N°: 2009 ENAM XXXX 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 présentée et soutenue pu bliquement par Fang FANG Le 15 Avril 2016 Susceptibility to acaricides and genetic diversity of Sarcoptes scabiei from animals Directeur de thèse : Pr Jacques GUILLOT Unité de Pa rasitologie, 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 1 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. 2 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. 3 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 4 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 5 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. 6 Résumé Sarcoptes scabiei est un acarien ectoparasite obligatoire. Sa présence dans la couche cornée de l’épiderme est à l’origine d’une gale dite sarcoptique. Cette ectoparasitose a été décrite chez 104 espèces de mammifères représentant 27 familles distinctes. Les traitements actuels de la gale sarcoptique ne sont pas toujours satisfaisants et il n’existe pas de produits qui permettent d’éliminer S. scabiei dans l’environnement. Par ailleurs, la diversité génétique de S. scabiei n’est pas clairement définie et l’unicité de l’espèce fait toujours l’objet de controverses. L’objectif de cette thèse a été d’évaluer l’efficacité d’acaricides vis-‐à-‐vis de S. scabiei en utilisant un modèle animal ou par le biais de tests in vitro. La diversité génétique d’isolats d’origine animale a également été étudiée. La première partie du travail de thèse a concerné un essai thérapeutique L’efficacité d’une administration orale unique d’afoxolaner, une molécule du groupe des isoaxazolines, a été évaluée sur des porcs expérimentalement infestés. Le critère principal d’évaluation a été la réduction du nombre de sarcoptes mis en évidence dans les raclages cutanés. Huit jours après le traitement, aucun sarcopte n’a été détecté sur les 4 porcs ayant reçu l’afoxolaner alors que des sarcoptes étaient toujours présents sur les 3 porcs ayant reçu de l’ivermectine. Tous les porcs traités étaient guéris à la fin de l’essai (J35) alors que les animaux non traités sont demeurés infestés. Les autres critères d’évaluation étaient l’évolution du score clinique et de prurit. Les lésions cutanées ont rapidement régressé dans le groupe traité par l’afoxolaner alors qu’elles étaient encore présentes à J14 dans le groupe traité avec l’ivermectine. La deuxième partie du travail de thèse a porté sur l’évaluation in vitro de différentes molécules ou produits acaricides. Plusieurs concentrations d’une solution d’ivermectin ou de moxidectine ainsi 11 huiles essentielles ont été testées. Après 24h de contact avec l’ivermectine et la moxidectine, la dose létale 50% étaient de 150,2±31,4 µg/mL et 608,3±88,0 µg/mL, respectivement. Une concentration inférieure à 1 ng/mL (pour l’ivermectine) ou à 10 ng/mL (pour la moxidectine) n’a aucune activité acaricide. Pour les huiles essentielles, des tests par fumigation et par immersion ont été réalisés. Parmi Lavandula augustifolia, Melaleuca altenifolia, Pelargonium asperum, Eucalyptus radiate, Leptospermum scoparium, Cryptomeria japonica, Citrus aurantium ssp amara et 3 l’huile essentielle identifiée (BOB4, BOB5, BOB9) testés par immersion, l’huile essentielle identifiée BOB4 s’est révélée la plus efficace (une solution à 1% tue tous les acariens en 20 min). Parmi les 10 huiles essentielles énumérées avant, plus Juniperus oxycedrus testés par immersion, l’huile essentielle de Melaleuca altenifolia s’est révélée la plus efficace (tous les acariens sont morts en 4 min). Pour le contrôle de S. scabiei dans l’environnement, différents biocides ou répulsifs ont été examinés. La durée moyenne de survie a été calculée pour les produits comportant de la perméthrine, de l’esdépallethrine et de la bioresmethrine, de la bifenthrine, de la cyperméthrine et de l’imiprothrine, de la cyfluthrine, de la tétramethrine et de la sumithrine, du DEET, de l’icaridine et le produit IR3535. La deuxième partie du travail de thèse a porté sur la diversité génétique d’isolats de S. scabiei provenant d’animaux. Une partie du gène cox1 a été amplifiée. L’analyse des séquences ainsi obtenues semble montrer que les sarcoptes circulant chez le Chien sont issus de population de sarcoptes d’origine humaine. Mots clés : Sarcoptes scabiei, acaricides, modèle animal, tests in vitro, diversité génétique. I. Background 7 I. Background and outline of the thesis I. Background 8 1. Sarcoptes scabiei 1.1 Classification Sarcoptes scabiei is an arthropod, subphylum Chelicerata, class Arachnida, order Acarina, suborder Astigmata (Sarcoptiformes) and family Sarcoptidae (figure 1). The word arthropod comes from the Greek words arthro that means joint and podos that means foot. Arthropods are characterized by their jointed limbs and cuticle made of chitin, often mineralized with calcium carbonate. The Phylum Arthropoda includes the insects, myriapods, crustaceans, chelicerates and trilobites. There are around 1.3 million different kinds arthropods that have been found, which is the most numerous phylum of all living organisms (Averof and Akam, 1995; Mangowi, 2014). Arachnida are a class of arthropods with 8 legs. The order Acarina (or Acari), including mites and ticks, contains numerous economically and medically important species that are parasitic for humans, domestic or wild animals, and crops, food, etc. The sub-‐order Astigmata is a large group of relatively slow moving, similar mites with thinly sclerotized integument and no detectable spiracles or tracheal system. The families Sarcoptidae, Psoroptidae and Cnemidocoptidae are of major veterinary importance. Sarcoptidae are characterized by short legs and short capitulum. Psoroptidae are characterized by long legs and long capitulum; the size of these parasites is relatively bigger than that of Sarcoptidae. Cnemidocoptidae (or Knemidocoptidae) are parasites of birds. The family Sarcoptidae includes three genera: Sarcoptes, Notoedres and Trixacarus. All of them are parasites in mammals (Mehlhorn and Armstrong, 2001; Taylor et al., 2007). I. Background 9 Figure 1. Simplified classification of mites of veterinary importance 1.2 Morphology Sarcoptes scabiei has a characteristic oval, ventrally flattened and dorsally convex, tortoise-‐like body. The most striking parts of the ventral surface are the chitinous bars (called epimeres), which strengthen the places where forelegs and hindlegs are inserted in the body. On the dorsal surface of the mite, there are transversely arranged thorns and 10 pairs of spines arranged on two sides, 3 pairs on the anterior part and 7 pairs on the posterior part of the dorsal surface. The female is 300 to 500 µm long by 230-‐420 µm wide, and the male is 210 to 285 µm long by 160-‐210 µm wide, around two-‐thirds the size of the female. Larvae have six legs, nymphs and adults have eight legs, with suckers present on legs 1 and 2 in both sexes and leg 4 only in male (figures 2 & 3). The anus is terminal in both sexes. The eggs are oval, whitish and glossy, with slightly tapering at the pole lying anteriorly in the female mite, and this pole is attached to the floor of the burrow by means of sticky substance, which may fasten the egg to the burrow securely. The dimensions of the eggs are 167-‐175 µm by 88-‐97 µm, and increase during development (figure 4) (Heilesen, 1946).
Description: