treated  and   the   IVM-­‐treated  groups  were   found   in   the   scrapings.  

These  data  suggest  that  both  ivermectin  and  afoxolaner  had  no  ovicidal  activity.  

 

Table  7.  Hatchability  of  the  eggs  collected  before  (D0)  and  after  (D2,  4,  8,  14)  the  treatments  from  

IVM-­‐  treated,  AFX-­‐treated  and  control  groups.  

 

Group   D0   D2   D4   D8   D14  

Ivermectin   12/12   7/8   3/3   *   *  

Afoxolaner   11/12   10/10   1/1   *   *  

Control   11/12   8/8   12/12   12/12   11/12  

 

 

 

 

 

 

 

 

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4.  Discussion  

Afoxolaner  is  a  new  systemic  insecticide  and  acaricide  which  has  been  proved  to  have  

good  insecticide  effect  against  ticks  and  fleas  (Frédéric  Beugnet  et  al.,  2014;  Dumont  et  al.,  

2014).  This  is  the  first  study  to  demonstrate  its  acaricidal  effect  against  S.  scabiei.   In  this  

study,   a   single   dose   of   afoxolaner   (2.5mg/kg)   was   found   superior   to   two   dose   of  

ivermectin   (0.2mg/kg)   against   sarcoptic  mange   in  pigs.   The  efficacy  of  AFX   increased   to  

63%   (range  37−87%),  94%   (range  77−100%)  at  day  2  and  4  post-­‐treatment   respectively,  

and  at  day  8  post-­‐treatment,  no  mites  were  found  from  AFX-­‐treated  pigs  (4/4  pigs)  while  

mites  were  still  observed  in  the  IVM-­‐treated  pig  (3/3  pigs),  and  one  pig  out  of  three  was  

still  infected  with  live  mites  at  the  end  of  the  study  (45  days  after  treatment).  Concerning  

clinical  scores,  both  AFX  and  IVM-­‐treated  pigs  demonstrated  a  strong  recovery  rate.  At  the  

end   of   the   study,   no   cutaneous   lesions   could   be   observed   for   all   the   AFX-­‐treated   pigs  

whereas   IVM-­‐treated   group   still   had   few   lesions.   Moreover,   the   AFX-­‐treated   pigs  

recovered  faster  than  the  IVM  ones.  As  for  the  pruritus  scores,  2  days  after  the  treatments,  

we  observed  an  increase  of  the  pruritus  in  the  two  treated  groups,  and  also  just  after  the  

second   dose   in   the   IVM-­‐treated   pigs.   Those   observations   may   due   to   the   release   of  

antigens  by  dead  mites.  Then  a  marked  decrease   in   the  pruritus   scores  was  observed   in  

both  treated  groups,  but  greater  in  the  AFX-­‐treated  pigs.  These  findings  are  in  accordance  

with   the   related   literatures   (Lee   et   al.,   1980;   Loewenstein   et   al.,   2006a).   The  pruritus  

persisted  several  days  after  treatments,  which  in  accordance  with  observations  in  humans  

with  the  so-­‐called  “post-­‐scabies  syndrome”  (Chosidow,  2006b).  

Despite   the   fact   that   the   number   of   pigs   involved   in   the   study   was   low   (a   major  

limitation  of  our  study),  the  analyses  of  primary  and  secondary  outcomes  highlighted  that  

all  results  converged  to  demonstrate  that  AFX  was  more  effective  than  IVM  at  every  time  

point.  The  primary  end-­‐point  was  relevant  and  the  analyses  attested  that  our  cohort  (even  

small)  was  a  reliable  representative  sample.  Concerning  the  pigs  at  baseline,   intensity  of  

the  infection,  distribution  of  the  cutaneous  lesions  and  number  of  mites  differed  between  

the   pigs   the   day   of   treatment.   Some   were   heavily   infected   whereas   others   had   fewer  

lesions.  We  used  randomization  to  exclude  this  systematic  bias,  but   it  doesn’t  necessary  

II.  In  vivo  evaluation  of  afoxolaner  

 72  

create  perfectly  homogenous  groups  at  baseline,   especially   for   small   groups.  One  other  

limitation   of   the   study   was   the   pruritus   assessment,   which   was   difficult   to   evaluate.  

Numerous   factors  could  modify   the  scratching  and   itching  behaviour  of   the  pigs  such  as  

the   presence   of   observers   in   the   room,   environmental   factors   (e.g.   room   temperature,  

humidity),  dryness  of  the  skin,  time  of  the  day  (pruritus  is  most  frequent  at  evenings  and  

middays)  (Davis  and  Moon,  1990).  The  use  of  cameras  and  video  recordings  to  observe  the  

animals   could  be  a   good  alternative   to  analyse   the  pruritus  and  may  be  use   for   further  

studies  (Davis  and  Moon,  1990;  Loewenstein  et  al.,  2006b).    

The   main   study   strength   was   the   robustness   and   the   trustworthiness   of   the  

experimental  model.  Scabies  has  always  been  difficult  to  study.  Studies  are  hampered  by  

the   lack   of   mite   sources   and   natural   infection   models.   Sarcoptes   mites   cannot   be  

maintained   alive   away   from   their   host   for   more   than   a   few   days   and   no   established  

methods  are  available  to  propagate  mites  in  vitro  (Arlian  et  al.,  1984).  To  overcome  these  

limits,  the  establishment  of  an  animal  model  for  scabies  was  necessary.  In  this  study,  we  

have  successfully  established  a  pig  model  previously  described  by  (Mounsey  et  al.,  2010).  

Our   observations   replicated   the   Australian   reports  with   regards   to   the   development   of  

disease,  e.g.  the  timescale  for  the  appearance  of  lesions  (erythema  and  crusts),  pruritus  as  

well  as   the  severity  of   the   infection.  The  observations  of   this   study  were  also   rigorously  

similar  in  a  previous  study  on  pigs  made  in  our  laboratory.  We  tempted  to  achieve  similar  

infection  in  all  pigs  by  challenging  the  pigs  with  the  same  amount  of  mite-­‐infested  crusts  

into   the   ear   canal   of   every   animal.   We   created   a   new   scoring   system   for   the   clinical  

monitoring   of   cutaneous   lesions   and   pruritus,   and   performed   standardized   scrapings   of  

the  skin.  

In  the  IVM-­‐treated  group,  it  is  interesting  to  look  at  the  proportion  of  the  different  life  

stages  after  treatments.  Before  treatments,  we  observed  a  quite  homogenous  repartition  

of  the  immature  and  adults,  with  a  little  more  of  the  immature  stages  in  all  groups.  After  

treatment,  at  day  2  and  especially  at  day  4,  we  observed  a  dramatic  decrease  of  the  adult  

mites   that  must  have  been  killed  by  the  drug.  At  day  10,  we  observed  a  modification   in  

II.  In  vivo  evaluation  of  afoxolaner  

 73  

the   proportion,   with  more   immature  mites.   This   could   be   explained   by   the   absence   of  

ovicidal  activity  of   IVM  and  concord  with  the   life  cycle  of  the  mite(Currie  and  McCarthy,  

2010).  Also,  due  to  short  duration  of  effective  doses  of   IVM   in   the  plasma  and  the  skin,  

newly  hatched  mites  could  not  be  killed,  confirming  that  the  second  administration  of  IVM  

is   dramatically   important.   The   second   dose   should   be   given   as   soon   as   all   eggs   have  

hatched,  between  day  7  and  day  10  (if  not  sooner),  and  before  newly  hatched  mites  have  

time   to  mate  and  produce  a  new  generation  of  eggs.  The  optimal   interval  of   treatment  

has   to   be   optimized   and   additional   studies   about   the   survival   of   eggs   and   their  

hatchability  are   required.  On   the  other  hand,   in  AFX   treated-­‐pigs,  a   rapid  and  definitive  

decrease  of  mite  count  was  shown.    

    As  the  isoxazolines  act  on  a  specific  site  of  GABA  channels,  it  is  unlikely  that  Sarcoptes  

mites,  which  exhibit   resistance  to  commonly  used  acaricides,  will   show  cross-­‐resistance.  

Afoxolaner   is  a  member  of   isoxazoline  compounds  which  control   insects  by   inhibition  of  

GABA-­‐gated  chloride  ion  channels  at  the  NCA-­‐II  site  (García-­‐Reynaga  et  al.  2013;  Zhao  and  

Casida   2014).   Although   these   channels   are   also   the   target   of   ivermectin   and   fipronil;  

ivermectin  activates  rather  than  blocks  GABA-­‐gated  chloride  channels  and  fipronil  binds  to  

another  site  in  the  chloride  channel  (Casida,  2015).    

    Afoxolaner   was   shown   to   have   advantages   in   pharmacokinetic   profiles   of   rapid  

absorption,   high   bioavailability,   moderate   distribution   into   tissues   and   low   systemic  

clearance.   In   dogs,   the   pharmacokinetics   of   afoxolaner   was   evaluated   following   oral  

administration  (Letendre  et  al.,  2014a).  Peak  plasma  concentrations  of  1655  ±  332  ng/mL  

were  observed  2–6h   after   treatment  dosing   at   2.5  mg/kg.   The   terminal   plasma  half-­‐life  

was  found  as  long  as  15.5  ±  7.8  days.  Moreover,  the  systemic  clearance  of  afoxolaner  was  

4.95  ±  1.20  mL/h/kg,  with  the  biliary  clearance  estimated  to  be  30%  of  the  total  clearance,  

and   renal   clearance  was   calculated   to  be   less   than  0.01%.  Given   these  data,   afoxolaner  

could  not  only  be  a  treatment  for  scabies/mange,  but  also  possess  potentially  protective  

efficacy  protecting  patients  from  infection/re-­‐infection.      

II.  In  vivo  evaluation  of  afoxolaner  

 74  

Afoxolaner   is   a   relatively   safe   drug.   So   far,   no   adverse   clinical   signs   have   been  

observed  in  the  previous  studies  in  dogs,  even  orally  administrated  six  times  with  up  to  5×  

the  maximum  exposure  dose  (Drag  et  al.,  2014;  Shoop  et  al.,  2014).  Mammalian  chloride  

channels,  as  study  in  rat  brain  membrane,  showed  no  significant  response  to  isoxazolines  

in  binding  assays  (Ozoe  et  al.,  2010).  The  binding  site  (NCA-­‐II)  of  afoxolaner  is  apparently  

not  present  or  is  of  low  sensitivity  in  mammals  (Casida,  2015).    

III.  In  vitro  evaluation  of  acaricides,  repellents  and  essential  oils  

 75  

       

III.  In  vitro  evaluation  of  acaricides,  repellents  and  essential  oils  for  the  control  

of  Sarcoptes  scabiei    

 

III.  In  vitro  evaluation  of  acaricides,  repellents  and  essential  oils  

 76  

1.  Introduction    

 

    For   scabies   or   mange,   the   in   vivo   evaluation   of   treatments   remains   a   difficult   task.  

Complications  include  difficulties  in  identifying  mites  or  eggs  and  thus  inaccuracies  in  both  

diagnosis   and  assessment  of   cure  and  differences   in   the   rapidity  with  which   symptoms,  

such  as   itching,  may  disappear.  These  complications  make  the  evaluation  of   the  relative  

efficacy  of  acaricides  in  both  individuals  and  controlled  clinical  trials  troublesome,  costly,  

and  time  consuming  (Walton  et  al.,  2000).  In  vitro  assays,  which  would  provide  evidence  

of  direct  killing,  make  the  experiments  on  the  comparison  of  drugs  efficacies,  detection  of  

mite  resistance  possible  and  effective.  However,   in  vitro  assays  have  been  limited  by  the  

lack  of   a   regular   supply  of   adequate  numbers  of  mites.   The   successful   establishment  of  

rabbit  and  pig  models  made  these  tests  possible  (L.  G.  Arlian  et  al.,  1984b;  Casais  et  al.,  

2014a;  Mounsey  et  al.,  2010).  

    In  a  previous  study  conducted  in  our  laboratory,  it  was  demonstrated  that  a  single  dose  

of  oral  moxidectin  (at  0.3  mg/kg)  was  more  effective  than  two  doses  of  oral  ivermectin  (at  

0.2   mg/kg)   for   the   treatment   of   sarcoptic   mange   in   the   pig   model   (Bernigaud   et   al.,  

submitted).   To   date,   ivermectin   is   the   only   macrocyclic   lactone   approved   for   the  

treatment  of  scabies  in  humans  (Geary  et  al.,  2010).  Because  of  its  relatively  shot  half-­‐life  

and  limited  ovicidal  effect,  the  treatment  with  oral  ivermectin  usually  needs  two  or  more  

doses  (Currie  et  al.,  2004).  Moxidectin  is  much  more  lipophilic  and  tends  to  accumulate  in  

fat  tissue  (Scott  and  McKellar,  1992),  which  could  be  a  potential  alternative  to  ivermectin  

for  the  treatment  of  scabies  in  human.    

    Sarcoptes  scabiei  burrows  in  the  stratum  corneum  and  stratum  granulosum  of  the  skin  

in  both  humans  and  animals  (Hengge  et  al.,  2006;  McCarthy,  2004)  and  the  transmission  

of  scabies/mange  acts  through  direct  and  indirect  contact.  Arlian  et  al.  demonstrated  that  

away  from  their  hosts,  mites  are  able  to  survive  and  remain  infective  for  24-­‐36h  at  21°C  

and  40-­‐80%  relative  humidity.  Mites  can  even  survive  longer  at  lower  temperatures  with  

higher  levels  of  humidity  (Arlian,  1989).  Generally,  female  and  nymph  mites  survive  longer  

than  larvae  and  males  in  comparable  conditions  (Arlian  et  al.,  1989).  Studies  on  pigs  and  

III.  In  vitro  evaluation  of  acaricides,  repellents  and  essential  oils  

 77  

foxes  showed  that  the  transmission  of  S.  scabiei  occurred  when  uninfected  animals  were  

exposed  to  fomites  (Samuel  et  al.,  2001;  Smith,  1986).  Mites  were  found  from  fomites  in  a  

survey   in   a   home   and   nursing   home   environment   with   scabies   patients   (Arlian   et   al.,  

1988a).  These  data  coupled  with  the  survival  and  infectivity  of  mites  suggest  that  fomites  

could  be  a  source  of  infection,  especially  in  cases  of  crusted  scabies  which  is  characterized  

by  the  presence  of  thousands  of  mites  (Chosidow,  2000;  Thomas  et  al.,  1987;  Walton  et  al.,  

1999b).  

    Currently,   treatments   for   scabies   or   mange   are   mainly   from   4   families   (macrocyclic  

lactones,   pyrethroids,   formamidins   and   organophosphates).   With   the   extensive   use   of  

these   acaricides,   drug   resistance   might   become   a   significant   issue   (Currie  et   al.,   2004;  

Heukelbach   and   Feldmeier,   2006;   Mounsey   et   al.,   2009b,   2008;   Roth,   1991;   Shelley   F.  

Walton   et   al.,   2004a;   Walton   et   al.,   2000).   In   addition,   these   acaricides   have   been  

reported  resulting  in  mild  to  severe  adverse  effects  (Boussinesq  et  al.,  2003;  Fujimoto  et  

al.,   2014).  With   the   trend   of   “green”   consumerism,   an   increasing   importance   has   been  

attached  to  botanical  products.  Aromatic  plants  and  their  essential  oils  have  been  showed  

manifold.   They   have   been   demonstrated   to   possess   fumigant   and   topical   toxicity   to   a  

number  of  insect  and  mite  pests,  as  well  as  to  fungi  and  bacteria  (Regnault-­‐Roger,  1997).  

 

    The  objectives  of  this  part  of  the  thesis  were:  

-­‐ to   assess   the   efficacy   of   moxidectin   and   ivermectin   against   S.   scabiei   with   a  

concentration  gradient  in  vitro  test;  

-­‐ to  assess  the  efficacy  of  biocides  or  repellents  for  the  environmental  control  of  S.  

scabiei;    

-­‐ to  assess  the  potential  acaricidal  efficacy  of  11  selected  essential  oils  which  can  be  

used  as  alternative  treatment  or  environmental  control  of  S.  scabiei.  

 

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 78  

2.  Materials  and  methods  

 

2.1  Sarcoptes  mites  

    Sarcoptes   mites   were   collected   from   pigs   maintained   at   the   CRBM   (Centre   de  

Recherche   Bio   Médicale),   Maisons-­‐Alfort,   France.   Pigs   were   experimentally-­‐infected   as  

described  by  Mounsey  (2010).  For  all  in  vitro  tests,  mites  were  collected  from  crusts  in  the  

external  ear  canal;  they  were  gently  removed  and  placed  in  a  Petri  dish.  Mites  crawled  out  

of   the   crusts   in   about   half   an   hour.  Mites   were   picked   one   by   one   with   a   needle   and  

placed  into  a  small  Petri  dish  (3  cm  in  diameter).  All  the  tests  were  performed  in  triplicate.    

 

2.2  Solutions  preparation  and  bioassays  of  ivermectin  and  moxidectin  

    Ivermectine  and  moxidectin  were  purchased  from  Sigma  Life  Science.  The  two  powders  

were   dissolved   with   pure   DMSO   into   stock   solutions   of   1000  µg/mL,   500  µg/mL,  

100  µg/mL,  10  µg/mL,  1  µg/mL,  respectively.    

    Mites  were  tested  within  3  h  after  collection  from  the  pigs.  Nymphs  and  females  (n=10)  

were  placed  in  a  plastic  Petri  dish.  Five  mL  of  distilled  water  were  added  to  each  Petri  dish,  

and   then   5  µL   of   each   stock   solution   was   added   to   the   Petri   dishes,   respectively.   The  

definitive   concentrations   of   drugs   were   1000  ng/mL,   500  ng/mL,   100  ng/mL,   10  ng/mL  

and  1  ng/mL,  respectively.  For  the  negative  control,  5  µL  of  pure  DMSO  was  added  to  5  mL  

distilled  water.  The  dishes  were  placed  at  room  temperature  (20°C)  for  24h.  The  solutions  

were   taken   out   with   a   pipet   before   careful   inspection   of   the   mites   under   a  

stereomicroscope.    

 

2.3  Products  and  bioassays  for  environmental  control  

    The   tested   products   were   chosen   from   the   products   that   were   used   for   the  

environmental  control  of  mites,   lice,  fleas  and  other   insects.  A  repellent  for  mosquitoes,  

ticks   and   flies   was   also   tested.   These   products   are   available   in   pharmacies,   veterinary  

clinics  or  supermarkets  in  France.  Brand  names  like  Insect  Ecran®  and  Pyréflor®  may  have  

III.  In  vitro  evaluation  of  acaricides,  repellents  and  essential  oils  

 79  

different   targets   and   consequently   a   single   brand   name   may   correspond   to   different  

chemical  compositions.  All  the  products  are  sprays  or  aerosols  (table  8).    

    Live  mites  of  all  motile  stages  (n  =  20)  were  placed  in  a  plastic  sterile  Petri  dish  (3  cm  in  

diameter).   In   each  Petri   dish,  mites  were   sprayed  uniformly  until   they  were   completely  

covered  by  the  tested  product.  A  control  Petri  dish  was  sprayed  by  distilled  water.  All  Petri  

dishes  were  placed  at  room  conditions  (25°C,  30-­‐70%  relative  humidity).  The  mites  were  

examined  under  a  stereomicroscope  after  5,  10,  15,  20,  25,  30,  40,  50,  60  min,  2,  3,  4,  5,  

24h.  Persistent  immobility,  even  when  stimulated  with  a  needle  was  considered  as  death  

(Pasay  et  al.,  2010).  

 

Table  8.  Active  compounds  of  the  products  tested  

 

 

 

No.   Active  compounds  and  

concentration  

Targets   Brand  names   Companies  

1   IR3535  20%   Repellent  for  lice   Paranix®  100  mL   Omega  Pharma  

Barcelona,  Spain  

2   DEET  25%   Repellent  for  mosquitoes,  

ticks  and  flies  

Insect  Ecran®  100  mL   Cooper,  Melun,  France  

3   DEET  50%   Repellent  for  mosquitoes,  

ticks  and  flies  

Insect  Ecran®  100  mL   Cooper,  Melun,  France  

4   icaridin  20%   Repellent  of  mosquitoes   Insect  Ecran®  75  mL   Cooper,  Melun,  France  

5   permethrin  4%   Insecticide   Insect  Ecran®  100  mL   Cooper,  Melun,  France  

6   esdepallethrin  2.1g/L,  

bioresmethrin  0.45g/L  

Environmental  control  of  

lice  

Pyréflor®  150  mL   Clément-­‐Thékan,  France  

7   bifenthrin  0.67g/L   Environmental  control  of  

lice  

Pyréflor®  150  mL   Ferlux,  Cournon  

d’Auvergne,  France  

8   cypermetrin  0.10%  

imiprothrin  0.10%  

Insecticide   Raid®  400  mL   S.C.  Johnson,  Mijdrecht,  

The  Netherlands  

9   permethrin  0.6%,  

pyriproxyfen  0.05%  

Environmental  control  of  

fleas  

Parastop®  500  mL   Virbac,  Carros,  France  

10   cyfluthrin  0.16g/L,    

pyriproxyfen  0.2g/L  

Environmental  control  of  

fleas  

Advanthome®  250  mL   Bayer,  Puteaux,  France  

11   tetramethrin  0.95g/L,  

sumithrin  0.95g/L  

Disinfectant  against  

Sarcoptes  mites,  lice,  fleas  

and  bedbug  

A-­‐PAR®  200  mL   Omega  Pharma,  

Châtillon,  France  

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 80  

2.4  Essential  oils  and  bioassays  

    Eleven   pure   essential   oils  were   gently   provided   by   Dr.   Arezki   Izri   (Avicenne  Hospital,  

Bobigny,   France).   They  were   Lavandula   augustifolia,  Melaleuca   altenifolia,   Pelargonium  

asperum,  Eucalyptus  radiate,  Leptospermum  scoparium,  Juniperus  oxycedrus,  Cryptomeria  

japonica,  Citrus  aurantium  amara  and  3  other  unknown  oils  (BOB4,  BOB5,  BOB9).  Each  of  

them   was   diluted   with   paraffin   to   10%,   5%   and   1%   right   before   testing   for   contact  

bioassays,  whereas  for  fumigant  bioassays,  pure  essential  oils  were  used.  

  Live  mites  of  all  motile  stages  (n=20)  were  placed  in  a  3  cm  in  diameter  plastic  Petri  dish.  

In   a   preliminary   experiment,   11   pure   essential   oils   were   tested   against   mites;   further  

bioassays   were   conducted   if   the   essential   oils   had   killed   all   the   mites   within   1h.   The  

selected  essential  oils  were  diluted  with  paraffin  to  10%,  5%  and  1%  (for  the  most  efficient  

ones).   In  each  Petri  dish,  1  mL  of  each  essential  oil  was  added  in  direct  contact  with  the  

mites.   A   control   Petri   dish  was   added  with   1mL   paraffin   oil.   The  mites  were   inspected  

under   a   stereomicroscope   after   10,   20,   30,   40,   50,   60,   90,   120,   150,   and   180  min.  

Persistent   immobility,   even   when   stimulated   with   a   needle   was   considered   as   death  

(Pasay  et  al.,  2010).    

    In   a   separate   experiment,   vapor   phase   toxicity   of   the   oils   was   investigated.   In   these  

tests,  10  mites  of  all  motile  stage  were  placed  on  the  bottom  of  a  small  Petri  dish.  A  filter  

paper  was  put  on  the  lid  of  the  petri  dish  and  treated  with  100  µl  of  each  pure  essential  oil.  

The  Petri  dishes  were  closed  and  turned  over.  Therefore,  the  mites  were  at  the  top,  which  

was  convenient  for  inspection.  Mites  were  inspected  constantly  under  a  stereomicroscope  

for   the   first   5   min,   and   then   every   5  min   until   1  h.   Persistent   immobility,   even   no  

movement  in  capitulum  and  inner  organ  was  considered  as  death.  The  tests  were  held  at  

room  temperature  (20±3°C  and  65±5%  relative  humidity).    

 

III.  In  vitro  evaluation  of  acaricides,  repellents  and  essential  oils  

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2.5  Statistical  analyses    

    To   analyze   the   data   obtained   from   the   experiments,   the   software   JMP   12.0   was  

employed.  For   the   test  of   ivermectin  and  moxidectine,   the  differences   in  mite  mortality  

between  control  group  and  different  concentrations  were  calculated  by  t-­‐test.  A  p-­‐value  

of  <0.05  was  assumed  to  indicate  a  statistically  significant  difference.  A  probit  analysis  was  

used   to   calculate   median   lethal   concentrations.   The   data   of   environment   control   and  

essential   oils   were   analyzed   by   Kaplan  Meier   survival   curves;   median   survival   times   of  

mites  and   significant  differences  between   survival   curves  were   calculated  by  a   Log-­‐rank  

test.    

 

 

3.  Results  

 

3.1  In  vitro  evaluation  of  ivermectin  and  moxidectin  efficacy    

    The   results   showed   that   S.   scabiei   mites   were   more   sensitive   to   ivermectin   than   to  

moxidectin.   The   lethal   concentrations   after   24  h   were   150.2±31.4   µg/mL   and  

608.3±88.0  µg/mL  for  ivermectin  and  moxidectin,  respectively.    

    Mortalities  for  the  mites  exposed  to  five  concentrations  of  ivermectin  or  moxidectin  are  

shown   in   table   9.  When   compared   to   the   control,   ivermectin   (1  ng/mL)   and  moxidectin  

(1  ng/mL  and  10  ng/mL)  were  found  to  have  no  significant  effect  against  S.  scabiei,  which  

demonstrated   that   there   was   no   scabicidal   effect   after   24h   exposure   to   doses   of  

ivermectin   lower   than   1  ng/mL   and   to   doses   of   moxidectin   lower   than   10  ng/mL.   This  

result   was   confirmed   by   the   condition   of   the   mites   when   observed   under  

stereomicroscope   after   24h.   The  mites   (treated  with   ivermectin   1  ng/mL   or  moxidectin  

1  ng/mL  or  10  ng/mL)  still  crawled  well  as  the  mites  in  the  control  group.  On  the  contrary,  

mites   treated   with   higher   concentrations   can   only   slightly   move   their   legs,   showing  

paralysis  by  the  drugs.    

 

 

III.  In  vitro  evaluation  of  acaricides,  repellents  and  essential  oils  

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Table  9.  Mortality  rate  of  S.  scabiei  var.  suis  exposed  to  ivermectin  or  moxidectin    

Drug   Concentration  (µg/mL)   Mortality  (mean±SD  %)   p-­‐value  (with  control)  

1000   100±0   <0.01  

500   91.7±12.8   <0.01  

100   42.2±8.7   <0.01  

10   31.3±10.2   <0.05  

Ivermectin  

1   28.5±16.9   0.1222  

1000   65.6±14.0   <0.01  

500   51.9±9.7   <0.01  

100   46.3±9.2   <0.01  

10   13.5±6.9   0.5856  

Moxidectin  

1   10.9±3.9   0.8186  

Control   0.1%  DMSO   14.6±4.9    

 

3.2  Evaluation  of  products  for  environmental  control  of  S.  scabiei    

    With   the   in   vitro   test,  we  were   able   to  demonstrate   the   scabicidal   activity   of   several  

biocides   and   repellents.  During   the   tests,  we  also  observed   that   larvae  and  males  were  

usually  killed  before   females  and  nymphs.  The  survival  curves   for   the  different  products  

are  presented  in  figure  32.  The  median  survival  time  of  mites  varied  from  10  to  1440  min  

(table   10).   Except   A-­‐PAR®   (product   No.   11),   all   the   products   killed   all  mites  within   24h  

(figure   37A   and   B).   Significant   differences   in   mite   survival   time   were   observed   for   all  

tested  products  with  the  distilled  water  used  as  control.  Although  the  main  components  

of  7  products  were  pyrethroids,  the  variability  in  survival  times  of  the  mites  was  obvious.  

All   mites   were   killed   within   2h   when   using   Insect   Ecran®   (product   No.   5),   Pyréflor®  

(product  No.  6),  Pyréflor®  (product  No.  7)  and  Raid®  (product  No.  8).  Of  these,  Pyréflor®  

(product  No.  7)  and  Raid®  (product  No.  8),  with  a  median  survival  time  of  10  ±  5.87  min  

and  15  ±  7.31  min,  showed  a  strong  scabicidal  effect.  In  contrast,  approximately  5,  10  and  

70%   of   mites   were   still   alive   after   5h   sprayed   with   Parastop®   (product   No.   9),  

Advanthome®   (product   No.   10)   and   A-­‐PAR®   (product   No.   11),   respectively.   A  

dose-­‐dependent   change   in   median   survival   time   of   permethrin-­‐based   products   was  

observed,   with   the   median   survival   time   of   50   ±   30.4   min   and   120   ±   309   min   for  

permethrin  4  (product  No.  5)  and  permethrin  0.6%  (product  No.  9),  respectively.    

III.  In  vitro  evaluation  of  acaricides,  repellents  and  essential  oils  

 83  

    The   four   repellent   products   were   active   against   S.   scabiei   mites   (figure   37C   and   D).  

DEET  and  IR3535  products  killed  all  the  mites  within  1h,  while  icaridine  took  3h  to  kill  all  

the   mites.   DEET   revealed   small-­‐scale   dose-­‐dependent   scabicidal   activity,   the   median  

survival  times  were  20±6.5  min  and  15±4.3  min  for  25  and  50%  of  DEET,  respectively.    

 

 

Table  10.  Comparisons  of  Log-­‐rank  test  pairwise  survival  time  of  S.  scabiei  var  suis  sprayed  with  

different  products  in  comparison  with  distilled  water  (negative  control).  

Product  name   Median  survival  

time  (min)  

Standard  

deviation  

p-­‐value  

Pyréflor®  esdepallethrin   10   5.9   <.001  

Insect  Ecran®  DEET  50%   15   4.3   <.001  

Paranix®   15   4.9   <.001  

Raid®   15   7.3   <.001  

Insect  Ecran®  DEET  25%   20   6.5   <.001  

Insect  Ecran®  icaridin   30   42.1   <.001  

Pyréflor®  bifenthrin   40   36.8   <.001  

Insect  Ecran®  permethrin   50   30.4   <.001  

Parastop®   120   309.0   <.001  

Advanthome®   180   417.0   <.001  

A-­‐PAR®   1440   600.0   <.002  

Distilled  water   1440   -­‐   -­‐  

 

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Figure  37.  Survival  curves  of  Sarcoptes  mites  exposed  to  biocides  or  repellents.  

A.  Survival  curves  with  fast-­‐acting  biocides  (all  the  mites  were  killed  within  40  min);  B.  Survival  curves  with  

other  biocides.  C.  Survival  curves  with  fast-­‐acting  repellents  (all  the  mites  were  killed  within  40  min);  D.  

Survival  curve  with  the  other  repellent,  icaridin.  

 

  3.3  In  vitro  evaluation  of  essential  oils    

    The   survival   curves   of  mites   exposed   to   essential   oils   in   direct   contact   and   in   vapor  

phase  are  showed  in  figure  38.  The  product  BOB4  was  the  most  effective  essential  oil  (it  

was   able   to   kill   all   the   mites   within   20min   with   only   1%   concentration).   BOB4   was  

followed   by   BOB9,   BOB5,   Pelargonium   asperum,   Melaleuca   altenifolia,   Lavandula  

augustifolia,   Leptospermum  scoparium,  Citrus  aurantium  amara,   Eucalyptus   radiate  and  

Cryptomeria   japonica.  Essential  oils  were  more  effective   in   fumigation  bioassays   than   in  

immersion   ones.   However,   the   fumigation   effect   of   these   oils   was   not   always  

corresponding  to  their   immersion  effect.   In   fumigation  assays,  Melaleuca  altenifolia  was  

III.  In  vitro  evaluation  of  acaricides,  repellents  and  essential  oils  

 85  

the  most  active  oil   (it  was  able   to  kill   the  mites  within  4min).  Melaleuca  altenifolia  was  

followed  by  BOB4,  Eucalyptus  radiate,  BOB5,  Lavandula  augustifolia,  BOB9,  Pelargonium  

asperum,  Cryptomeria   japonica,  Citrus  aurantium  amara,  Leptospermum  scoparium,  and  

Juniperus  oxycedrus  (which  demonstrated  no  activity  against  the  mites).  

 

 

Figure  38.  Survival  curves  of  Sarcoptes  scabiei  exposed  to  essential  oils.    

A.  Fumigation  test  of  11  essential  oils;  B.  Immersion  test  with  10  essential  oils  (10%  concentration).  

C.  Immersion  test  with  10  essential  oils  (5%  concentration);    

D.  Immersion  test  with  4  essential  oils  (1%  concentration).  

 

 

 

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4.  Discussion  

 

    Although  both  ivermectin  and  moxidectin  act  on  ligand-­‐gated  chloride  channels  (GluCls)  

and  share  similar  spectrum  of  activity  (Wolstenholme  and  Rogers,  2005),  we  found  that  S.  

scabiei   var   suis   was   more   sensitive   to   ivermectin   than   to   moxidectin.   This   result   is   in  

accordance  with  previous  studies.  In  a  study  about  the  nematode  Caenorhabditis  elegans,  

the  larval  development  ceased  at  0.61  nM  ivermectin  and  39.0  nM  moxidectin;  threshold  

of  motility  was  at  19.5  nM  ivermectin  and  39.0  nM  moxidectin;  pharyngeal  pumping  was  

paralyzed  at  4.9  nM  ivermectin  but  required  78  nM  moxidectin  to  reach  a  similar  paralysis  

(Ardelli   et   al.,   2009).   Ivermectin   was   also   found   more   effective   than   moxidectin   on  

microfilariae  released  from  females  of  Brugia  malayi  (Tompkins  et  al.,  2010).  

    For  the  scabicidal  efficacy  of   ivermectin,  a  range  of  concentrations  has  been  tested   in  

vitro.   The   threshold   for   scabicidal   effect   was   found   to   be   between   50   and   100  ng/mL  

(Brimer  et  al.,  1995).  In  the  present  study,  a  significant  difference  was  found  between  the  

control  group  and  ivermectin  group  at  the  concentration  of  10  ng/mL,  which  is  a  little  bit  

lower   than   50  ng/mL.   However,   the   assays   in   previous   study   were   performed   on   agar  

plates  while  our  tests  were  performed  in  distilled  water.  

    Even   though   moxidectin   showed   less   scabicidal   effect   than   ivermectin   in   vitro,  

moxidectin   has   advantage   in   pharmacokinetic   and   safety   profiles   as   a   treatment   for  

mange  or  scabies.  In  livestock  and  humans,  ivermectin  has  a  half-­‐life  of  about  0.6-­‐2.8  days  

(Fink   and   Porras,   1989),   while   the   half-­‐life   of   moxidectin   is   in   the   range   of   20   days  

(Cotreau  et  al.,  2003).  The  long  half-­‐life  of  moxidectin  ensures  one  dose  treatment  enough  

for  S.  scabiei  infection.  Moxidectin  was  found  to  be  safer  than  ivermectin.  A  comparative  

toxicity  study  of  subcutaneous  administration  in  MDR1  (−/−)  mice  demonstrated  that  the  

LD50  for   ivermectin  was  0.46  µmol/kg,  while  the  LD50  for  moxidectin  was  2.3  µmol/kg.   In  

addition,   moxidectin   has   been   administered   to   about   1700   human   as   alternative  

treatment   for  onchocercosis,  without  evidence  of  any  serious  adverse  effects   (Ménez  et  

al.,  2012).  

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    For   the   environmental   control   of   S.   scabiei,   the   present   study   was   the   first   one   to  

assess   so  many   products.   The   finding   that   larvae   and  males  were   killed   before   females  

and   nymphs   is   in   accordance   with   Arlian’s   observation   that   female   and   nymph   mites  

survived  longer  in  comparable  conditions  (Arlian,  1989).  Since  we  tested  all  motile  stages  

of  mites   in   the   study,   the   variable   number   of   females   and   nymphs   could   influence   the  

results,  especially  for  the  products  with   low  activity.  Moreover,  this  can  explain  why  the  

standard  deviation  values  of  some  tested  products  were  high.  Distilled  water  was  used  as  

control,  which   is  probably  not  always  a  good  control   to  determine  the  specific  action  as  

solvents  might  also  be   involved.  However,   in  biocides  or  repellents  products  used   in  the  

present  study,  the  solvents  were  not  mentioned  by  the  manufacturers.  

    The   active   components   of   all   the   biocide   products   tested   belong   to   the   pyrethroid  

family   that   is   widely   used   in   public   health   and   agriculture   throughout   the   world   and  

generally   considered   to   be   the   safest   class   of   insecticides/acaricides   available   so   far.  

Pyrethroids   are   known   to   act   on   voltage   dependent   sodium   channels   in   the   nerve  

membrane  (Vijverberg  and  vanden  Bercken,  1990).  The  present  study  demonstrated  that  

pyrethroids   differed   a   lot   regarding   the   speed   of   activity   against   S.  scabiei.   Pyréflor®  

(product   No.   6)   was   the   most   effective   of   all.   It   includes   the   active   component  

esdepallethrin   which   is   used   for   the   treatment   of   human   scabies   in   France  

(Andriantsoanirina  et  al.,  2014).  Permethrin  is  not  only  a  treatment  of  scabies,  but  is  also  

recommended  for  environmental  control  of  S.  scabiei.  In  previous  studies  of  in  vitro  tests  

against   S.  scabiei,   the   lethal   time   of   5%   permethrin   was   480  min   and   1320  min,  

respectively  (Shelley  F.  Walton  et  al.,  2004b;  Walton  et  al.,  2000),  whereas  it  was  120  min  

with  4%  permethrin  in  the  present  study.  This  may  be  attributed  to  the  method  used  for  

efficacy  assessment  of  permethrin.   In  the  current  study  permethrin  was  sprayed  directly  

on  the  mites  while  Walton  et  al.  applied  0.1  g  of  permethrin  in  a  thin  layer,  using  cotton  

swabs,  on  the  bottom,  top  and  sides  of  Petri  dishes  and  then  placed  a  mite  into  each  Petri  

dish.  Another  explanation  would  be   that   the  widespread  use  of  permethrin   in  Australia  

since   1994   induced   tolerance   to   this   compound   (Mounsey   et   al.,   2008).   However,   a  

previous   study  on  KDR   gene  did  not  detect   resistance   to  pyrethroids   in  S.   scabiei  mites  

from  French  patients  (Andriantsoanirina  et  al.,  2014).  Compared  to  other  biocide  products,  

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permethrin-­‐based  products  were  not   so  efficient   in   the  present   study.  Raid®   (No.8)   is   a  

common   and   cheap   product   used   for   household   control   of   insects.   Even   though   the  

concentration  of  its  active  components  cypermetrin  and  imiprothrin  are  as  low  as  0.10%,  

the   efficacy   on   mites   was   notable.   This   result   suggested   that   mites   might   be   more  

sensitive   to   some  pyrethroids.   A-­‐PAR®   (No.11)   is   used   in   some   French  hospitals   for   the  

environmental   control   of   S.   scabiei.   However,   the   present   study   showed   that   A-­‐PAR®  

should  not  be  recommended  for  this  indication.  

    Repellents  are  supposed  to  prevent  arthropods  from  landing  on  the  surface  where  they  

are   applied   (without   a   necessary   killing   effect).   However,   previous   studies   showed   that  

DEET   and   IR3535   display   insecticidal   as   well   as   acaricidal   activity   (Faulde   et   al.,   2010;  

Licciardi   et   al.,   2006).   In   the   present   study,   the   repellents   DEET,   IR3535   and   icaridin  

demonstrated  acaricidal  activity  and  differed  in  their  effects  on  mites.  Although  the  exact  

mode  of  action  and  molecular  target  of  repellents  remain  controversial,  there  is  evidence  

that   repellents   exert   their   effects   through   interactions   with   odorant   receptors   and  

gustatory  receptors  in  insects  (Dickens  and  Bohbot,  2013).  It  was  also  demonstrated  that  

DEET  induced  a  neurotoxic  effect  on  insects  by  disrupting  the  calcium  equilibrium  in  the  

nerve  cells  (Lapied  et  al.,  2006).  Faulde  et  al.  showed  that  Both  DEET  and  IR3535  revealed  

a  dose-­‐dependent   insecticidal   as  well   as   acaricidal   activity,   and  DEET  exhibited  a  higher  

knockdown  effect  and  mortality  than  IR3535  (Faulde  et  al.,  2010).  From  the  present  study,  

DEET  also  showed  a  small-­‐scale  dose  dependent  scabicidal  activity,  but  IR3535  may  work  

better  than  DEET  against  S.  scabiei.  Although  the  repellent  products,  especially  DEET  and  

IR3535,   caused   relatively   high   scabicidal   effect,   one   cannot   infer   that   repellents   work  

better  than  pyrethroids.  

    Since   commercial   products   are   complex   in   their   composition   and   varied   in  

concentration,   further   investigations   are   required   to   determine   the   scabicidal   effect   of  

individual  chemicals.  

  In  vitro  bioassays  of  immersion  and  fumigation  were  conducted  to  screen  the  scabicidal  

activity   of   essential   oils.   The   differences   observed   in   the  mite   killing   properties   can   be  

attributed  to  variation   in  essential  oils  chemical  compositions.  Essential  oils  usually  have  

versatile   effects   due   to   the   complex   constituents.   The   use   of   them   as   alternative   or  

III.  In  vitro  evaluation  of  acaricides,  repellents  and  essential  oils  

 89  

complementary   acaricide   could   also   lower   the   risk   of   secondary   infection.   A   notable  

finding  of  this  study  is  that  the  essential  oils  possess  strong  fumigant  toxicity  against  mites.  

This  property  of  essential  oils  plus  their  toxicity  in  direct  contact  on  mites  makes  them  not  

only  an  alternative  treatment  for  scabies/mange,  but  also  good  products  for  the  control  of  

S.  scabiei  in  the  environment.  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

III.  In  vitro  evaluation  of  acaricides,  repellents  and  essential  oils  

 90  

 

 

 

IV.  Characterization  of  the  genetic  diversity  of  Sarcoptes  scabiei    

 91  

 

 

 

 

 

 

 

IV.  Characterization  of  the  genetic  diversity  of  Sarcoptes  scabiei    

 

 

 

 

 

 

 

IV.  Characterization  of  the  genetic  diversity  of  Sarcoptes  scabiei    

 92  

1.  Introduction    

For  many   years,   host-­‐associated   populations   of   S.   scabiei   have   been   taxonomically  

divided   into  morphologically   indistinguishable   varieties   (Currier  et   al.,   2011;   Fain,   1978;  

Mounsey   et   al.,   2013).   The   monospecificity   of   these   host-­‐specific   varieties   is   still  

controversial,   and   current   studies   are   investigating   whether   they   belong   or   not   to  

different   species.   Cross-­‐infectivity   was   observed   experimentally   on   some   occasions  

(Alasaad   et   al.,   2013;   L.   G.   Arlian   et   al.,   1984b;   Bornestein,   1991).   Natural   apparent  

cross-­‐infectivity   has   been   recently   reported   in   sympatric   wild   animal   host   populations  

(Holz  et  al.,  2011;  Makouloutou  et  al.,  2015;  Matsuyama  et  al.,  2015;  Rentería-­‐Solís  et  al.,  

2014).   Transmission   of   scabies  mites   between   other   species   and   humans   are   common,  

leading  to  usually  clinically  moderate  and  self-­‐limiting  forms,  though  they  may  persist  for  

several  weeks  or  in  rare  cases,  until  treated  (Andriantsoanirina  et  al.,  2015c;  Barker,  1974;  

Bazargani  et  al.,  2007;  Fain,  1978;  Menzano  et  al.,  2004;  Skerratt  et  al.,  2002;  Skerratt  and  

Beveridge,   1999).   In   particular,   the   domestic   dogs   (Canis   lupus   familiaris   or   Canis  

familiaris)   are   reportedly   the   most   frequent   non   human   reservoir   of   mites   infecting  

humans,  which  may  have  great  implications  in  term  of  transmission  and  control  of  scabies  

(Aydıngöz  and  Mansur,  2011;  Emde,  1961;  Smith  and  Claypoole,  1967;  Thomsett,  1968).    

    A   widely   accepted   hypothesis,   though   never   substantiated   by   factual   data,   suggests  

that   humans   and   protohumans   were   the   initial   source   of   the   animal   sarcoptic   mange;  

dogs   and   other   domestic   animals   being   infested   by   human   contacts   and   themselves   a  

source  for  other  species  of  wildlife  (Alasaad  et  al.,  2013;  Amer  et  al.,  2014;  Currier  et  al.,  

2011;  Fain,  1978).   In  this  study  we  performed  phylogenetic  analyses  of  populations  of  S.  

scabiei  in  humans  and  in  canids  to  validate  or  not  the  hypothesis  of  a  human  origin  of  the  

mites  infecting    

 

 

IV.  Characterization  of  the  genetic  diversity  of  Sarcoptes  scabiei    

 93  

2.  Materials  and  methods  

2.1  Collection  of  S.  scabiei  mites  

    Sarcoptes   mites   were   obtained   from   13   individual   10   dogs   and   3   foxes   during   the  

period  from  April  2014  to  July  2015.  The  animals  were  either  wild  animals,  farm  animals  

or  as  pet.  Overall,  mites  were  collected  from  3  wild  fox  in  Île-­‐de-­‐France,  and  9  dogs  from  

five   locations:  France  (2),   Italy   (1),  Thailand  (1),  South  Africa   (1)  and  Southern  China  (5).  

The   mites   from   humans   were   collected   in   23   scabies   patients   consulting   at   Avicenne  

Hospital,   Bobigny,   France,   between   January   2013   and   March   2014   (table   11).   All   the  

specimens   were   conserved   at   -­‐20°C.   All   cases   were   independent;   only   one   mite   per  

different  dog  was  included  in  the  study.  

 

Table  11.  Sarcoptes  scabiei  sequences  used  in  this  study  

 

IV.  Characterization  of  the  genetic  diversity  of  Sarcoptes  scabiei    

 94  

 

IV.  Characterization  of  the  genetic  diversity  of  Sarcoptes  scabiei    

 95  

2.2  DNA  extraction  and  gene  amplification  

    Mite   genomic   DNA   was   individually   extracted   with   NucleoSpin   Tissue   kit,  

Macherey-­‐Nagel,   Germany   (Soglia   et   al.   2009;   Andriantsoanirina   et   al.   2014).   A   part   of  

cytochrome  c  oxidase  subunit  1   (cox1)  gene  was  amplified.  PCR  was  carried  out   in  50  µl  

and  reaction  mixture  contained  1X  PCR  buffer,  2.5  mM  MgCl2,  1mM  of  dNTPs,  1.25U  DNA  

polymerase   AmpliTaq   Gold   (Applied   Biosystems,   Courtaboeuf,   France)     and   0.25µM   of  

primer   (NavF:   5’-­‐TGATTTTTTGGTCACCCAGAAG-­‐3’;   NavR:   5’-­‐TACAGCTCCTATAGATAA  

AAC-­‐3’)   (Fournier   et   al.   1994).   Amplifications  conditions   were   as   follows:   an   initial  

denaturation  step  at  94°C  for  5min,  followed  by  35  cycles  of  denaturing  at  94°C  for  30s,  

annealing  at  51°C  for  30s,  and  extending  at  72°C  for  40  s  and  a  5min  of  final  extension  at  

72°C.    

2.3  Sequence  and  phylogenetic  analyses  

    The   PCR-­‐amplified   products   of   400   bp   were   purified   and   directly   sequenced.   The  

Otodectes   cynotis   cox1   sequence   (KF891933)   was   retrieved   from   GenBank.   Multiple  

sequence  alignments  of  nucleotide  sequences  in  this  study  and  sequences  available  from  

GenBank   (n=81)   were   generated   using   MAFFT   v.6.951.   The   dataset   was   analyzed   with  

Maximum  Likelihood  using  MEGA5  and  RAxML-­‐HPC  v7.0.4  under  General  Time-­‐Reversible  

(GTR+G)   model   and   Bayesian   Inference   analysis.   Support   of   internal   branches   was  

evaluated   by   non-­‐parametric   bootstrapping   with   500   replicates.   Bayesian   Inference  

analysis  was  performed  with  MrBayes  v.3.2.1  conducting   in   two  simultaneous   runs  with  

four   parallel   Markov   chains   (one   cold   and   three   heated)   for   1   million   generations,  

sampling   every   1000   generations   and   discarding   the   first   25%   of   samples   as   burn-­‐in.  

Potential   Scale  Reduction  Factor  approached  1.0  and  average  of   split   frequencies  under  

0.01  were  used   for  examining   convergence.  All   trees  were  visualized  using  FigTree  with  

Otodectes   cynotis   as   outgroup   (figure   39).   To   visualize   the   relationship   between  

haplotypes,  a  median  joining  haplotype  network  of  cox1  sequence  was  constructed  using  

Network  v4.6  according  to  host.    

IV.  Characterization  of  the  genetic  diversity  of  Sarcoptes  scabiei    

 96  

3.  Results  

The   sequences   of   cox1   fragment   were   obtained   in  mites   from   9   dogs   and   3   foxes  

(table   11).   All   sequences   were   deposited   [GenBank:   KT961021-­‐KT961032].   Other  

sequences   corresponding   to   50   mites   from   humans,   raccoon   dogs   (Nyctereutes  

procyonoides)  (n=6),  fox  (n=1),   jackal  (Canis  aureus)  (n=1)  and  domestic  dogs  (n=11)  and  

from  various  geographical  areas  were  retrieved  from  GenBank  and  from  a  previous  study  

(table  11).  

    All  of  the  successfully  sequenced  samples  were  assigned  to  only  one  haplotype.  In  all,  

21   haplotypes   were   observed   among   mites   collected   from   5   different   host   species,  

including  humans  and  canids,  and  9  geographical  areas  (table  11).  Seven  haplotypes  were  

observed   among  mites   collected   in   humans   (H12-­‐H18);   2   haplotypes  were   shared  with  

mites  collected  from  canids  and  human  (H3  and  H11)  and  12  haplotypes  (H1-­‐H2,  H4-­‐H10,  

H20-­‐H21)  were  observed  among  mites  collected  from  canids.  

Sequences  from  dogs  (n  =  20),  raccoon  dogs  (n  =  6),  foxes  (n=4),  Jackal  (n=1)  and  humans  

(n=50)  were  used  to  construct  the  phylogenetic  trees  based  on  Maximum  Likelihood  and  

Bayesian  Inference  analyses.  They  showed  similar  topologies  with  few  differences  in  node  

support  values  (figure  39).    

    The  haplotype  network  showed   two  distinct  populations  of  mites,  a   relatively  diverse  

population   from   dogs   and   other   canids,   and   a   more   homogeneous   population   from  

humans  (figure  40).  In  addition,  values  of  haplotype  diversity  (Hd)  and  nucleotide  diversity  

(π)  indicated  a  larger  genetic  diversity  for  S.  scabiei  mites  collected  in  dogs  than  for  those  

collected  in  humans  (table  12).  

 

1/84 0,95/87

1/

0,76/89 1/89

0,98/85

0,66/77

0,91/91

0,77/87 0,98/88

0,86/90 0,99/90

IV.  Characterization  of  the  genetic  diversity  of  Sarcoptes  scabiei    

 98  

   Figure  40.  Haplotype  map  of  Sarcoptes  scabiei  from  canids  and  humans  inferred  under  median  

joining.  Size  of  circles  is  proportional  to  haplotype  frequency.  Median  vectors  correspond  to  

possibly  extant  unsampled  sequences  or  extinct  ancestral  sequences.  

 

 Table   12.   Estimates   of   genetic   diversity   of   Sarcoptes   scabiei   mites   from   humans   and  canids  

 

 No.  

sequences  No.  haplotypes  

Haplotype  diversity    

(Hd)  (±  sd)  

Nucleotide  diversity        

(π)  ±  (sd)  

Humans     50   9   0.606  (0.056)   0.0022  (0.00041)  

Dogs   20   13   0.942  (0.034)   0.011  (0.0012)  

Canids  (including  dogs)   31   14   0.871  (0.046)   0.0087  (0.0011)  

raccoon dog jackal

human dog

fox median vectors

IV.  Characterization  of  the  genetic  diversity  of  Sarcoptes  scabiei    

 99  

4.  Discussion  

    The  historical  hypothesis  about  the  origin  of  S.  scabiei  in  dogs  is  a  transfer  of  parasites  

from  humans   to   their  domestic  dogs.  Under   this   scenario,   the  population  of  mites   from  

humans   should  be  basal   in   the  phylogenetic   tree.   This   is   not  what  was  observed   in   the  

present   phylogenetic   analyses.  Our   data  were   not   consistent  with   a   human   origin   of   S.  

scabiei   in  dogs.  On  the  contrary  our  results  did  not  exclude  the  opposite  hypothesis  of  a  

host   switch   from   dogs   to   humans.   The   haplotype   network   showed   also   that,   in   two  

occasions,  haplotypes   from  dogs,  H19  and  H5,  H1,  H2,  seemed  to  derive   from  S.   scabiei  

mites  in  humans.  Being  possibly  from  canine  origin,  mites  infecting  humans  may  in  some  

occasions  return  to  canine  hosts.  

    The  fact  that  nonhuman  primates  are  not  affected  by  scabies  (or  the  few  times  it  was  

described   it   was   considered   that   this   was   via   a   human   contamination)   (Bernstein   and  

Didier   2009)   while   the   brother   genera   of   Sarcoptes   (Otodectes   and   Psoroptes)   infect  

carnivores   or   sheep   (phylogenetically   closer   to   dogs   than   human)   reinforces   the  

hypothesis  of  a  canine  origin  of  scabies  and  a  host  transfer  to  humans  (Amer  et  al.  2015).  

    According   to   the   historical   hypothesis,   behavioral   transmission   between   humans   and  

dogs  occurred  when  humans  domesticated  various  species  of  animals  at  the  beginning  of  

agriculture  and  sedentarization  (Currier  et  al.  2011).  The  origin  of  the  domestic  dog  is  still  

debated.  Recent  data   indicate   that   domestic   dogs   evolved   from  a   group  of  wolves   that  

came   into   contact   with   hunter-­‐gatherers   between   18,800   and   32,100   years   ago  

(Thalmann  et  al.  2013).  Those  data  contradict  the  historical  hypothesis  as  agriculture  was  

developed  later,  around  11,500  years  ago.    

    We   included  all   the  cox1  nucleotide  sequences  of  S.  scabiei  available   in  GenBank  that  

were  from  canids  and  from  all  human  mites  sharing  the  same  clade  than  canids  mites  in  

published   phylogenetic   studies   (table   11).   Cox1   gene,   including   a   very   high   number   of  

polymorphisms,   was   found   to   be   valid   and   best   suited   for   this   type   of   phylogenetic  

IV.  Characterization  of  the  genetic  diversity  of  Sarcoptes  scabiei    

 100  

analysis   according   to   previous   studies   on   the   same   topic   (Thalmann   et   al.   2013;  

Andriantsoanirina  et  al.  2015c).  

    Mites  of  human  origin  were  collected   in  only   two  countries,  mostly   in  France.   It  does  

not  necessarily  mean  that  patients  acquired  their  mites  in  France.  Indeed,  various  ethnic  

communities   are   represented   among   the   outpatients   that   visit   the   hospital   (about   one  

third   are   immigrants)   and   it   is   likely   that   a   not-­‐insignificant   number   of   cases   of   scabies  

were  acquired  abroad.    

    Host   switching   promotes   S.   scabiei   diversification   and   reflects   the   exceptional  

dissemination   potential   of   these   mites   among   various   species   of   mammals.   Scabies  

spreading  in  wild  populations  may  occur  on  an  epidemic  mode  and  may  be  devastating  for  

naive   populations   because   of   the   lack   of   immunity   (Skerratt   et   al.   1998).   It   may   be  

underlined   that   transmission   between   dogs   and   humans   still   occurs.   In   a   recent   study,  

Zhao  et  al.,  using  cox1  for  phylogenetic  analysis,  reported  that  mites  from  dogs  in  China,  

Australia  and  USA  clustered  with  mites  collected  from  Australian  people  (Zhao  et  al.  2015).  

Those   authors   concluded   that   humans   could   be   infected   with   mites   from   dogs.   The  

present  data  and  our  previous  results  on  this  point  are  in  agreement  with  those  authors  

(Andriantsoanirina  et  al.  2014).  Thoses  authors  also  conclude  that  geographical   isolation  

was  observed  between  human  mites.  The  aim  of  our  study  was  not  to  explore  a  possible  

geographic   effect   on   Sarcoptes   evolution   but   to   present   documented   data   on   the  

possibility  that  humans  are  the  initial  source  of  canine  mange.  We  agree  that  geographic  

clustering  occurs   in  human  Sarcoptes   evolution   (Andriantsoanirina  et  al.   2015c)  but   this  

seems   not   to   be   the   case   for   canids   Sarcoptes.   Indeed,   our   phylogenetic   tree   argues  

against  any  geographical  effect  on  canids  Sarcoptes  evolution  because  most  of  the  clades  

are  made  of   taxa   from  different   localizations   (for   example   a   clade   show   that   foxes   and  

dogs  from  France  clustered  with  dogs  from  china  in  figure39).  Nevertheless,  other  studies  

including   more   S.   scabiei   mites   from   canids   originating   from   different   locations   are  

needed  to  answer  to  this  question.    

  Two  mites  collected   in  humans,  S16  and  S42,  belonging   to  haplotypes  shared  by  mites  

IV.  Characterization  of  the  genetic  diversity  of  Sarcoptes  scabiei    

 101  

from   humans   and   canids,   clustered  with  mites   collected   in   canids   in   the   present   study  

(figure  39  and  table  11).   In  addition,  some  other  haplotypes  may  be  shared  by  different  

hosts,   as   shown   in   this   study   and   in   other   works   (Andriantsoanirina   et   al.   2014;  

Andriantsoanirina  et  al.  2015b).  Thus,  the  historical  hypothesis  of  the  "high  degree  of  host  

specificity   and   low   degree   of   cross-­‐infectivity   of   S.   scabiei"   (Bornestein   1991)   is  

challenged.  

V.  Conclusion  and  perspectives  

 102  

V.  Conclusions  and  perspectives  

 

     

The   pig   model   was   very   useful   throughout   the   thesis.   It   allowed   us   to   test   a   new  

molecule  and  to  collect  mites  for  in  vitro  tests.  In  order  to  further  study  the  mite  S.  scabiei,  

Arlian   et   al.   developed   the   first   animal  model   in   1984   by   infecting   naive   New   Zealand  

rabbits  with  mites   from  dogs   (Arlian  et  al.  1984).  Thirty  years   later,  an  experimental  pig  

model  was  successfully  developed   in  Australia  by  Mounsey  et  al.   (2010).  More  recently,  

Casais  et   al.   (2014)   used   rabbit  mites   (Sarcoptes   scabiei   var.   cuniculi)   to   experimentally  

infect   New   Zealand   white   rabbits.   Comparing   to   other   animals   models,   pig   is   usually  

considered  as  the  best  animal  model  to  study  human  dermatological  diseases  (Meyer  et  al.  

1979).   Pigs   are   natural   hosts   for   S.   scabiei   var.   suis,   developing   similar   clinical   and  

immunological   responses  with   human   scabies   (Van  Neste   and   Staquet   1986).   A   further  

potential  advantage  of  this  host-­‐parasite  system  is  the  fact  that  the  complement  system  in  

pigs  closely   resembles  humans   (Salvesen  and  Mollnes  2009).  The  pig  model  consistently  

provides   large   numbers   of   mites,   facilitating   researches   about   S.   scabiei.   A   major  

limitation   of   this   model   is   the   high   expense   of   keeping   pigs.   As   a   consequence,   the  

number  of  pigs  involved  in  the  studies  was  relatively  small.  

 

The   first   part   of   the   experimental   work   demonstrated   that   orally   administered  

afoxolaner  at  a  single  dose  (2.5  mg/kg)  was  more  effective  than  two  doses  of  ivermectin  

(0.2   mg/kg)   for   the   treatment   of   sarcoptic   infection   in   the   experimental   pig   model.  

However,  both  afoxolaner  and  ivermectin  were  found  to  have  no  ovicidal  activity.  A  single  

dose  of  afoxolaner,   therefore,   seems   to  be  a  promising  alternative   for   the   treatment  of  

mange   and   maybe   scabies.   Further   studies   are   required   to   better   understand   the  

pharmacokinetics   of   afoxolaner   in   pigs.   Tissue   and   blood   samples   have   already   been  

collected   and   the   analyses   are   about   to   be   completed   in   a   laboratory   in   USA.   The  

pharmacokinetics   of   afoxolaner   in   the   blood   of   dogs   was   evaluated   following   oral  

administration   (Letendre  et  al.   2014).  Peak  plasma  concentrations  of  1655  ±  332  ng/mL  

V.  Conclusion  and  perspectives  

 103  

were  observed  2–6h   after   treatment  dosing   at   2.5  mg/kg.   The   terminal   plasma  half-­‐life  

was  found  as   long  as  15.5  ±  7.8  days   in  dog.  Afoxolaner  has  a   low  systemic  clearance  of  

4.95   ±   1.20   mL/h/kg,   with   the   biliary   clearance   is   estimated   to   be   30%   of   the   total  

clearance,   and   renal   clearance   was   calculated   to   be   less   than   0.01%.   Therefore,   the  

pharmacokinetic  properties  can  be  expected  to  be  rapid  absorption,  long  half-­‐life  and  low  

systemic  clearance   in  pigs.  Letendre  et  al.   (2014)  also  demonstrated  that  the  EC90  values  

of   afoxolaner   for   fleas   and   ticks   of   23ng/ml   and   ≥100   ng/ml   respectively.   A   study   on  

Sarcoptes   mites   has   to   be   done   to   obtain   this   data   so   as   to   optimize   the   dosage   of  

afoxolaner.    

    Moxidectin,   a   member   of   macrocyclic   lactones,   has   been   shown   a   good   alternative  

treatment  for  scabies  in  a  previous  study  made  in  our  laboratory  (submitted).  A  single  oral  

administration  of  0.3  mg/kg  moxidectin  led  to  a  peak  concentration  of  70  ±  42.3  ng/mL  in  

plasma  occurring   at   about   half   a   day   after   treatment,  while   the   peak   concentrations   in  

skin   were   602.7±   68.2   ng/mL   which   is   about   9   times   higher   than   those   measured   in  

plasma.  The  half-­‐life  of  moxidectin  was  7.2  ±  1.1  in  plasma  ng/mL  and  8.6  ±  2.8  ng/mL  in  

skin  respectively.  Afoxolaner  has  been  shown  lipophilic  and  remanent  in  tissues  (Letendre  

et   al.   2014),   thus,   a   higher   concentration   of   afoxolaner   in   skin   than   in   plasma   can   be  

expected.   Moreover,   a   longer   half-­‐life   of   afoxolaner   than   ivermectin   is   anticipated.  

Nevertheless,  for  the  treatment  of  mange/scabies,  a  very  long  half-­‐life  of  drug  may  not  be  

a  necessity.   To  date,   there   is   no  drug  with   an  ovicidal   effect   (Mounsey  et  al.   2008)  but  

drugs  which  last  in  plasma  and  skin  for  more  than  3  days  until  the  hatching  of  eggs  (Arlian  

and  Vyszenski-­‐Moher  1988)  might  achieve  a  curative  effect.    

A  preventive  treatment  will  be  useful  to  control  the  disease,  especially  in  regions  with  

high  prevalence  of   infection  (e.g.   in  the  Indigenous  population  of  Northern  Australia,  Fiji  

island)   (Romani   et   al.),   or   in   institutions   (e.g.   school,   aged   care   centers   or   hospitals)  

(Hewitt  et  al.   2015).   In  next   future,   the  preventive  effect  of   afoxolaner   could  be   tested  

with  2  groups  (A  &  B)  of  five  pigs.  At  day  0,  two  pigs  per  group  could  be  experimentally  

infected  by  introducing  mite-­‐infected  crusts  deep  into  the  ear  canals.  In  group  A,  the  three  

V.  Conclusion  and  perspectives  

 104  

non-­‐infected  pigs  could  receive  oral  afoxolaner,  at  a  dose  of  2.5  mg/kg  on  day  2.  Group  B  

could   be   a   control   group   with   no   treatment.   The   outcome   for   the   evaluation   of   the  

protective  efficacy  of  afoxolaner  will  be  the  monitoring  of  skin  lesions  and  pruritus  based  

on   clinical   scores,   and   regular   detection   of   anti-­‐Sarcoptes   antibodies   (Sarcoptes-­‐ELISA  

2001®  Afosa  GmbH)  during  2  months.  

Other  isoxazolines  (fluralaner,  sarolaner)  are  also  worth  testing  with  the  pig  model  for  

both  treatment  and  preventive  purposes.   In  dogs,   fluralaner  has  been  shown  to  provide  

persistent   efficacy   against   fleas   and   ticks   for   3  months  with   the   half-­‐life   of   12–15   days  

(Kilp  et  al.  2014;  Rohdich  et  al.  2014).  As   for  sarolaner,   the  peak  concentration   in  blood  

was   reached   3   h   after   oral   administration   in   dogs.   Its   half-­‐life   in   blood   after   oral  

administration  was  12  days.    

        In  vitro   tests  are  required  for  the  calculation  of  minimum  efficacy  concentrations.  It  

is  worthwhile  to  note  that  the  methods  used  for  in  vitro  tests  must  be  modified  according  

to  the  different  purposes  of  the  studies.  In  vitro  test  can  be  used  for  the  assessment  of  the  

efficacy  of  new  drugs  or  the  surveillance  of  resistance  to  already  commercialized  drugs.  

 

The   second   part   of   the   thesis  was   about   in   vitro   tests   to   determine   the   scabicidal  

effect  of  ivermectin  and  moxidectin,  various  acaricides  and  repellent  products,  as  well  as  

essential   oils.   The   mites   were   found   more   sensitive   to   ivermectin   than   to   moxidectin.  

Additional  mite  strains   (from  pigs  and  also   from  humans)  must  be  evaluated   to  confirm  

these   results.   As   for   the   environmental   control   of   S.   scabies,   5   products   (containing  

esdepallethrine,   cypermetrin   and   imiprothrin,   DEET   or   IR3535)   demonstrated   relatively  

good  efficacy,  whereas  one  product  (containing  sumithrine  of  A-­‐PAR®)  was  shown  not  be  a  

good  choice  for  the  environment  control  of  S.  scabiei.   It  would  be  interesting  to  test  the  

susceptibility  of  wild  isolates  from  humans  and  animals  with  these  products.  In  regard  to  

essential  oils,  among  Lavandula  augustifolia,  Melaleuca  altenifolia,  Pelargonium  asperum,  

Eucalyptus  radiate,  Leptospermum  scoparium,  Cryptomeria  japonica,  Citrus  aurantium  ssp  

amara  and  3  other  unknown  oils   (BOB4,  BOB5,  BOB9)   tested  with   the  contact  bioassay,  

V.  Conclusion  and  perspectives  

 105  

the  essential  oil   identified  as  BOB4  demonstrated  the  best  scabicidal  effect   (1%  solution  

killed   all   the   mites   in   20   min).   Among   10   essential   oils   listed   before   plus   Juniperus  

oxycedrus  tested  with  the  fumigation  bioassay,  the  oil  Melaleuca  altenifolia  demonstrated  

the  best  scabicidal  effect  (all  the  mites  died  in  only  4  min).  Melaleuca  altenifolia  took  90  

min  in  contact  bioassay  to  kill  all  the  mites,  which  showed  that  some  essential  oils  might  

possess   a   strong   fumigant   effect   and   a   much   lower   contact   efficacy   against   mites.  

Unfortunately,   some  essential   oils  were  not   identified  and  we  need   to  obtain   complete  

information   for   further   experiments.   As   natural   products   are   attaching   increasing  

importance   by   the   public   as   an   alternative   or   complementary   approach   to   synthetic  

acaricides,  essential  oils  could  be  very  promising  treatments.  In  further  investigations,  the  

efficacy   of   individual   components   or   potential   synergistic   effects   of   a   combination   of  

these   components   should   be   explored.   The   tests   should   be   done   against  motile   stages  

(larvae,  nymphs  and  adults)  as  well  as  against  eggs.  Previous  studies   in   lice  showed  that  

essential   oils   and   their   constituents   such   as   nerolidol,   linalool,   methyl   salicylate   and  

eugenol  exibited  potent  ovicidal  activity  (Yang  et  al.  2003;  Priestley  et  al.  2006;  Yang  et  al.  

2009).   In  addition,  a  number  of  essential  oils  and  several  of  their   individual  components  

exhibit  versatile  effects  including  antibacterial  activity.  As  scabies  is  known  to  predispose  

to   secondary   bacterial   infections,   in   particular   by   Streptococcus   pyogenes   and  

Staphylococcus  aureus   (Swe  and  Fischer  2014),   the  use  of  essential  oils  as  alternative  or  

complementary  acaricide  can  not  only  kill  the  mites,  but  also  lower  the  risk  of  secondary  

infection.  

     

The   third   part   of   the  experimental  work  provided  new   findings  and   challenged   the  

widely  accepted  hypothesis  that  Sarcoptes  mite  originated  from  human  (Fain  1978).    

Our  phylogenetic  analysis  of  humans  and  canids  showed  that  dogs  might  be  the  origin  of  S.  

scabiei   in   humans   and   in   some   occasions,   sarcoptes   infecting   humans   may   return   to  

canine   hosts.   Given   the   gene   flow   observed   between  mites   from   humans   and   animals,  

scabies  may  be  considered  as  a  zoonosis.    

    At  present,  the  controversy  remains  whether  genus  Sarcoptes  contains  several  species  

or  only  one,  as  Sarcoptes  mites  from  different  hosts  or  different  geographic  areas  tend  to  

V.  Conclusion  and  perspectives  

 106  

exhibit  variable  morphologic  characteristics  and  host  specificity.  The  same  question  also  

exists   in   closely   related   mites   of   family   Psoroptidae.   In   Psoroptes  mites,   at   least   five  

species   had   been   recognized,   based   on   the   host   infected,   the   infection   site   and  

differences   in   length  of   the  opisthosomal  setae  of  males.  However,  despite  host-­‐related  

differences   in   setae   length,   molecular   characterization   using   sequence   from   ITS-­‐2   and  

microsatellite   markers   found   little   or   no   consistent   host-­‐related   variation   between   the  

mite  population  samples,  suggesting  there  is  no  case  for  considering  Psoroptes  mites  from  

the  different  hosts  as  separate  species  (Pegler  et  al.  2005).  In  the  case  of  Chorioptes  mites,  

two   species   (C.   bovis   and  C.   texanus)  were   identified   and  have  been   confirmed  by   ITS2  

sequencing   (Essig   et   al.   1999).   However,   the   two   species   appear   to   show   lack   of   host  

specificity  or  geographic  separation;  they  are  geographically  widespread  and  found  on  a  

wide   and   overlapping   host   range   (Essig   et   al.   1999;   Lusat   et   al.   2011).   These   studies  

suggested  that  there  is  a  large  degree  of  phenotypic  plasticity  and  that  the  morphological  

variation   may   represent   phenotypic   adaptation   to   the   local   microenvironment   on  

particular  species  of  host  (Pegler  et  al.  2005).  In  order  to  elucidate  the  taxonomic  status  of  

Sarcoptes  mites  as  well  as  to  confirm  the  findings  of  the  present  PhD  work,  more  genetic  

markers   have   to   be   investigated.   Further   research   should   include   the   analysis   of  

microsatellites,   which   have   been   demonstrated   to   be   a   good   marker   for   S.   scabiei   in  

previous  studies  (Soglia  et  al.  2007;  Rasero  et  al.  2010;  Gakuya  et  al.  2011;  Gakuya  et  al.  

2011).  Moreover,  experiments  on  cross  transmissions  of  Sarcoptes  mites  between  animals  

and   human,   animals   and   animals   need   to   be   explored.   Additional   samples   from   more  

animals/humans  hosts  and  more  distributions  should  be  collected.  We  should  also  collect  

mites  from  dogs  and  humans  in  case  of  zoonotic  transmission.  

Bibliographic  references   107  

References  

 

Abdel-­‐Ghaffar   F,   Al-­‐Quraishy   S,   Sobhy   H,   Semmler   M   (2008)   Neem   seed   extract   shampoo,   Wash   Away  

Louse®,   an   effective   plant   agent   against   Sarcoptes   scabiei   mites   infesting   dogs   in   Egypt.   Parasitol   Res  

104:145–148.  

Abdel-­‐Hafez   K,   Abdel-­‐Aty   MA,   Hofny   ERM   (2003)   Prevalence   of   skin   diseases   in   rural   areas   of   Assiut  

Governorate,  Upper  Egypt.  Int  J  Dermatol  42:887–892.  doi:  10.1046/j.1365-­‐4362.2003.01936.x  

Abu-­‐Samra  MT,  Ibrahim  KE,  Aziz  MA  (1984)  Experimental  infection  of  goats  with  Sarcoptes  scabiei  var.  ovis.  

Ann  Trop  Med  Parasitol  78:55–61.  

Akers  RM,  Denbow  DM  (2013)  Anatomy  and  physiology  of  domestic  animals.  Am  Vet  Med  Assoc  

Alasaad  S,  Oleaga  Á,  Casais  R,  et  al   (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.  

Parasit  Vectors  4:151.  

Alasaad   S,   Rossi   L,   Heukelbach   J,   et   al   (2013)   The   neglected   navigating   web   of   the   incomprehensibly  

emerging  and  re-­‐emerging  Sarcoptes  mite.  Infect  Genet  Evol  17:253–259.  

Alasaad  S,  Sarasa  M,  Heukelbach  J,  et  al   (2014)  Advances   in  studies  of  disease-­‐navigating  webs:  Sarcoptes  

scabiei  as  a  case  study.  Parasit  Vectors  7:16.  

Alasaad   S,   Soglia  D,   Spalenza  V,   et   al   (2009)   Is   ITS-­‐2   rDNA   suitable  marker   for   genetic   characterization  of  

Sarcoptes  mites  from  different  wild  animals   in  different  geographic  areas?  Vet  Parasitol  159:181–185.  doi:  

10.1016/j.vetpar.2008.10.001  

Al-­‐Rawashdeh  OF,  Al-­‐Ani  FK,  Sharrif   LA,  et  al   (2000)  A  survey  of  camel   (Camelus  dromedarius)  diseases   in  

Jordan.  J  Zoo  Wildl  Med  31:335–338.  

Amer  S,  Abd  El  Wahab  T,  El  Naby  Metwaly  A,  et  al   (2015)  Morphologic  and  Genotypic  Characterization  of  

Psoroptes  Mites  from  Water  Buffaloes  in  Egypt.  PLoS  ONE  10:e0141554.  doi:  10.1371/journal.pone.0141554  

Amer  S,  Wahab  TAE,  Metwaly  AEN,  et  al  (2014)  Preliminary  molecular  characterizations  of  Sarcoptes  scaibiei  

(Acari:  Sarcoptidae)  from  farm  animals  in  Egypt.  PLoS  ONE  9:e94705.  doi:  10.1371/journal.pone.0094705  

Andriantsoanirina  V,  Ariey  F,  Izri  A,  et  al  (2015a)  Wombats  acquired  scabies  from  humans  and/or  dogs  from  

outside  Australia.  Parasitol  Res.  doi:  10.1007/s00436-­‐015-­‐4422-­‐2  

Andriantsoanirina  V,  Ariey  F,  Izri  A,  et  al  (2015b)  Wombats  acquired  scabies  from  humans  and/or  dogs  from  

outside  Australia.  Parasitol  Res  114:2079–2083.  doi:  10.1007/s00436-­‐015-­‐4422-­‐2  

Andriantsoanirina  V,  Ariey  F,  Izri  A,  et  al  (2015c)  Sarcoptes  scabiei  mites  in  humans  are  distributed  into  three  

genetically  distinct  clades.  Clin  Microbiol  Infect  21:1107–1114.  doi:  10.1016/j.cmi.2015.08.002  

Andriantsoanirina  V,  Izri  A,  Botterel  F,  et  al  (2014)  Molecular  survey  of  knockdown  resistance  to  pyrethroids  

in  human  scabies  mites.  Clin  Microbiol  Infect  20:O139–O141.  doi:  10.1111/1469-­‐0691.12334  

Ardelli  BF,  Stitt  LE,  Tompkins  JB,  Prichard  RK  (2009)  A  comparison  of  the  effects  of  ivermectin  and  moxidectin  

on  the  nematode  Caenorhabditis  elegans.  Vet  Parasitol  165:96–108.  doi:  10.1016/j.vetpar.2009.06.043  

Arends  JJ,  Stanislaw  CM,  Gerdon  D  (1990)  Effects  of  sarcoptic  mange  on  lactating  swine  and  growing  pigs.  J  

Bibliographic  references   108  

Anim  Sci  68:1495–1499.  

Arlian   LG   (1989)   Biology,   host   relations,   and   epidemiology   of   Sarcoptes   scabiei.   Annu   Rev   Entomol  

34:139–161.  doi:  10.1146/annurev.en.34.010189.001035  

Arlian  LG,  Estes  SA,  Vyszenski-­‐Moher  DL  (1988a)  Prevalence  of  Sarcoptes  scabiei   in  the  homes  and  nursing  

homes  of  scabietic  patients.  19:806–811.  

Arlian  LG,  MOHER  DV,  Cordova  D  (1988b)  Host  specificity  of  S.  scabiei  var.  canis  (Acari:  Sarcoptidae)  and  the  

role  of  host  odor.  J  Med  Entomol  25:52–56.  

Arlian  LG,  Runyan  RA,  Achar  S,  Estes  SA  (1984a)  Survival  and  infestivity  of  Sarcoptes  scabiei  var.  canis  and  var.  

hominis.  J  Am  Acad  Dermatol  11:210–215.  

Arlian  LG,  Runyan  RA,  Achar  S,  Estes  SA  (1984b)  Survival  and  infectivity  of  Sarcoptes  scabiei  var.  canis  and  var.  

hominis.  J  Am  Acad  Dermatol  11:210–215.  

Arlian   LG,   Runyan   RA,   Estes   SA   (1984c)   Cross   infestivity   of   Sarcoptes   scabiei.   J   Am   Acad   Dermatol  

10:979–986.  

Arlian   LG,   Vyszenski-­‐Moher   DL   (1988)   Life   cycle   of   Sarcoptes   scabiei   var.   canis.   J   Parasitol   74:427.   doi:  

10.2307/3282050  

Arlian   LG,   Vyszenski-­‐Moher   DL,   Pole  MJ   (1989)   Survival   of   adults   and   developmental   stages   of   Sarcoptes  

scabiei  var.  canis  when  off  the  host.  Exp  Appl  Acarol  6:181–187.  

Averof  M,   Akam  M   (1995)   Insect-­‐crustacean   relationships:   insights   from   comparative   developmental   and  

molecular  studies.  Philos  Trans  R  Soc  B  Biol  Sci  347:293–303.  

Aydıngöz   İE,   Mansur   AT   (2011)   Canine   scabies   in   humans:   a   case   report   and   review   of   the   literature.  

Dermatology  223:104–106.  doi:  10.1159/000327378  

Bajgar   J   (2004)  Organophosphates⧸   Nerve  Agent   Poisoning:  Mechanism  of  Action,  Diagnosis,   Prophylaxis,  

And  Treatment.  Adv  Clin  Chem  38:151–216.  

Barker   IK   (1974)   Sarcoptes   Scabiei   Infestation   of   a   Koala   (phascolarctos   Cinereus),   with   Probable   Human  

Involvement.  Aust  Vet  J  50:528–528.  doi:  10.1111/j.1751-­‐0813.1974.tb14068.x  

Bazargani  TT,  Hallan  JA,  Nabian  S,  Rahbari  S  (2007)  Sarcoptic  mange  of  gazelle  (Gazella  subguttarosa)  and  its  

medical  importance  in  Iran.  Parasitol  Res  101:1517–1520.  doi:  10.1007/s00436-­‐007-­‐0669-­‐6  

Beck  AL  (1965)  Animal  scabies  affecting  man.  Arch  Dermatol  91:54–55.  

Beech   R,   Levitt   N,   Cambos  M,   et   al   (2010)   Association   of   ion-­‐channel   genotype   and  macrocyclic   lactone  

sensitivity   traits   in   Haemonchus   contortus.   Mol   Biochem   Parasitol   171:74–80.   doi:  

10.1016/j.molbiopara.2010.02.004  

Bernstein   JA,   Didier   PJ   (2009)   Nonhuman   primate   dermatology:   a   literature   review.   Vet   Dermatol  

20:145–156.  doi:  10.1111/j.1365-­‐3164.2009.00742.x  

Berrilli  F,  D’Amelio  S,  Rossi  L  (2002)  Ribosomal  and  mitochondrial  DNA  sequence  variation  in  Sarcoptes  mites  

from  different  hosts  and  geographical  regions.  Parasitol  Res  88:772–777.  

Beugnet  F,  deVos  C,  Liebenberg  J,  et  al   (2014a)  Afoxolaner  against   fleas:   immediate  efficacy  and  resultant  

mortality  after  short  exposure  on  dogs.  Parasite.  doi:  10.1051/parasite/2014045  

Bibliographic  references   109  

Beugnet   F,   Halos   L,   Larsen   D,   et   al   (2014b)   The   ability   of   an   oral   formulation   of   afoxolaner   to   block   the  

transmission  of  Babesia  canis  by  Dermacentor  reticulatus  ticks  to  dogs.  Parasit  Vectors  7:283.  

Bloomquist   JR  (2003)  Chloride  channels  as  tools   for  developing  selective   insecticides.  Arch   Insect  Biochem  

Physiol  54:145–156.  doi:  10.1002/arch.10112  

Bornestein  S   (1991)  Experimental   infection  of  dogs  with  Sarcoptes   scabiei  derived   from  naturally   infected  

wild  red  foxes  (Vulpes  vulpes):  clinical  observations.  Vet  Dermatol  2:151–159.  

Bornstein  S,  de-­‐Verdier  K  (2010)  Some  important  ectoparasites  of  Alpaca  (vicugna  pacos)  and  Llama  (Lama  

glama).  J  Camelid  Sci  3:49–61.  

Bornstein   S,   Frössling   J,   Näslund   K,   et   al   (2006)   Evaluation   of   a   serological   test   (indirect   ELISA)   for   the  

diagnosis   of   sarcoptic   mange   in   red   foxes   (Vulpes   vulpes).   Vet   Dermatol   17:411–416.   doi:  

10.1111/j.1365-­‐3164.2006.00548.x  

Bornstein  S,  Zakrisson  G,  Thebo  P  (1994)  Clinical  picture  and  antibody  response  to  experimental  Sarcoptes  

scabiei  var.  vulpes  infection  in  red  foxes  (Vulpes  vulpes).  Acta  Vet  Scand  36:509–519.  

Boussinesq  M,  Gardon  J,  Gardon-­‐Wendel  N,  Chippaux  J-­‐P  (2003)  Clinical  picture,  epidemiology  and  outcome  

of   Loa-­‐associated   serious   adverse   events   related   to   mass   ivermectin   treatment   of   onchocerciasis   in  

Cameroon.  Filaria  J  2:S4.  

Bouvresse   S,   Chosidow   O   (2010)   Scabies   in   healthcare   settings:   Curr   Opin   Infect   Dis   23:111–118.   doi:  

10.1097/QCO.0b013e328336821b  

Brimer  L,  Bønløkke  L,  Pontoppidan  C,  et  al  (1995)  A  method  for  in  vitro  determination  of  the  acaricidal  effect  

of  ivermectin  using<  i>  Sarcoptes  scabiei</i>  var.<  i>  suis</i>  as  test  organism.  Vet  Parasitol  59:249–255.  

Brites  C,  Weyll  M,  Pedroso  C,  Badaró  R  (2002)  Severe  and  Norwegian  scabies  are  strongly  associated  with  

retroviral  (HIV-­‐1/HTLV-­‐1)  infection  in  Bahia,  Brazil.  Aids  16:1292–1293.  

Bronaugh  RL,  Stewart  RF,  Congdon  ER  (1982)  Methods  for  in  vitro  percutaneous  absorption  studies  II.  Animal  

models  for  human  skin.  Toxicol  Appl  Pharmacol  62:481–488.  

Brook   I   (1995)   Microbiology   of   secondary   bacterial   infection   in   scabies   lesions.   J   Clin   Microbiol  

33:2139–2140.  

Brown  AS,  Seawright  AA,  Wilkinson  GT  (1982)  The  use  of  amitraz  in  the  control  of  an  outbreak  of  sarcoptic  

mange  in  a  colony  of  koalas.  Aust  Vet  J  58:8–10.  

Brown  DDR,  Siddiqui  SZ,  Kaji  MD,  Forrester  SG  (2012)  Pharmacological  characterization  of  the  Haemonchus  

contortus  GABA-­‐gated  chloride  channel,  Hco-­‐UNC-­‐49:  Modulation  by  macrocyclic  lactone  anthelmintics  and  

a  receptor  for  piperazine.  Vet  Parasitol  185:201–209.  doi:  10.1016/j.vetpar.2011.10.006  

Buehlmann  M,  Beltraminelli  H,  Strub  C,  et  al   (2009)  Scabies  outbreak   in  an   intensive  care  unit  with  1,659  

exposed  individuals—key  factors  for  controlling  the  outbreak.  Infect  Control  30:354–360.  

Buffet  M,  Dupin  N  (2003)  Current  treatments  for  scabies.  Fundam  Clin  Pharmacol  17:217–225.  

Burkhart  CG,  Burkhart  CN,  Burkhart  KM,  others   (2000)  An  epidemiologic  and   therapeutic   reassessment  of  

scabies.  CUTIS-­‐N  Y-­‐  65:233–242.  

Capobussi  M,   Sabatino  G,  Donadini   A,   et   al   (2014)   Control   of   scabies   outbreaks   in   an   Italian   hospital:   An  

information-­‐centered   management   strategy.   Am   J   Infect   Control   42:316–320.   doi:  

Bibliographic  references   110  

10.1016/j.ajic.2013.10.006  

Carapetis   JR,   Connors   C,   Yarmirr   D,   et   al   (1997)   Success   of   a   scabies   control   program   in   an   Australian  

aboriginal  community.  Pediatr  Infect  Dis  J  16:494–499.  

Cargill  CF,  Pointon  AM,  Davies  PR,  Garcia  R  (1997)  Using  slaughter  inspections  to  evaluate  sarcoptic  mange  

infestation  of  finishing  swine.  Vet  Parasitol  70:191–200.  

Carslaw  JW,  Dobson  RM,  Hood  AJ,  Taylor  RN  (1975)  Mites  in  the  environment  of  cases  of  Norwegian  scabies.  

Br  J  Dermatol  92:333–337.  

Casais   R,   Dalton   KP,   Millán   J,   et   al   (2014)   Primary   and   secondary   experimental   infestation   of   rabbits  

(Oryctolagus   cuniculus)   with   Sarcoptes   scabiei   from   a   wild   rabbit:   Factors   determining   resistance   to  

reinfestation.  Vet  Parasitol  203:173–183.  doi:  10.1016/j.vetpar.2014.03.001  

Casais  R,  Prieto  M,  Balseiro  A,  et  al  (2007)  Identification  and  heterologous  expression  of  a  Sarcoptes  scabiei  

cDNA  encoding  a  structural  antigen  with  immunodiagnostic  potential.  Vet  Res  38:435–450.  

Casida   JE   (2015)  Golden  Age   of   RyR   and  GABA-­‐R  Diamide   and   Isoxazoline   Insecticides:   Common  Genesis,  

Serendipity,  Surprises,  Selectivity,  and  Safety.  Chem  Res  Toxicol  28:560–566.  doi:  10.1021/tx500520w  

CDC  (2011)  CDC  Health  Information  for  International  Travel  2012  :  The  Yellow  Book:  The  Yellow  Book.  Oxford  University  Press  

Charlesworth  EN,  Johnson  JL  (1974)  An  epidemic  of  canine  scabies  in  man.  Arch  Dermatol  110:572–574.  

Chen  Y-­‐Z,  Liu  G-­‐H,  Song  H-­‐Q,  et  al  (2014)  Prevalence  of  Sarcoptes  scabiei  Infection  in  Pet  Dogs  in  Southern  

China.  Sci  World  J  2014:1–3.  doi:  10.1155/2014/718590  

Chosidow  O  (2000)  Scabies  and  pediculosis.  The  Lancet  355:818.  

Chosidow  O  (2006a)  Scabies.  N  Engl  J  Med  354:1718–1727.  

Chosidow  O  (2006b)  Clinical  practices.  Scabies.  N  Engl  J  Med  354:1718–1727.  doi:  10.1056/NEJMcp052784  

Colebrook  E,  Wall  R  (2004)  Ectoparasites  of  livestock  in  Europe  and  the  Mediterranean  region.  Vet  Parasitol  

120:251–274.  doi:  10.1016/j.vetpar.2004.01.012  

Costa   LG   (2006)   Current   issues   in   organophosphate   toxicology.   Clin   Chim   Acta   366:1–13.   doi:  

10.1016/j.cca.2005.10.008  

Cotreau   MM,   Warren   S,   Ryan   JL,   et   al   (2003)   The   Antiparasitic   Moxidectin:   Safety,   Tolerability,   and  

Pharmacokinetics  in  Humans.  J  Clin  Pharmacol  43:1108–1115.  doi:  10.1177/0091270003257456  

Cruickshank  RH  (2002)  Molecular  markers  for  the  phylogenetics  of  mites  and  ticks.  Syst  Appl  Acarol  7:3–14.  

Curole   JP,  Kocher  TD  (1999)  Mitogenomics:  digging  deeper  with  complete  mitochondrial  genomes.  Trends  

Ecol  Evol  14:394–398.  

Currie  BJ,  Harumal  P,  McKinnon  M,  Walton  SF  (2004)  First  documentation  of  in  vivo  and  in  vitro  ivermectin  

resistance  in  Sarcoptes  scabiei.  Clin  Infect  Dis  39:e8–e12.  

Currie   BJ,   McCarthy   JS   (2010)   Permethrin   and   ivermectin   for   scabies.   N   Engl   J   Med   362:717–725.   doi:  

10.1056/NEJMct0910329  

Currier  RW,  Walton  SF,  Currie  BJ  (2011)  Scabies  in  animals  and  humans:  history,  evolutionary  perspectives,  

and   modern   clinical   management:   Currier   et   al.   Ann   N   Y   Acad   Sci   1230:E50–E60.   doi:  

Bibliographic  references   111  

10.1111/j.1749-­‐6632.2011.06364.x  

Curtis  CF  (2004)  Current  trends  in  the  treatment  of  Sarcoptes,  Cheyletiella  and  Otodectes  mite  infestations  

in  dogs  and  cats.  Vet  Dermatol  15:108–114.  

Damriyasa   IM,   Failing   K,   Volmer   R,   et   al   (2004)   Prevalence,   risk   factors   and   economic   importance   of  

infestations  with  Sarcoptes  scabiei  and  Haematopinus  suis  in  sows  of  pig  breeding  farms  in  Hesse,  Germany.  

Med  Vet  Entomol  18:361–367.  

Davies  PR,  Bahnson  PB,  Grass   JJ,  et  al   (1996)  Evaluation  of   the  monitoring  of  papular  dermatitis   lesions   in  

slaughtered  swine  to  assess  sarcoptic  mite  infestation.  Vet  Parasitol  62:143–153.  

Davis   DP,  Moon   RD   (1990)   Pruritus   and   behavior   of   pigs   infested   by   itch  mites,   Sarcoptes   scabiei   (Acari:  

Sarcoptidae).  J  Econ  Entomol  83:1439–1445.  

Deng  Y,  Shi  D,  Yin  Z,  et  al  (2012)  Acaricidal  activity  of  petroleum  ether  extract  of  neem  (Azadirachta  indica)  

oil  and  its  four  fractions  separated  by  column  chromatography  against  Sarcoptes  scabiei  var.  cuniculi  larvae  

in  vitro.  Exp  Parasitol  130:475–477.  doi:  10.1016/j.exppara.2012.02.007  

De   Vega   FA,   de   Vigo   JM,   Sanchez   JO,   et   al   (1998)   Evaluation   of   the   prevalence   of   sarcoptic   mange   in  

slaughtered  fattening  pigs  in  southeastern  Spain.  Vet  Parasitol  76:203–209.  

Dickens   JC,  Bohbot   JD   (2013)  Mini   review:  Mode  of  action  of  mosquito   repellents.  Pestic  Biochem  Physiol  

106:149–155.  doi:  10.1016/j.pestbp.2013.02.006  

Drag  M,  Saik  J,  Harriman  J,  Larsen  D  (2014)  Safety  evaluation  of  orally  administered  afoxolaner  in  8-­‐week-­‐old  

dogs.  Vet  Parasitol  201:198–203.  doi:  10.1016/j.vetpar.2014.02.022  

Dumont  P,  Blair  J,  Fourie  JJ,  et  al  (2014)  Evaluation  of  the  efficacy  of  afoxolaner  against  two  European  dog  

tick   species:   Dermacentor   reticulatus   and   Ixodes   ricinus.   Vet   Parasitol   201:216–219.   doi:  

10.1016/j.vetpar.2014.02.017  

Dupuy  A,  Dehen   L,   Bourrat   E,   et   al   (2007)  Accuracy  of   standard  dermoscopy   for   diagnosing  scabies.   J  Am  

Acad  Dermatol  56:53–62.  doi:  10.1016/j.jaad.2006.07.025  

Du  Y-­‐H,  Jia  R-­‐Y,  Yin  Z-­‐Q,  et  al  (2008)  Acaricidal  activity  of  extracts  of  neem  (Azadirachta  indica)  oil  against  the  

larvae   of   the   rabbit   mite   Sarcoptes   scabiei   var.   cuniculi   in   vitro.   Vet   Parasitol   157:144–148.   doi:  

10.1016/j.vetpar.2008.07.011  

Du   Y-­‐H,   Li   J-­‐L,   Jia   R-­‐Y,   et   al   (2009)   Acaricidal   activity   of   four   fractions   and   octadecanoic  

acid-­‐tetrahydrofuran-­‐3,4-­‐diyl   ester   isolated   from   chloroform   extracts   of   neem   (Azadirachta   indica)   oil  

against   Sarcoptes   scabiei   var.   cuniculi   larvae   in   vitro.   Vet   Parasitol   163:175–178.   doi:  

10.1016/j.vetpar.2009.04.002  

Du  Y.,  Jia  R.,  Pu  Z.,  Li  J.   (2007)  Acaricidal  activity  of  neem  oil  against  Sarcoptes  scabiei  var.cuniculi   in  vitro.  

VetSciChin  37:1086–1089.  

Ejidokun  OO,  Aruna  OS,  O’neill  B  (2007)  A  scabies  outbreak  in  a  further  education  college  in  Gloucestershire.  

Epidemiol  Infect  135:455–457.  doi:  10.1017/S0950268806007072  

Emde  RN  (1961)  Sarcoptic  mange  in  the  human:  a  report  of  an  epidemic  of  10  cases  of  infection  by  Sarcoptes  

scabiei,  variety  canis.  Arch  Dermatol  84:633–636.  

Emodi   IJ,   Ikefuna  AN,  Uchendu  U,  Duru  A   (2010)   Skin   diseases   among   children   attending   the   out   patient  

Bibliographic  references   112  

clinic  of  the  University  of  Nigeria  teaching  hospital,  Enug.    

Emtenan  M.H  NAM,  Manal  M.R,  Mona  A.M,  Hewida  M.E  (2010)  Aromatherapy  of  Cinnamomum  zeylanicum  

bark  oil   for   treatment  of   scabies   in   rabbits  with   emphasis   on   the  productive  performance.  Am-­‐Eurasian   J  

Agric  Env  Sci  7:719–727.  

Erster  O,  Roth  A,  Pozzi  PS,  et  al  (2015)  First  detection  of  Sarcoptes  scabiei  from  domesticated  pig  (Sus  scrofa)  

and   genetic   characterization   of   S.   scabiei   from   pet,   farm   and   wild   hosts   in   Israel.   Exp   Appl   Acarol.   doi:  

10.1007/s10493-­‐015-­‐9926-­‐z  

Estes  SA,  Kummel  B,  Arlian  L  (1983)  Experimental  canine  scabies  in  humans.  J  Am  Acad  Dermatol  9:397–401.  

Fain  A  (1978)  Epidemiological  problems  of  scabies.  Int  J  Dermatol  17:20–30.  

Fain  A  (1968)  Etude  de  la  variabilite  de  Sarcoptes  scabiei  avec  une  revision  des  Sarcoptidae.  Acta  Zool  Pathol  

Antverp  47:1–196.  

Fanneau   de   la   Horie   P   (1990)   Importance   de   la   gale   chez   le   porc   en   croissance.   Bull   Soc   Vét   Prat   Fr  

74:341–345.  

Faulde   MK,   Albiez   G,   Nehring   O   (2010)   Insecticidal,   acaricidal   and   repellent   effects   of   DEET-­‐   and  

IR3535-­‐impregnated   bed   nets   using   a   novel   long-­‐lasting   polymer-­‐coating   technique.   Parasitol   Res  

106:957–965.  doi:  10.1007/s00436-­‐010-­‐1749-­‐6  

Faust  EC,  Beaver  PC,  Jung  RC  (1962)  Animal  Agents  and  Vectors  of  Human  Disease.  Acad  Med  37:804.  

Feldmeier  H,  Jackson  A,  Ariza  L,  et  al  (2009)  The  epidemiology  of  scabies  in  an  impoverished  community  in  

rural  Brazil:  Presence  and  severity  of  disease  are  associated  with  poor  living  conditions  and  illiteracy.  J  Am  

Acad  Dermatol  60:436–443.  doi:  10.1016/j.jaad.2008.11.005  

Fernández-­‐Morán  J,  Gómez  S,  Ballesteros  F,  et  al  (1997)  Epizootiology  of  sarcoptic  mange  in  a  population  of  

cantabrian  chamois  (Rupicapra  pyrenaica  parava)  in  Northwestern  Spain.  Vet  Parasitol  73:163–171.  

Fink  DW,  Porras  AG  (1989)  Pharmacokinetics  of   Ivermectin   in  Animals  and  Humans.   In:  Campbell  WC  (ed)  

Ivermectin  and  Abamectin.  Springer  New  York,  pp  113–130  

Fournier  D,  Bride  JM,  Navajas  M  (1994)  Mitochondrial  DNA  from  a  spider  mite:  isolation,  restriction  map  and  

partial  sequence  of  the  cytochrome  oxidase  subunit  I  gene.  Genetica  94:73–75.  doi:  10.1007/BF01429222  

Fox   G   (2009)   Diagnosis   of   scabies   by   dermoscopy.   BMJ   Case   Rep   2009:bcr0620080279.   doi:  

10.1136/bcr.06.2008.0279  

Fthenakis  GC,  Karagiannidis  A,  Alexopoulos  C,  et  al   (2001)  Effects  of   sarcoptic  mange  on   the   reproductive  

performance  of  ewes  and  transmission  of  Sarcoptes  scabiei  to  newborn  lambs.  Vet  Parasitol  95:63–71.  

Fujimoto  K,  Kawasaki  Y,  Morimoto  K,  et  al  (2014)  Treatment  for  Crusted  Scabies:  Limitations  and  Side  Effects  

of  Treatment  with  Ivermectin.  J  Nippon  Med  Sch  81:157–163.  

Fukuto   TR   (1990)  Mechanism   of   action   of   organophosphorus   and   carbamate   insecticides.   Environ   Health  

Perspect  87:245–254.  

Fuller   LC   (2013)   Epidemiology   of   scabies:   Curr   Opin   Infect   Dis   26:123–126.   doi:  

10.1097/QCO.0b013e32835eb851  

Gakuya  F,  Ombui  J,  Maingi  N,  et  al  (2012)  Sarcoptic  mange  and  cheetah  conservation  in  Masai  Mara  (Kenya):  

Bibliographic  references   113  

epidemiological  study  in  a  wildlife/livestock  system.  Parasitology  139:1587–1595.  

Gakuya   F,   Rossi   L,  Ombui   J,   et   al   (2011)   The   curse  of   the  prey:   Sarcoptes  mite  molecular   analysis   reveals  

potential   prey-­‐to-­‐predator   parasitic   infestation   in   wild   animals   from  Masai   Mara,   Kenya.   Parasit   Vectors  

4:193.  

García-­‐Reynaga   P,   Zhao   C,   Sarpong   R,   Casida   JE   (2013)   New   GABA/Glutamate   Receptor   Target   for  

[3H]Isoxazoline  Insecticide.  Chem  Res  Toxicol  26:514–516.  doi:  10.1021/tx400055p  

Gassel  M,  Wolf  C,  Noack  S,  et  al  (2014)  The  novel  isoxazoline  ectoparasiticide  fluralaner:  Selective  inhibition  

of   arthropod   γ-­‐aminobutyric   acid-­‐   and   l-­‐glutamate-­‐gated   chloride   channels   and   insecticidal/acaricidal  

activity.  Insect  Biochem  Mol  Biol  45:111–124.  doi:  10.1016/j.ibmb.2013.11.009  

Geary   TG,   Woo   K,   McCarthy   JS,   et   al   (2010)   Unresolved   issues   in   anthelmintic   pharmacology   for  

helminthiases  of  humans.  Int  J  Parasitol  40:1–13.  doi:  10.1016/j.ijpara.2009.11.001  

Golant  AK,  Levitt  JO  (2012)  Scabies:  a  review  of  diagnosis  and  management  based  on  mite  biology.  Pediatr  

Rev  Acad  Pediatr  33:e1–e12.  

Goldust  M,  Babae  Nejad  S,  Rezaee  E,  Raghifar  R  (2013)  Comparative  trial  of  permethrin  5%  versus   lindane  

1%  for  the  treatment  of  scabies.  J  Dermatol  Treat  1–3.  

Goldust   M,   Rezaee   E,   Hemayat   S   (2012)   Treatment   of   scabies:   Comparison   of   permethrin   5%   versus  

ivermectin:   Permethrin   versus   ivermectin   in   scabies.   J   Dermatol   39:545–547.   doi:  

10.1111/j.1346-­‐8138.2011.01481.x  

Goldust  M,  Rezaee  E,  Raghiafar  R  (2014)  Topical  ivermectin  versus  crotamiton  cream  10%  for  the  treatment  

of  scabies.    

Gortázar  C,  Villafuerte  R,  Blanco   JC,  Fernández-­‐De-­‐Luco  D   (1998)  Enzootic  sarcoptic  mange   in   red   foxes   in  

Spain.  Z  Für  Jagdwiss  44:251–256.  

Gray  DF  (1937)  Sarcoptic  mange  affecting  wild  fauna  in  New  South  Wales.  Aust  Vet  J  13:154–155.  

Gregorini  M,   Castello  M,   Rampino   T,   et   al   (2012)   Scabies   Crustosa   in   a   61-­‐Year-­‐Old   Kidney-­‐Transplanted  

Patient.  J  Gen  Intern  Med  27:257–257.  doi:  10.1007/s11606-­‐011-­‐1758-­‐x  

Gu   X-­‐B,   Yang   G-­‐Y   (2008)   A   study   on   the   genetic   relationship   of   mites   in   the   genus   Sarcoptes   (Acari:  

Sarcoptidae)  in  China.  Int  J  Acarol  34:183–190.  doi:  10.1080/01647950808683722  

Gu   X,   Fang   C,   Yang   G,   et   al   (2014)   Acaricidal   properties   of   an   Ailanthus   altissima   bark   extract   against  

Psoroptes   cuniculi   and   Sarcoptes   scabiei   var.   cuniculi   in   vitro.   Exp   Appl   Acarol   62:225–232.   doi:  

10.1007/s10493-­‐013-­‐9736-­‐0  

Harumal  P,  Morgan  M,  Walton  SF,  et  al  (2003)  Identification  of  a  homologue  of  a  house  dust  mite  allergen  in  

a   cDNA   library   from   Sarcoptes   scabiei   var.   hominis   and   evaluation   of   its   vaccine   potential   in   a   rabbit/S.  

scabiei  var.  canis  model.  Am  J  Trop  Med  Hyg  68:54–60.  

Hay   RJ,   Steer   AC,   Engelman   D,   Walton   S   (2012)   Scabies   in   the   developing   world-­‐-­‐its   prevalence,  

complications,  and  management.  Clin  Microbiol  Infect  Off  Publ  Eur  Soc  Clin  Microbiol  Infect  Dis  18:313–323.  

doi:  10.1111/j.1469-­‐0691.2012.03798.x  

Heilesen  B  (1946)  Studies  on  Acarus  scabiei  and  scabies.    

Hengge  UR,  Currie  BJ,   Jäger  G,  et  al   (2006)  Scabies:  a  ubiquitous  neglected  skin  disease.   Lancet   Infect  Dis  

Bibliographic  references   114  

6:769–779.  doi:  10.1016/S1473-­‐3099(06)70654-­‐5  

Heukelbach  J,  Feldmeier  H  (2004)  Ectoparasites—the  underestimated  realm.  The  Lancet  363:889–891.  doi:  

10.1016/S0140-­‐6736(04)15738-­‐3  

Heukelbach  J,  Feldmeier  H  (2006)  Scabies.  The  Lancet  367:1767–1774.  

Hilbe  JM  (2011)  Negative  Binomial  Regression,  2nd  Edition,  Cambridge  University  Press.  New  York  

Hollingworth  RM  (1976)  Chemistry,  biological  activity,  and  uses  of   formamidine  pesticides.  Environ  Health  

Perspect  14:57–69.  

Holz  P,  Orbell  G,  Beveridge  I  (2011)  Sarcoptic  mange  in  a  wild  swamp  wallaby  (Wallabia  bicolor).  Aust  Vet  J  

89:458–459.  doi:  10.1111/j.1751-­‐0813.2011.00830.x  

Hong   M-­‐Y,   Lee   C-­‐C,   Chuang   M-­‐C,   et   al   (2010)   Factors   related   to   missed   diagnosis   of   incidental   scabies  

infestations  in  patients  admitted  through  the  emergency  department  to  inpatient  services.  Acad  Emerg  Med  

17:958–964.  

James  WD,   Berger   T,   Elston  D   (2015)   Andrews’   diseases   of   the   skin:   clinical   dermatology.   Elsevier  Health  

Sciences  

Kalema   G,   Kock   RA,   Macfie   E   (1998)   An   outbreak   of   sarcoptic   mange   in   free-­‐ranging   mountain   gorillas  

(Gorilla   gorilla   berengei)   in   Bwindi   Impenetrable   National   Park,   South   Western   Uganda.   In:   ANNUAL  

CONFERENCE-­‐AMERICAN   ASSOCIATION   OF   ZOO   VETERINARIANS.   AMERICAN   ASSOCIATION   OF   ZOO  

VETERINARIANS,  pp  438–438  

Karthikeyan   K   (2005)   Treatment   of   scabies:   newer   perspectives.   Postgrad   Med   J   81:7–11.   doi:  

10.1136/pgmj.2003.018390  

Kessler   E,  Matthes   H-­‐F,   Schein   E,  Wendt  M   (2003)   Detection   of   antibodies   in   sera   of   weaned   pigs   after  

contact   infection  with  Sarcoptes  scabiei  var.   suis  and  after   treatment  with  an  antiparasitic  agent  by   three  

different  indirect  ELISAs.  Vet  Parasitol  114:63–73.  doi:  10.1016/S0304-­‐4017(03)00098-­‐0  

Kiki-­‐Mvouaka   S,   Menez   C,   Borin   C,   et   al   (2010)   Role   of   P-­‐Glycoprotein   in   the   Disposition   of  Macrocyclic  

Lactones:   A   Comparison   between   Ivermectin,   Eprinomectin,   and  Moxidectin   in  Mice.   Drug  Metab   Dispos  

38:573–580.  doi:  10.1124/dmd.109.030700  

Kilp   S,   Ramirez  D,   Allan  MJ,   et   al   (2014)   Pharmacokinetics   of   fluralaner   in   dogs   following   a   single   oral   or  

intravenous  administration.  Parasit  Vectors  7:85.  

Kim  KW,  Oh  YJ,  Cho  BK,  et  al  (1990)  Norwegian  scabies  -­‐  dissemination  of  mites  by  medical  intruments.  Ann  

Dermatol  2:50–54.  

Lapied   B,   Pennetier   C,   Stankiewicz   M,   et   al   (2006)   The   insect   repellent   DEET   exerts   neurotoxic   effects  

through  alterations  of  both  neuronal  function  and  synaptic  transmission.  Vienna,  Austria,    

Lavín  S,  Ruiz-­‐Bascaran  M,  Marco  I,  et  al  (2000)  Experimental  infection  of  chamois  (Rupicapra  pyrenaica  parva)  

with  Sarcoptes  scabiei  derived  from  naturally  infected  goats.  J  Vet  Med  Ser  B  47:693–699.  

Lee   RP,   Dooge   DJ,   Preston   JM   (1980)   Efficacy   of   ivermectin   against   Sarcoptes   scabiei   in   pigs.   Vet   Rec  

107:503–505.  

Lefevre   PC,   Blancou   J,   Chermette   R,   Uilenberg   G   (2010)   Infectious   and   parasitic   diseases   of   livestock.  

Lavoisier  

Bibliographic  references   115  

Leguía  G  (1991)  The  epidemiology  and  economic  impact  of  llama  parasites.  Parasitol  Today  7:54–56.  

Lespine  A,  Alvinerie  M,  Sutra  J-­‐F,  et  al  (2005)  Influence  of  the  route  of  administration  on  efficacy  and  tissue  

distribution  of  ivermectin  in  goat.  Vet  Parasitol  128:251–260.  doi:  10.1016/j.vetpar.2004.11.028  

Letendre  L,  Huang  R,  Kvaternick  V,  et  al   (2014a)  The   intravenous  and  oral  pharmacokinetics  of  afoxolaner  

used   as   a   monthly   chewable   antiparasitic   for   dogs.   Vet   Parasitol   201:190–197.   doi:  

10.1016/j.vetpar.2014.02.021  

Letendre  L,  Huang  R,  Kvaternick  V,  et  al   (2014b)  The   intravenous  and  oral  pharmacokinetics  of  afoxolaner  

used   as   a   monthly   chewable   antiparasitic   for   dogs.   Vet   Parasitol   201:190–197.   doi:  

10.1016/j.vetpar.2014.02.021  

Liao   F,   Hu   Y,   Tan   H,   et   al   (2014)   Acaricidal   activity   of   9-­‐oxo-­‐10,11-­‐dehydroageraphorone   extracted   from  

Eupatorium  adenophorum  in  vitro.  Exp  Parasitol  140:8–11.  doi:  10.1016/j.exppara.2014.02.009  

Licciardi  S,  Herve  JP,  Darriet  F,  et  al  (2006)  Lethal  and  behavioural  effects  of  three  synthetic  repellents  (DEET,  

IR3535  and  KBR  3023)  on  Aedes  aegypti  mosquitoes  in  laboratory  assays.  Med  Vet  Entomol  20:288–293.  doi:  

10.1111/j.1365-­‐2915.2006.00630.x  

Lifschitz  A,  Virkel  G,   Imperiale  F,  et  al   (1999)  Moxidectin   in  cattle:   correlation  between  plasma  and   target  

tissues  disposition.  J  Vet  Pharmacol  Ther  22:266–273.  

Lin  AN,  Reimer  RJ,  Carter  DM  (1988)  Sulfur  revisited.  J  Am  Acad  Dermatol  18:553–558.  

Lindström  ER,  Andren  H,  Angelstam  P,  et  al   (1994)  Disease  reveals   the  predator:  sarcoptic  mange,  red  fox  

predation,  and  prey  populations.  Ecology  1042–1049.  

Little   SE,  Davidson  WR,  Howerth  EW,  et   al   (1998)  Diseases  diagnosed   in   red   foxes   from   the   southeastern  

United  States.  J  Wildl  Dis  34:620–624.  

Liu   X,   Walton   S,   Mounsey   K   (2014)   Vaccine   against   scabies:   necessity   and   possibility.   Parasitology  

141:725–732.  doi:  10.1017/S0031182013002047  

Lloyd  HG,  others  (1980)  The  red  fox.  BT  Batsford.  

Loewenstein   M,   Ludin   A,   Schuh   M   (2006a)   Comparison   of   scratching   behaviour   of   growing   pigs   with  

sarcoptic  mange  before  and  after  treatment,  employing  two  distinct  approaches.  Vet  Parasitol  140:334–343.  

doi:  10.1016/j.vetpar.2006.04.001  

Loewenstein   M,   Ludin   A,   Schuh   M   (2006b)   Comparison   of   scratching   behaviour   of   growing   pigs   with  

sarcoptic  mange  before  and  after  treatment,  employing  two  distinct  approaches.  Vet  Parasitol  140:334–343.  

doi:  10.1016/j.vetpar.2006.04.001  

Lower   KS,  Medleau   LM,   Hnilica   K,   Bigler   B   (2001)   Evaluation   of   an   enzyme-­‐linked   immunosorbant   assay  

(ELISA)  for  the  serological  diagnosis  of  sarcoptic  mange  in  dogs.  Vet  Dermatol  12:315–320.  

Luo  B,  Liao  F,  Hu  Y,  et  al  (2015)  Acaricidal  activity  of  extracts  from  Ligularia  virgaurea  against  the  Sarcoptes  

scabiei  mite  in  vitro.  Exp  Ther  Med.  doi:  10.3892/etm.2015.2503  

Mahmud  R  (2010)  Prevalence  of  scabies  and  head   lice  among  children   in  a  welfare  home  in  Pulau  Pinang,  

Malaysia.  Trop  Biomed  27:442–446.  

Makouloutou  P,  Suzuki  K,  Yokoyama  M,  et  al  (2015)  Involvement  of  two  genetic  lineages  of  sarcoptes  scabiei  

mites  in  a  local  mange  epizootic  of  wild  mammals  in  japan.  J  Wildl  Dis  51:69–78.  doi:  10.7589/2014-­‐04-­‐094  

Bibliographic  references   116  

Mangowi  AL   (2014)  Effect  of  Agriculture  on  Abundance  and  Diversity  of  Arthropods  with  Chewing  Mouth  

Parts  at  Sokoine  University  of  Agriculture  Main  Campus.  J  Nat  Sci  Res  4:55–62.  

Martin  RW,  Handasyde  KA,  Skerratt  LF  (1998)  Current  distribution  of  sarcoptic  mange  in  wombats.  Aust  Vet  J  

76:411–414.  

Matsuyama   R,   Yabusaki   T,   Kuninaga  N,   et   al   (2015)   Coexistence   of   two   different   genotypes   of   Sarcoptes  

scabiei   derived   from   companion   dogs   and   wild   raccoon   dogs   in   Gifu,   Japan:   The   genetic   evidence   for  

transmission   between   domestic   and   wild   canids.   Vet   Parasitol   212:356–360.   doi:  

10.1016/j.vetpar.2015.06.023  

McCarthy   JS   (2004)   Scabies:   more   than   just   an   irritation.   Postgrad   Med   J   80:382–387.   doi:  

10.1136/pgmj.2003.014563  

McKellar  QA,  Benchaoui  HA  (1996)  Avermectins  and  milbemycins.  J  Vet  Pharmacol  Ther  19:331–351.  

Mehlhorn  H,   Armstrong   PM   (2001)   Encyclopedic   reference   of   parasitology:   Diseases,   treatment,   therapy.  

Springer  Science  &  Business  Media  

Mellanby  K  (1941)  Transmission  of  scabies.  Br  Med  J  2:405.  

Mellanby   K   (1944)   The   development   of   symptoms,   parasitic   infection   and   immunity   in   human   scabies.  

Parasitology  35:197–206.  

Ménez   C,   Sutra   J-­‐F,   Prichard   R,   Lespine   A   (2012)   Relative   Neurotoxicity   of   Ivermectin   and  Moxidectin   in  

Mdr1ab  (−/−)  Mice  and  Effects  on  Mammalian  GABA(A)  Channel  Activity.  PLoS  Negl  Trop  Dis  6:e1883.  doi:  

10.1371/journal.pntd.0001883  

Menzano   A,   Rambozzi   L,   Rossi   L   (2004)   Outbreak   of   scabies   in   human   beings,   acquired   from   chamois  

(Rupicapra  rupicapra).  Vet  Rec  155:568–568.  doi:  10.1136/vr.155.18.568  

Miller  WH,  Griffin  CE,  Campbell  KL,  Muller  GH  (2013a)  Muller  and  Kirk’s  Small  Animal  Dermatology7:  Muller  

and  Kirk’s  Small  Animal  Dermatology.  Elsevier  Health  Sciences  

Miller  WH,  Griffin  CE,  Campbell  KL,  Muller  GH  (2013b)  Muller  and  Kirk’s  Small  Animal  Dermatology,  7th  edn.  

Elsevier  Health  Sciences  

Mimouni  D,  Ankol  O  e.,  Davidovitch  N,  et  al  (2003)  Seasonality  trends  of  scabies  in  a  young  adult  population:  

a  20-­‐year  follow-­‐up.  Br  J  Dermatol  149:157–159.  doi:  10.1046/j.1365-­‐2133.2003.05329.x  

Mitra  M,  Mahanta  SK,  Sen  S,  et  al   (1992)  Sarcoptes  scabiei   in  animals  spreading  to  man.  Trop  Geogr  Med  

45:142–143.  

Mörner  T  (1992)  Sarcoptic  mange  in  Swedish  wildlife.  Rev  Sci  Tech  Int  Off  Epizoot  11:1115–1121.  

Mörner  T,  Christensson  D  (1984)  Experimental  infection  of  red  foxes  (Vulpes  vulpes)  with  Sarcoptes  scabiei  

var.  vulpes.  Vet  Parasitol  15:159–164.  doi:  10.1016/0304-­‐4017(84)90031-­‐1  

Mosallanejad  B,  Alborzi  A,  Katvandi  N   (2012)  A  Survey  on  Ectoparasite   Infestations   in  Companion  Dogs  of  

Ahvaz  District,  South-­‐west  of  Iran.  J  Arthropod-­‐Borne  Dis  6:70–78.  

Mounsey  K,  Bernigaud  C,  Chosidow  O,  McCarthy  J  (2015)  Prospects  for  Moxidectin  as  a  new  oral  treatment  

for  human  scabies.    

Mounsey  KE,  Holt  DC,  McCarthy  J,  et  al  (2008)  Scabies:  molecular  perspectives  and  therapeutic  implications  

Bibliographic  references   117  

in  the  face  of  emerging  drug  resistance.  Future  Microbiol  3:57–66.  doi:  10.2217/17460913.3.1.57  

Mounsey  KE,  Holt  DC,  McCarthy   JS,   et   al   (2009a)   Longitudinal  evidence  of   increasing   in   vitro   tolerance  of  

scabies  mites  to  ivermectin  in  scabies-­‐endemic  communities.  Arch  Dermatol  145:840–841.  

Mounsey  KE,  Holt  DC,  McCarthy   JS,  et  al   (2009b)   Longitudinal  evidence  of   increasing   in  vitro   tolerance  of  

scabies  mites  to  ivermectin  in  scabies-­‐endemic  communities.  Arch  Dermatol  145:840–841.  

Mounsey  KE,  McCarthy   JS   (2013)  Treatment  and  control  of   scabies.  Curr  Opin   Infect  Dis  26:133–139.  doi:  

10.1097/QCO.0b013e32835e1d57  

Mounsey   KE,  McCarthy   JS,  Walton   SF   (2013)   Scratching   the   itch:   new   tools   to   advance   understanding   of  

scabies.  Trends  Parasitol  29:35–42.  doi:  10.1016/j.pt.2012.09.006  

Mounsey   KE,   Pasay   CJ,   Arlian   LG,   et   al   (2010a)   Increased   transcription   of   Glutathione   S-­‐transferases   in  

acaricide  exposed  scabies  mites.    

Mounsey  K,  Ho  M-­‐F,  Kelly  A,  et  al   (2010b)  A  tractable  experimental  model  for  study  of  human  and  animal  

scabies.  PLoS  Negl  Trop  Dis  4:e756.  doi:  10.1371/journal.pntd.0000756  

Mueller   RS,   Bettenay   SV,   Shipstone  M   (2001)   Value   of   the   pinnal-­‐pedal   reflex   in   the   diagnosis   of   canine  

scabies.  Vet  Rec  148:621–623.  

Narahashi   T,   Zhao   X,   Ikeda   T,   et   al   (2010)   Glutamate-­‐activated   chloride   channels:   Unique   fipronil   targets  

present   in   insects   but   not   in   mammals.   Pestic   Biochem   Physiol   97:149–152.   doi:  

10.1016/j.pestbp.2009.07.008  

Nathanson   JA   (1985)  Characterization  of   octopamine-­‐sensitive   adenylate   cyclase:   elucidation  of   a   class  of  

potent   and   selective   octopamine-­‐2   receptor   agonists   with   toxic   effects   in   insects.   Proc   Natl   Acad   Sci  

82:599–603.  

Nayel  NM,  Abu-­‐Samra  MT  (1986)  Experimental  infection  of  the  one-­‐humped  camel  (Camelus  dromedarius)  

and  goats  with  Sarcoptes  scabiei  var.  Cameli  and  S.  scabiei  var.  caprae.  Br  Vet  J  142:264–269.  

Neveu-­‐Lemaire  M,  others  (1938)  Traité  d’entomologie  medicale  et  vétérinaire.    

Nolan   K,   Kamrath   J,   Levitt   J   (2012)   Lindane   Toxicity:   A   Comprehensive   Review   of   the  Medical   Literature.  

Pediatr  Dermatol  29:141–146.  doi:  10.1111/j.1525-­‐1470.2011.01519.x  

Nong  X,  Fang  C-­‐L,  Wang  J-­‐H,  et  al  (2012)  Acaricidal  activity  of  extract  from  Eupatorium  adenophorum  against  

the   Psoroptes   cuniculi   and   Sarcoptes   scabiei   in   vitro.   Vet   Parasitol   187:345–349.   doi:  

10.1016/j.vetpar.2011.12.015  

Odueko  O,  Onayemi  O,  Onayemi  G  (2000)  A  prevalence  survey  of  skin  diseases  in  Nigerian  children.  Niger  J  

Med  J  Natl  Assoc  Resid  Dr  Niger  10:64–67.  

Oladimeji  FA,  Orafidiya  OO,  Ogunniyi  TAB,  Adewunmi  TA  (2000)  Pediculocidal  and  scabicidal  properties  of<  

i>  Lippia  multiflora</i>  essential  oil.  J  Ethnopharmacol  72:305–311.  

Oleaga  A,  Alasaad  S,  Rossi   L,  et  al   (2013)  Genetic  epidemiology  of  Sarcoptes   scabiei   in   the   Iberian  wolf   in  

Asturias,  Spain.  Vet  Parasitol  196:453–459.  doi:  10.1016/j.vetpar.2013.04.016  

Ozoe   Y,   Asahi   M,   Ozoe   F,   et   al   (2010)   The   antiparasitic   isoxazoline   A1443   is   a   potent   blocker   of   insect  

ligand-­‐gated   chloride   channels.   Biochem   Biophys   Res   Commun   391:744–749.   doi:  

10.1016/j.bbrc.2009.11.131  

Bibliographic  references   118  

Pannell   RS,   Fleming   DM,   Cross   KW   (2005)   The   incidence   of   molluscum   contagiosum,   scabies   and   lichen  

planus.  Epidemiol  Infect  133:985.  doi:  10.1017/S0950268805004425  

Pasay   C,   Arlian   L,   Morgan  M,   et   al   (2008)   High-­‐resolution  melt   analysis   for   the   detection   of   a   mutation  

associated  with  permethrin  resistance  in  a  population  of  scabies  mites.  Med  Vet  Entomol  22:82–88.  

Pasay  C,  Arlian   L,  Morgan  M,   et   al   (2009)   The  Effect  of   Insecticide   Synergists   on   the  Response  of   Scabies  

Mites  to  Pyrethroid  Acaricides.  PLoS  Negl  Trop  Dis  3:e354.  doi:  10.1371/journal.pntd.0000354  

Pasay   C,   Mounsey   K,   Stevenson   G,   et   al   (2010)   Acaricidal   Activity   of   Eugenol   Based   Compounds   against  

Scabies  Mites.  PLoS  ONE  5:e12079.  doi:  10.1371/journal.pone.0012079  

Pasay  C,  Walton  S,  Fischer  K,  et  al  (2006)  Pcr-­‐Based  Assay  to  Survey  for  Knockdown  Resistance  to  Pyrethroid  

Acaricides  in  Human  Scabies  Mites  (sarcoptes  Scabiei  Var  Hominis).  Am  J  Trop  Med  Hyg  74:649–657.  

Pence  DB,  Ueckermann  E  (2002)  Sarcoptic  mange  in  wildlife.  Rev  Sci  Tech  Int  Off  Epizoot  21:385–398.  

Pence  DB,  Windberg  LA  (1994)  Impact  of  a  sarcoptic  mange  epizootic  on  a  coyote  population.  J  Wildl  Manag  

624–633.  

Pence  DB,  Windberg  LA,  Pence  BC,  Sprowls  R  (1983)  The  epizootiology  and  pathology  of  sarcoptic  mange  in  

coyotes,  Canis  latrans,  from  south  Texas.  J  Parasitol  1100–1115.  

Potkay  S  (1977)  Diseases  of  marsupials.  Biol  Marsupials  2:415–506.  

Pouplard  L,  Losson  B,  Detry  M,  Hollanders  W  (1990)  Les  gales  bovines.  Ann  Méd  Vét  134:531–539.  

Prichard  R,  Ménez  C,  Lespine  A  (2012)  Moxidectin  and  the  avermectins:  Consanguinity  but  not  identity.  Int  J  

Parasitol  Drugs  Drug  Resist  2:134–153.  doi:  10.1016/j.ijpddr.2012.04.001  

Pruksachatkunakorn  C,  Wongthanee  A,  Kasiwat  V  (2003)  Scabies  in  Thai  orphanages.  Pediatr  Int  45:719–723.  

doi:  10.1111/j.1442-­‐200X.2003.01811.x  

Rahbari  S,  Nabian  S,  Bahonar  AR  (2009)  Some  observations  on  sheep  sarcoptic  mange   in  Tehran  province,  

Iran.  Trop  Anim  Health  Prod  41:397–401.  doi:  10.1007/s11250-­‐008-­‐9203-­‐9  

Rasero  R,  Rossi  L,  Soglia  D,  et  al  (2010)  Host  taxon-­‐derived  Sarcoptes  mite  in  European  wild  animals  revealed  

by  microsatellite  markers.  Biol  Conserv  143:1269–1277.  doi:  10.1016/j.biocon.2010.03.001  

Regnault-­‐Roger  C  (1997)  The  potential  of  botanical  essential  oils  for  insect  pest  control.  Integr  Pest  Manag  

Rev  2:25–34.  

Rehbein  S,  Visser  M,  Winter  R,  et  al  (2003)  Productivity  effects  of  bovine  mange  and  control  with  ivermectin.  

Vet  Parasitol  114:267–284.  

Rentería-­‐Solís   Z,  Min  AM,  Alasaad  S,   et   al   (2014)  Genetic  epidemiology  and  pathology  of   raccoon-­‐derived  

Sarcoptes  mites  from  urban  areas  of  Germany.  Med  Vet  Entomol  28:98–103.  

Roth  WI   (1991)   Scabies   resistant   to   lindane   1%   lotion   and   crotamiton   10%   cream.   J   Am   Acad   Dermatol  

24:502–503.  

Ruiz-­‐Maldonado  R,  Tamayo  L,  Domin̆guez  J  (1977)  Norwegian  scabies  due  to  sarcoptes  scabiei  var  canis.  Arch  

Dermatol  113:1733–1733.  doi:  10.1001/archderm.1977.01640120101038  

Ryser-­‐Degiorgis  M-­‐P,  Ryser  A,  Bacciarini  LN,  et  al  (2002)  Notoedric  and  sarcoptic  mange  in  free-­‐ranging  lynx  

from  Switzerland.  J  Wildl  Dis  38:228–232.  

Bibliographic  references   119  

Sachs  R,  Sachs  C  (1968)  survey  of  parasitic  infestation  of  wild  herbivores  in  the  Serengeti  region  in  northern  

Tanzania  and  the  Lake  Rukwa  region  in  southern  Tanzania.    

Salifou  S,  Attindehou  S,   Salifou  CFA,  Pangui   L-­‐J   (2013)  Prevalence  and   zoonotic   aspects  of   small   ruminant  

mange  in  the  lateritic  and  waterlogged  zones,  southern  Benin.  Rev  Bras  Parasitol  Veterinária  22:243–247.  

Salvesen   B,   Mollnes   TE   (2009)   Pathway-­‐specific   complement   activity   in   pigs   evaluated   with   a   human  

functional  complement  assay.  Mol  Immunol  46:1620–1625.  

Samuel   WM   (1981)   Attempted   experimental   transfer   of   sarcoptic   mange   (Sarcoptes   scabiei,   Acarina:  

Sarcoptidae)  among  red  fox,  coyote,  wolf  and  dog.  J  Wildl  Dis  17:343–347.  doi:  10.7589/0090-­‐3558-­‐17.3.343  

Samuel  WM,  Kocan  AA,  Pybus  MJ,  Davis  JW  (2001)  Parasitic  diseases  of  wild  mammals.  Iowa  State  University  

Press,  Ames  

Schmeller  W,  Dzikus  A   (2001)  Skin  diseases   in   children   in   rural  Kenya:   long-­‐term  results  of  a  dermatology  

project   within   the   primary   health   care   system.   Br   J   Dermatol   144:118–124.   doi:  

10.1111/j.1365-­‐2133.2001.03962.x  

Scott  EW,  McKellar  QA  (1992)  The  distribution  and  some  pharmacokinetic  parameters  of  ivermectin  in  pigs.  

Vet  Res  Commun  16:139–146.  

Seddiek  SA,  Khater  HF,  El-­‐Shorbagy  MM,  Ali  AM  (2013)  The  acaricidal  efficacy  of  aqueous  neem  extract  and  

ivermectin   against   Sarcoptes   scabiei   var.   cuniculi   in   experimentally   infested   rabbits.   Parasitol   Res  

112:2319–2330.  doi:  10.1007/s00436-­‐013-­‐3395-­‐2  

Senthilkumaran   B   (2015)   Pesticide-­‐   and   sex   steroid   analogue-­‐induced   endocrine   disruption   differentially  

targets  hypothalamo–hypophyseal–gonadal  system  during  gametogenesis  in  teleosts  –  A  review.  Gen  Comp  

Endocrinol  219:136–142.  doi:  10.1016/j.ygcen.2015.01.010  

Sharma  R,  Singal  A,  others  (2011)  Topical  permethrin  and  oral  ivermectin  in  the  management  of  scabies:  a  

prospective,  randomized,  double  blind,  controlled  study.  Indian  J  Dermatol  Venereol  Leprol  77:581.  

Sharma  RS,  Mishra  RS,  Pal  D,  et  al  (1984)  An  epidemiological  study  of  scabies  in  a  rural  community  in  India.  

Ann  Trop  Med  Parasitol  78:157–164.  

Shoop  WL,  Hartline  EJ,  Gould  BR,  et  al  (2014)  Discovery  and  mode  of  action  of  afoxolaner,  a  new  isoxazoline  

parasiticide  for  dogs.  Vet  Parasitol  201:179–189.  doi:  10.1016/j.vetpar.2014.02.020  

Shoop  WL,  Mrozik   H,   Fisher  MH   (1995)   Structure   and   activity   of   avermectins   and  milbemycins   in   animal  

health.  Vet  Parasitol  59:139–156.  doi:  10.1016/0304-­‐4017(94)00743-­‐V  

Skerratt   L,   Beveridge   I   (1999)   Human   scabies   of   wombat   origin.   Aust   Vet   J   77:607–607.   doi:  

10.1111/j.1751-­‐0813.1999.tb13202.x  

Skerratt   L,   Campbell   N,   Murrell   A,   et   al   (2002)   The   mitochondrial   12S   gene   is   a   suitable   marker   of  

populations  of  Sarcoptes  scabiei  from  wombats,  dogs  and  humans  in  Australia.  Parasitol  Res  88:376–379.  doi:  

10.1007/s00436-­‐001-­‐0556-­‐5  

Skerratt  LF,  Martin  RW,  Handasyde  KA  (1998)  Sarcoptic  mange  in  wombats.  Aust  Vet  J  76:408–410.  

Smith  EB,  Claypoole  TF  (1967)  Canine  scabies  in  dogs  and  in  humans.  Jama  199:59–64.  

Smith  HJ  (1986)  Transmission  of  Sarcoptes  scabiei  in  swine  by  fomites.  Can  Vet  J  Rev  Vét  Can  27:252–254.  

Bibliographic  references   120  

Soderlund   DM,   Bloomquist   JR   (1989)   Neurotoxic   actions   of   pyrethroid   insecticides.   Annu   Rev   Entomol  

34:77–96.  

Soderlund  DM,  Clark   JM,  Sheets   LP,  et  al   (2002)  Mechanisms  of  pyrethroid  neurotoxicity:   implications   for  

cumulative  risk  assessment.  Toxicology  171:3–59.  

Soglia  D,  Rambozzi   L,  Maione  S,   et   al   (2009)  Two   simple   techniques   for   the   safe  Sarcoptes   collection  and  

individual  mite  DNA  extraction.  Parasitol  Res  105:1465–1468.  doi:  10.1007/s00436-­‐009-­‐1580-­‐0  

Soglia   D,   Rasero   R,   Rossi   L,   et   al   (2007)   Microsatellites   as   markers   for   comparison   among   different  

populations  of  Sarcoptes  scabiei.  Ital  J  Anim  Sci  6:214–216.  

Soulsbury  CD,  Iossa  G,  Baker  PJ,  et  al  (2007)  The  impact  of  sarcoptic  mange  Sarcoptes  scabiei  on  the  British  

fox  Vulpes  vulpes  population.  Mammal  Rev  37:278–296.  

Tannenbaum  MH  (1965)  Canine  scabies  in  man:  A  report  of  human  mange.  JAMA  193:321–322.  

Taplin   D,   Meinking   TL,   Chen   JA,   Sanchez   R   (1990)   Comparison   of   Crotamiton   10%   Cream   (Eurax)   and  

Permethrin   5%   Cream   (Elimite)   for   the   Treatment   of   Scabies   in   Children.   Pediatr   Dermatol   7:67–73.   doi:  

10.1111/j.1525-­‐1470.1990.tb01078.x  

Taplin  D,  Meinking   TL,   Porcelain   SL,   et   al   (1991)   Community   control   of   scabies:   a  model   based  on  use  of  

permethrin  cream.  The  Lancet  337:1016–1018.  doi:  10.1016/0140-­‐6736(91)92669-­‐S  

Taylor  MA,  Coop  RL,  Wall  RL  (2007)  Veterinary  Parasitology,  3rd  Edition.    

Terada   Y,   Murayama   N,   Ikemura   H,   et   al   (2010)   Sarcoptes   scabiei   var.   canis   refractory   to   ivermectin  

treatment   in   two   dogs:   Ivermectin-­‐refractory   canine   scabies.   Vet   Dermatol   21:608–612.   doi:  

10.1111/j.1365-­‐3164.2010.00895.x  

Thalmann  O,  Shapiro  B,  Cui  P,  et  al   (2013)  Complete  Mitochondrial  Genomes  of  Ancient  Canids  Suggest  a  

European  Origin  of  Domestic  Dogs.  Science  342:871–874.  doi:  10.1126/science.1243650  

Thomas   J,   Peterson  GM,  Walton   SF,   et   al   (2015)   Scabies:   an   ancient   global   disease  with   a   need   for   new  

therapies.  BMC  Infect  Dis.  doi:  10.1186/s12879-­‐015-­‐0983-­‐z  

Thomas  MC,  Giedinghagen  DH,  Hoff  GL  (1987)  Brief  report:  an  outbreak  of  scabies  among  employees   in  a  

hospital-­‐associated  commercial  laundry.  Infect  Control  8:427–429.  

Thomsett  LR  (1968)  Mite  infestations  of  man  contracted  from  dogs  and  cats.  BMJ  3:93–95.  

Tikaram  SM,  Ruprah  NS   (1986)   Incidence  of   sarcoptic  mange   in  buffaloes   in   India.  Trop  Anim  Health  Prod  

18:86–90.  doi:  10.1007/BF02359718  

Todd  AW,  Gunson  JR,  Samuel  WM  (1981)  Sarcoptic  mange:  an  important  disease  of  coyotes  and  wolves  of  

Alberta,  Canada.  In:  Worldwide  Furbearer  Conference  Proceedings,  Frostburg,  Maryland.  pp  706–729  

Tompkins  JB,  Stitt  LE,  Ardelli  BF  (2010)  Brugia  malayi:  In  vitro  effects  of  ivermectin  and  moxidectin  on  adults  

and  microfilariae.  Exp  Parasitol  124:394–402.  doi:  10.1016/j.exppara.2009.12.003  

Twomey  DF,  Birch  ES,  Schock  A  (2009)  Outbreak  of  sarcoptic  mange  in  alpacas  (Vicugna  pacos)  and  control  

with   repeated   subcutaneous   ivermectin   injections.   Vet   Parasitol   159:186–191.   doi:  

10.1016/j.vetpar.2008.10.023  

Ugbomoiko  US,  Ariza  L,  Heukelbach  J  (2008)  Parasites  of  importance  for  human  health  in  Nigerian  dogs:  high  

Bibliographic  references   121  

prevalence  and  limited  knowledge  of  pet  owners.  BMC  Vet  Res  4:49.  doi:  10.1186/1746-­‐6148-­‐4-­‐49  

Van  Neste  DJ,  Staquet  MJ  (1986)  Similar  epidermal  changes   in  hyperkeratotic  scabies  of  humans  and  pigs.  

Am  J  Dermatopathol  8:267–273.  

Vijverberg   HP,   vanden   Bercken   J   (1990)   Neurotoxicological   effects   and   the  mode   of   action   of   pyrethroid  

insecticides.  CRC  Crit  Rev  Toxicol  21:105–126.  

Vos   T,   Flaxman   AD,   Naghavi   M,   et   al   (2012)   Years   lived   with   disability   (YLDs)   for   1160   sequelae   of   289  

diseases   and   injuries   1990–2010:   a   systematic   analysis   for   the  Global   Burden  of  Disease   Study   2010.   The  

Lancet  380:2163–2196.  doi:  10.1016/S0140-­‐6736(12)61729-­‐2  

Walton  SF,  Choy  JL,  Bonson  A,  et  al  (1999a)  Genetically  distinct  dog-­‐derived  and  human-­‐derived  Sarcoptes  

scabiei  in  scabies-­‐endemic  communities  in  northern  Australia.  Am  J  Trop  Med  Hyg  61:542–547.  

Walton   SF,   Currie   BJ   (2007)   Problems   in   Diagnosing   Scabies,   a   Global   Disease   in   Human   and   Animal  

Populations.  Clin  Microbiol  Rev  20:268–279.  doi:  10.1128/CMR.00042-­‐06  

Walton  SF,  Currie  BJ,  Kemp  DJ   (1997)  A  DNA   fingerprinting   system   for   the  ectoparasite  Sarcoptes   scabiei.  

Mol  Biochem  Parasitol  85:187–196.  

Walton  SF,  Dougall  A,  Pizzutto  S,  et  al  (2004a)  Genetic  epidemiology  of  Sarcoptes  scabiei  (Acari:  Sarcoptidae)  

in  northern  Australia.  Int  J  Parasitol  34:839–849.  

Walton  SF,  Holt  DC,  Currie  BJ,  Kemp  DJ  (2004b)  Scabies:  New  Future  for  a  Neglected  Disease.  In:  Advances  in  

Parasitology.  Elsevier,  pp  309–376  

Walton  SF,  McBroom  J,  Mathews  JD,  et  al  (1999b)  Crusted  scabies:  a  molecular  analysis  of  Sarcoptes  scabiei  

variety  hominis  populations  from  patients  with  repeated  infestations.  Clin  Infect  Dis  29:1226–1230.  

Walton  SF,  McKinnon  M,  Pizzutto  S,  et  al  (2004c)  Acaricidal  activity  of  Melaleuca  alternifolia  (tea  tree)  oil:  in  

vitro  sensitivity  of  sarcoptes  scabiei  var  hominis  to  terpinen-­‐4-­‐ol.  Arch  Dermatol  140:563–566.  

Walton  SF,  Myerscough  MR,  Currie  BJ  (2000)  Studies  in  vitro  on  the  relative  efficacy  of  current  acaricides  for  

Sarcoptes  scabiei  var.  hominis.  Trans  R  Soc  Trop  Med  Hyg  94:92–96.  

Walton   SF,   Oprescu   FI   (2013)   Immunology   of   scabies   and   translational   outcomes:   identifying   the  missing  

links.  Curr  Opin  Infect  Dis  26:116–122.  doi:  10.1097/QCO.0b013e32835eb8a6  

Weber   T,   Selzer   PM   (2016)   Isoxazolines:   A   Novel   Chemotype   Highly   Effective   on   Ectoparasites.  

ChemMedChem  n/a–n/a.  doi:  10.1002/cmdc.201500516  

White  AV,  Hoy  WE,  McCredie  DA  (2001)  Childhood  post-­‐streptococcal  glomerulonephritis  as  a  risk  factor  for  

chronic  renal  disease  in  later  life.  Med  J  Aust  174:492–496.  

Wolstenholme   AJ   (2012)   Glutamate-­‐gated   Chloride   Channels.   J   Biol   Chem   287:40232–40238.   doi:  

10.1074/jbc.R112.406280  

Wolstenholme   AJ,   Rogers   AT   (2005)   Glutamate-­‐gated   chloride   channels   and   the   mode   of   action   of   the  

avermectin/milbemycin  anthelmintics.  Parasitology  131:S85–S95.  doi:  10.1017/S0031182005008218  

Wooldridge  WE   (1948)  The  gamma   isomer  of  hexachlorocyclohexane   in   the   treatment  of   scabies.   J   Invest  

Dermatol  10:363–366.  

Xhaxhiu  D,  Kusi  I,  Rapti  D,  et  al  (2009)  Ectoparasites  of  dogs  and  cats  in  Albania.  Parasitol  Res  105:1577–1587.  

Bibliographic  references   122  

doi:  10.1007/s00436-­‐009-­‐1591-­‐x  

Xu  J,  Fan  Q-­‐J,  Yin  Z-­‐Q,  et  al  (2010)  The  preparation  of  neem  oil  microemulsion  (Azadirachta  indica)  and  the  

comparison  of  acaricidal  time  between  neem  oil  microemulsion  and  other  formulations  in  vitro.  Vet  Parasitol  

169:399–403.  doi:  10.1016/j.vetpar.2010.01.016  

Young  E  (1975)  Some  important  parasitic  and  other  diseases  of  lion.  Panthera  Leo  181–183.  

Zahler   M,   Essig   A,   Gothe   R,   Rinder   H   (1999)   Molecular   analyses   suggest   monospecificity   of   the   genus  

Sarcoptes  (Acari:  Sarcoptidae).  Int  J  Parasitol  29:759–766.  

Zane  L,  Bargelloni  L,  Patarnello  T  (2002)  Strategies  for  Microsatellite  isolation:  a  review.  Mol  Ecol  11:1–16.  

Zhao  C,  Casida   JE   (2014a)   Insect   γ-­‐Aminobutyric  Acid  Receptors  and   Isoxazoline   Insecticides:   Toxicological  

Profiles  Relative  to  the  Binding  Sites  of  [  3  H]Fluralaner,  [  3  H]-­‐4′-­‐Ethynyl-­‐4-­‐  n   -­‐propylbicycloorthobenzoate,  and  [  3  H]Avermectin.  J  Agric  Food  Chem  62:1019–1024.  doi:  10.1021/jf4050809  

Zhao  C,  Casida   JE   (2014b)   Insect  γ-­‐Aminobutyric  Acid  Receptors  and   Isoxazoline   Insecticides:  Toxicological  

Profiles  Relative  to  the  Binding  Sites  of  [3H]Fluralaner,  [3H]-­‐4′-­‐Ethynyl-­‐4-­‐n-­‐propylbicycloorthobenzoate,  and  [3H]Avermectin.  J  Agric  Food  Chem  62:1019–1024.  doi:  10.1021/jf4050809  

Zhao  Y,  Cao  Z,  Cheng   J,  et  al   (2015)  Population   identification  of  Sarcoptes  hominis  and  Sarcoptes  canis   in  

China  using  DNA  sequences.  Parasitol  Res  114:1001–1010.  doi:  10.1007/s00436-­‐014-­‐4266-­‐1  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Skin  structure  in  mammals  

 

The  skin  is  composed  of  three  layers:  the  epidermis,  the  dermis,  and  hypodermis  (also  

known  as  subcutaneous  tissue)  (figure  41).  The  outermost  level,  the  epidermis  (from  the  

greek   epi,   on   top   and   derma,   the   skin),   is   a   stratified   squamous   epithelium   layer   with  

multiple   functions   (enclosing   barrier,   environmental   protection,   motion   and   shape,  

thermoregulation,  storage,  indicator  of  general  health,   immunoregulation,  pigmentation,  

antimicrobial  action,  sensory  perception,  secretion,  excretion,  vitamin  d  production).  The  

epidermis  is  commonly  divided  into  four  layers  according  to  keratinocyte  morphology  and  

position   as   they   differentiate   into   horny   cells,   including   stratum   corneum,   stratum  

granulosum,  stratum  spinosum  and  stratum  germinativum  (the  basal  cell  layer)  (James  et  

al.  2015).  In  addition,  the  stratum  lucidum  is  a  thin  layer  of  translucent  cells  only  seen  in  

thick   epidermis   (figure  42).   The   thickness  of   the  whole   skin,   epidermis   and   the   stratum  

corneum  varies  according  to  the  species  of  animals  (table  13).  

    The   epidermis   is  mainly   composed   of   keratinocytes.   They   originate   from   cells   in  

the   deepest   layer   of   the   epidermis   called   the   basal   layer.   New   keratinocytes   slowly  

migrate   up   toward   the   surface  of   the   epidermis.  Once   the   keratinocytes   reach   the   skin  

surface,  they  are  gradually  shed  and  are  replaced  by  newer  cells  pushed  up  from  below.    

    Hairs  are  generated  by  hair  follicles  and  are  flexible  strands  that  mostly  consist  of  

layers  of  dead  keratinized  cells.  However,  the  keratin  in  hair   is  harder  and  more  durable  

than  those  of  the  epidermis.  The  hair  has  two  parts:  the  shaft  projects  from  the  skin;  the  

root   is  embedded   in  the  skin.   In  horses  and  cattle,   the  hairs  occur  singly  and  are  evenly  

distributed  across  the  body.  In  the  copound  hair  follicule  arrangement  (dog,  cat,  pig),  hair  

follicules  occur  in  clusters  of  variable  composition.  In  general,  a  cluster  consists  of  two  to  

five   large   primary   hairs,   surrounded   by   groups   of   smaller   secondary   hairs.   Other  

specialized   hairs   include   tactile   or   sinus   hairs.   These   are   familiar   as   cat’s   whiskers,   for  

Annexes   124  

example.   These   hairs   are   usually   longer   and   larger   than   normal   but   share   similar  

structures   to  normal   guard  hairs.   The   roots  of   these  hairs   are  highly   innervated  by   free  

nerve  endings  and  Merkel’s  discs.  

    There  are  mainly   two   types  of   skin   glands:   sebaceous   glands   and   sweat   glands.  

The   sebaceous   glands   are  derived   from  epidermal   cells   and   are   closely   associated  with  

hair  follicles.  They  produce  an  oily  sebum  by  holocrine  secretion  in  which  the  cells  break  

down   and   release   their   lipid   cytoplasm.   Sebum   helps   keep   hair   from   becoming   brittle,  

prevents  excessive  evaporation  of  water  from  the  skin,  keeps  the  skin  soft,  and  contains  a  

bactericidal  agent  that   inhibits  the  growth  of  certain  bacteria  (Akers  and  Denbow  2013).  

Sweat  glands   in  primates  are  plentiful  and  widely  distributed.  The  most  common  type  is  

the  eccrine  gland.  They  produce  a  hypotonic  watery  secretion  that  derives  from  interstitial  

fluids.   It   is  mostly  water  with  some  dissolved  salts,   lactic  acid,  and  traces  of  other  waste  

products.  The  primary  function  of  sweat  is  to  cool  the  body  as  a  result  of  evaporation.  The  

rate  of  secretion  is  controlled  by  the  activity  of  the  sympathetic  nervous  system.  In  dogs  

and   cats,   for   example,   they   are   located   only   on   the   footpad.   The   excretory   duct   opens  

directly   to   the   footpad   surface.   This   limited   distribution  means   that   these   glands   have  

virtually   no   effect   on   heat   loss,   but   they   do   act   to   moisten   the   surface   and   improve  

traction  (  Akers  and  Denbow  2013,  Miller  et  al.  2013b).   A   second   type   of   sweat   gland,  

apocrine  glands,  makes  up  a  small  proportion  of  the  total.  They  are  primarily  confined  to  

the  axillary  and  anogenital  areas  of  the  primate  body.  The  secretions  in  addition  to  watery  

sweat   also   contain   fatty   acids   and   some   proteins.   These   glands   are   affected   by   sex  

steroids.  For  the  reason,  apocrine  glands  are  believed  to  be  analogous  to  the  scent  glands  

of  animals.    

    The   skin   immune   system   is   an   active   component   of   the   overall   immune   system   and  

helps   defend   the   host   against   many   environmental   insults,   including   microbial   and  

parasite  attack.   It   is   traditionally  divided   into   innate  and  adaptive   immunity.   The   innate  

immune  system   includes  physical  barriers   such  as   the  skin  or  gut;  protective   substances  

such  as  mucus,  enzymes,  or  peptide  antibiotics;  soluble  proteins  such  as  complement;  and  

Annexes   125  

phagocytic  cells  such  as  macrophages,  neutrophils,  and  eosinophils.  The  adaptive  immune  

system   is   highly   specific   and   shows   phenomenal  memory.   Adaptive   immune   responses  

usually   commence   in   response   to   an   antigen   and   eventuate   in   the   production   of  

antibodies  and  a  population  of  antigen-­‐specific  lymphocytes.  

 Figure  41.  Diagram  of  human  skin  structure  (from  University  of  Waikato)  

 

 

Figure  42.  The  structure  of  human  epidermis    

Annexes   126  

 

Table  13.  Human  and  animal  skin  thickness  (from  Bronaugh  et  al.  1982)  

Type  of  skin   Whole  skin  (mm)   Epidermis  (µm)   Stratum  corneum  (µm)  

Human   2.97±0.28   46.9±2.3   16.8±0.7  

Pig   3.43±0.05   65.8±1.8   26.8±0.4  

Rat   2.09±0.07   32.1±1.3   18.4±0.5  

Hairless  mouse   0.70±0.02   28.6±0.9   8.9±0.4  

Mouse   0.84±0.02   12.6±0.8   5.8±0.3  

 

 

 

 

 

 

 

 

 

 

 

Annexes   127  

Comparative  efficacy  of  botanical  extracts  in  vitro  tested  against  Sarcoptes  scabiei  

Scientific  name   Compound/solvent   Concentrations   Lethal  time  or  lethal  concentration   Source    Melaleuca  alternifolia   Tea  tree  oil   5%   LT50=60min  LT=180min     terpinen-­‐4-­‐ol   2.1%     LT50=35min  LT=690min     α-­‐terpineol   0.15%   LT50=690min  LT≈1400min     1,8-­‐cineole   0.1%   LT50=1020min  LT  >1400min     Combination  mix  of  terpinen-­‐4-­‐ol,α-­‐terpineol  

and  1,8-­‐cineole     LT50=20min  LT≈600min  

(Walton  et  al.  2004c)  

Azadirachta  indica   Azadirachtins   0.3-­‐0.5%     LT>1080min   (Walton  et  al.  2000)     Undiluted  neem  seed  oil       LT=25min     Neem  seed  oil   500,  250,  125ml/l   LT50=60,  120,  300min     Neem  seed  oil     LC50=2.908ml/l  

(Du  et  al.  2007)  

  petroleum  ether  extract,  chloroform  extract     LC50=1.3,  4.1ml/l     petroleum  ether  extract,  chloroform  extract   500.0ml/l   LT50=504,  576min  

(Du  et  al.  2008)  

  octadecanoic  acid-­‐tetrahydrofuran-­‐3,4-­‐diyl  ester  from  chloroform  extract  of  neem  seed  oil  

200  mg/ml     LT=270min  

  octadecanoic  acid-­‐tetrahydrofuran-­‐3,4-­‐diyl  ester  isolated  from  chloroform  extracts  of  neem  seed  

  LC50=0.1mg/ml  

  octadecanoic  acid-­‐tetrahydrofuran-­‐3,4-­‐diyl  ester  

7.5mg/ml     LT50=918min    

(Du  et  al.  2009)  

  neem  seed  oil  microemulsion   10%   LT50=81.75min  LT=192.5min       neem  seed  oil  aqueous  emulsion   10%   LT50=95.55min  LT=212.5min    

(Xu  et  al.  2010)  

  Undiluted  neem  seed  oil     LT=26mins     Petroleum  ether  extract  of  neem  seed  oil     LC50=70.9mg/ml     Petroleum  ether  extract  of  neem  seed  oil   500.0,  250.0,  125.0,  62.5  and  

31.2ml/l    LT50=522,  528,648,690  and  786mins  

(Deng  et  al.  2012)    

  Aqueous  leaf  extract  of  neem   20%,30%,40%     LT=2880,2880,1440min     Aqueous  leaf  extract  of  neem   5%,  10%     LT50=9417,  1999min       Aqueous  leaf  extract  of  neem     LC50=11.68%  

(Seddiek  et  al.  2013)  

Eupatorium  adenophorum   Ethanol  thermal  circumfluence  extract  from  E.adenophorum  

0.25,0.5,1g/ml   LT50=52,47,31min      

(Nong  et  al.  2012)  

  euptox  A   2,3  and  4mg/ml     LT50=  41,  32,  20  min   (Liao  et  al.  2014)  

Annexes   128  

Ailanthus  altissima      

Bark  extract   0.25,0.5  and1  g/ml    

LT50=89,  47,  36  min    

(Gu  et  al.  2014)  

Ligularia  virgaurea     0.25,  0.5,  1,  2  g/ml   LT50=4.84,  2.97,  1.74,  0.72h   (Luo  et  al.  2015)  

Eugenia  caryophyllata     Clove  oil   1.56%,  3.12%,  6.25%,  12.5%,  25%  

LT=15mins  for  permethrin-­‐sensitive  mites    

    6.25%,  12.5%,  25%   LT=15mins  for  permethrin-­‐resistant  mites  

  Eugenol   12,25,50,100mM   LT50=60mins  for  permethrin-­‐sensitive  mites  

    25,50,100mM   LT50=1440,60,60mins  for  permethrin-­‐resistant  mites  

  Isoeugenol   6,12,25,50,100mM   LT50=180,120,120,60,60mins  for  permethrin-­‐sensitive  mites  

    25,50,100mM   LT50=240,180,60mins  for  permethrin-­‐resistant  mites  

  Acetyleugenol   12,25,50,100mM   LT50=1440,60,60,60mins  for  permethrin-­‐sensitive  mites  

    100mM   LT50=240mins  for  permethrin-­‐resistant  mites  

  Methyleugenol   100mM   LT50=60mins  for  permethrin-­‐sensitive  mites  

    100mM   LT50=1440mins  for  permethrin-­‐resistant  mites  

Myristica  fragrans     Nugmeg  oil   6.25%,  12.5%,  25%   LT50=90,  30,  15mins  for  permethrin-­‐sensitive  mites  

    25%   LT50=240mins  for  permethrin-­‐resistant  mites  

Cananga  odorata     Ylang  ylang  oil   25%   LT50=60mins  for  permethrin-­‐sensitive  mites  

    25%   LT50=240mins  for  permethrin-­‐resistant  mites  

(Pasay  et  al.  2010)