Biomed Res Int. 2016;2016:7985104.

Development of a Murine Infection Model with Leishmania killicki, Responsible for Cutaneous Leishmaniosis in Algeria: Application in Pharmacology.

Eddaikra N, Kherachi Djenas I, Benikhlef R, Aït-Oudhia K, Moulti-Mati F, Oury B, Sereno D, Harrat Z.

 

– Laboratoire d’Eco-Épidemiologie Parasitaire et Génétique des Populations, Institut Pasteur d’Algerie, Route de Petit Staouéli, Dely Brahim, Algiers, Algeria;

Unité Mixte de Recherche IRD 224 MiVegec (Maladies Infectieuses et Vecteurs: Écologie, Génétique, – Évolution et Contrôle), Institut de Recherche pour le Développement (IRD), BP 64501, 34394 Montpellier Cedex 5, France;

– Laboratoire de Biochimie Analytique et Biotechnologies, Université Mouloud Mameri de Tizi-Ouzou, Algeria.

– Laboratoire d’Eco-Épidemiologie Parasitaire et Génétique des Populations, Institut Pasteur d’Algerie, Route de Petit Staouéli, Dely Brahim, Algiers, Algeria.

– Ecole Nationale Supérieure Vétérinaire, Hassan Badi, BP 161, El Harrach, Algiers, Algeria.

– Unité Mixte de Recherche IRD 177 InterTryp (“Interactions Hôtes-Vecteurs-Parasites-Environnement dans les Maladies Tropicales Négligées dues aux Trypanosomatides”), Institut de Recherche pour le Développement (IRD), BP 64501, 34394 Montpellier Cedex 5, France.

 

Abstract

In Algeria, Leishmania infantum, Leishmania major, and Leishmania killicki (Leishmania tropica) are responsible for cutaneous leishmaniosis. We established a murine model of L. killicki infection to investigate its infective capacity, some immunophysiopathological aspects, and its suitability for pharmacological purposes. Following the injection of L. major or L. killicki metacyclic promastigotes in the ear dermis of BALB/c mice, the course of infection was followed. The infection with L. killicki caused slower lesion formation than with L. major. The presence of L. killicki or L. major DNA and parasites was detected in the ear dermis and in lymph nodes, spleen, and liver. Lesions induced by L. killicki were nonulcerative in their aspect, whereas those caused by L. major were highly ulcerative and necrotic, which matches well with the lesion phenotype reported in humans for L. killicki and L. major, respectively. The treatment of L. killicki lesions by injection of Glucantime® significantly reduced the lesion thickness and parasite burden. Ear dermal injection of BALB/c mice constitutes a model to study lesions physiopathology caused by L. killicki and presents interest for in vivo screening of new compounds against this pathogen, emerging in Algeria.

PMID:26949705

 

Supplement :

Leishmania sp. are causative protozoal agents of various forms of leishmaniasis, which is a significant cause of morbidity and mortality in more than 98 countries and territories1.  The clinical manifestations of leishmaniasis range from various cutaneous forms to a fatal visceral form. In the Mediterranean Basin, anthroponotic cutaneous leishmaniasis (ACL) caused by Leishmania tropica, zoonotic cutaneous leishmaniasis (ZCL) caused by Leishmania major or, less frequently Leishmania infantum and zoonotic visceral leishmaniasis (ZVL) caused by L. infantum are endemic. Visceral leishmaniasis is widely distributed around the Mediterranean Basin, unlike ZCL and ACL, which are more restricted to the meridional and oriental regions. In South America, ZCL caused by various Leishmania species (e.g., Leishmania mexicana, Leishmania (Vianna) peruviana, Leishmania (Vianna) guyanensis, Leishmania (Vianna) panamensis and others), mucocutaneous leishmaniasis caused by Leishmania (Vianna) braziliensis, diffuse cutaneous leishmaniasis caused by Leishmania amazonensis and ZVL caused by Leishmania chagasi are all highly endemic. In the Indian subcontinent, anthroponotic visceral leishmaniasis (AVL) caused by Leishmania donovani poses a major health problem. In several regions of the world, the incidence of leishmaniasis outbreaks has been associated with urbanization, travel, climatic change and social conflicts1.

In Algeria, three distinct clinical forms of Leishmaniasis are encountered. In the Sahara and the highland regions, the zoonotic form of leishmaniases, caused by L major. In the southern part of the country and particularly in the oasis of Ghardaia, the chronic form of cutaneous leishmaniasis due to L killicki is present. L. killicki belongs to the L tropica complex and generally occurs in sympatry with L. major. In the northern part of the country, a zoonotic cutaneous form caused by L infantum is sporadic. The visceral form of leishmaniases caused by L. infantum occurs mainly in the northern part of the country. The active visceral leishmaniasis foci are located in the region of Kabylie in the north of the country as well in the east in the departments of Jijel and Constantine

In the absence of effective vaccines, the only feasible way to treat and control leishmaniasis is through the use of affordable medications. The current chemotherapeutic arsenal consists of molecules developed in the 1950s, including pentavalent antimony (SbV) compounds (e.g., Pentostam®, Glucantime®), pentamidine and various formulations of the antifungal Amphotericin B and, more recently, miltefosine.

Taking into account that cutaneous forms of Leishmaniasis caused by L. killicki are emerging in Algeria, we have decided to test the possibility of developing an in vivo model of infection with this pathogen and to probe the capability of this new model to be used in experimental pharmacology.

 

 

ds fig1-1Figure 1: New Animal model of cutaneous leishmaniasis by L. killicki infection of BALB/c mice. L. killicki develops chronic and non-healing lesions in BALB/c mice, which mimics the human lesion phenotype observed in endemic foci of L. killicki. L. killicki model of infection is responsive to the well-known antileishmanial drug, Glucantime®, which is in clinical use and has a good predictive efficacy.

 

The ideal in an animal model is that it should replicate to a great extent a human disease phenotype and its underlying causality. To set up this in vivo model of infection at to probe its adequacy for experimental pharmacology, we have performed several experiments aimed to investigate the lesion phenotype, the replicative and disseminative capacity of L. killicki in compasison with  L. major, an already well-known model in used in experimental leishmaniasis pharmacology (see figure 1).

We found that the onset, the type, and the severity of lesions are clearly different between the two leishmania species we tested. Firstly, striking differences in the clinical presentation of lesions is noticed (see Figure 1 upper panel). Infection with L. killicki never caused lesion ulceration, which was observed in L. major-infected mice. Secondly, mices infected with L. killicki developed a detectable lesion later than those infected by L. major. Thirdly, the ear thickness increased gradually during the time course of the experiment (Figure 1 lower panel right). Finally, replication of L. killicki is higher than for L. major (Figure 1 lower panel left). In human infected by L. killicki the clinical presentation generally described is as follow : a localized lesion, nonulcerative in nature that do not heal spontaneously. The appearance of lesions induced by L. killicki is different from those produced by L. major or by other already studied L. tropica strains. Therefore, all the characteristics we noticed during the in vivo infection of the BALBc mice, mimics what is observed in human lesions caused by L. killicki infection.

For drug discovery and development as well as to estimate clinical dosing regimens safety margins and toxicity or to validate targets and compounds, the model must respond to classical drugs and regimen in clinical used. In Algeria as well as in most part of the word, Pentavalent antimony (SbV) compounds are considered as the first line drug for all forms of leishmaniasis treatment. In Algeria the guidelines were implemented by the health ministry based on those of edicted by the World health organization. It consists of the administration of 20 mg/kg/day of Glucantime® for 15 days, by intramuscular injection when multiple lesions are observed or if the lesion is located on the face. We have therefore evaluated the efficiency of the protocol edicted by the World Health organization, to readily interrupt lesion development and to reduce the parasite proliferation in lesions. The protocol (20mg/kg/day) for 15 consecutive days was adapted to mice. To closely mimic in mice, what happen during the treatment of patients suffering from lesion caused by L. killicki, the injection of Glucantime was initiated when the lesions were obviously observed.  We observed that antimony treatment does not affect the outcome of lesions induced by L. major in contrast to L. killicki (figure 1 middle left and right panel). The treatment of mice infected with L. killicki resulted in a drastic diminution of the parasite load at the inoculation site (figure 1 middle left and right panel). All these characteristics were not observed in L. major treated mice (figure 1 middle right panel), where no reduction in lesion size and parasite burden at the inoculation site was observed.

Our study clearly demonstrates that, this new L. killicki model of infection present numerous practical advantages over the L. major model for experimental pharmacology. Firstly, we can evaluate and compare the drug regimen over a long time period (up to 30 weeks) as compared to L. major, where tissue loss and the ulcerative forms of the lesions strongly limit the time course of the experimentation. Second, the intense and continuous parasite multiplication at the inoculation site makes it possible to more easily assess to the in vivo leishmanicidal activity of new molecules, in a simple way. Finally, the continuous emergence of antimony resistance in Leishmania spp. in various parts of the world2,3 necessitates the development of new alternative antileishmanial drugs, like temporin4 of which this new in vivo model may help to define their efficiency, potential toxicity, and regimen.

 

References :

1: Akhoundi M, Kuhls K, Cannet A, Votýpka J, Marty P, Delaunay P, Sereno D. A Historical Overview of the Classification, Evolution, and Dispersion of Leishmania Parasites and Sandflies. PLoS Negl Trop Dis. 2016 Mar 3;10(3):e0004349.

2: Seblova V, Oury B, Eddaikra N, Aït-Oudhia K, Pratlong F, Gazanion E, Maia C, Volf P, Sereno D. Transmission potential of antimony-resistant leishmania field isolates. Antimicrob Agents Chemother. 2014 Oct;58(10):6273-6.

3: Sereno D, Maia C, Aït-Oudhia K. Antimony resistance and environment: Elusive links to explore during Leishmania life cycle. Int J Parasitol Drugs Drug Resist. 2012 Sep 3;2:200-3.

4: Abbassi F, Raja Z, Oury B, Gazanion E, Piesse C, Sereno D, Nicolas P, Foulon T, Ladram A. Antibacterial and leishmanicidal activities of temporin-SHd, a 17-residue long membrane-damaging peptide. Biochimie. 2013 Feb;95(2):388-99.

 

Contact :

Denis SERENO Ph. D

IRD (Institut de Recherche pour le Développement) Montpellier

Office 164

911 Avenue Agropolis

34 032 Montpellier Cedex

denis.sereno@ird.fr

 

 

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