Susceptibility to acaricides and genetic diversity of · Spécialité : Pathologie et recherche...
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
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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
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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
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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
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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
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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
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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
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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
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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
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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).
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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)
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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)
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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).
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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)
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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).
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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
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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
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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
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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
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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
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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,
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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).
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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)
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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
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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).
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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).
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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
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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.
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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
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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
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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
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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.
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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
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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).
II. In vivo evaluation of afoxolaner
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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
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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
II. In vivo evaluation of afoxolaner
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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.
II. In vivo evaluation of afoxolaner
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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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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
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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
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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
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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
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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
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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.
III. In vitro evaluation of acaricides, repellents and essential oils
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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
III. In vitro evaluation of acaricides, repellents and essential oils
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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).
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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.
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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 -‐ -‐
III. In vitro evaluation of acaricides, repellents and essential oils
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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).
III. In vitro evaluation of acaricides, repellents and essential oils
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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).
III. In vitro evaluation of acaricides, repellents and essential oils
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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,
III. In vitro evaluation of acaricides, repellents and essential oils
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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.
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
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).
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.
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Annexes 123
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)