# Development of a Murine Infection Model withLeishmania killicki, Responsible for Cutaneous Leishmaniosis in Algeria: Application in Pharmacology

Authors: Naouel Eddaikra; Ihcene Kherachi Djenad; Sihem Benbetka; Razika Benikhlef; Khatima Aït-Oudhia; Farida Moulti-Mati; Bruno Oury; Denis Sereno; Zoubir Harrat
Journal: BioMed Research International
 (2016)

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## Abstract

In Algeria,Leishmania infantum,Leishmania major, andLeishmania killicki (Leishmania tropica) are responsible for cutaneous leishmaniosis. We established a murine model ofL. killicki infection to investigate its infective capacity, some immunophysiopathological aspects, and its suitability for pharmacological purposes. Following the injection ofL. major orL. killicki metacyclic promastigotes in the ear dermis of BALB/c mice, the course of infection was followed. The infection withL. killicki caused slower lesion formation than withL. major. The presence ofL. killicki orL. major DNA and parasites was detected in the ear dermis and in lymph nodes, spleen, and liver. Lesions induced byL. killicki were nonulcerative in their aspect, whereas those caused byL. major were highly ulcerative and necrotic, which matches well with the lesion phenotype reported in humans forL. killicki andL. major, respectively. The treatment ofL. 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 byL. killicki and presents interest forin vivo screening of new compounds against this pathogen, emerging in Algeria.


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## Body

## 1. Introduction

Leishmania are obligate intracellular parasites, which cause different forms of leishmanioses in humans, ranging from dermal ulcers to fatal visceral forms. Cutaneous leishmanioses (CL) are caused by severalLeishmania species and display various clinical manifestations. In Algeria,L. killicki was discovered in 2005 in the southern province of Ghardaia and recently reported in the northern part of the country [1, 2]. Phylogenetic studies based on Multilocus Enzyme Electrophoresis showed thatL. killicki strains were included in clearly distinct clades within theL. tropica complex. A recent Multilocus Sequence Analysis further evidenced the substructuration ofL. tropica species and supported this proposal [3, 4]. In Algeria, up till now only strains belonging to theL. killicki subgroup within theL. tropicacomplex have been isolated. The pathogenicity and the infectivity of these strains have never been studied. Cutaneous lesions caused byL. tropica tend to form dry ulcers that require a long time to heal, typically one year or more. Healing is often associated with disfiguring scars and papules that can also recur at the periphery of the original lesion and are called recurrent type lesions [5, 6]. Cutaneous lesions caused byL. killicki are called chronic cutaneous lesions because they persist for several years [7]. So clinical signs of cutaneous leishmaniosis due toL. killickiare restricted to a chronic cutaneous lesion, resistant to standard treatment in opposite toL. tropicacutaneous leishmaniosis [8–11]. In addition, the transmission ofL. killicki seems to be strictly zoonotic, while it is anthroponotic or occasionally anthropozoonotic forL. tropica.Animal models have been used in the drug discovery and development process to characterize disease physiopathology and to estimate clinical dosing regimens safety margins and toxicity and of course to validate targets and compounds. The ideal in an animal model is that it should replicate to a great extent a human disease phenotype and its underlying causality. Many experimental models have been developed on rodents to study CL. Mouse models were established forL. major,L. tropica,L. amazonensis, andL. braziliensis, each with specific features in order to characterize the immune response, but none of them reproduces the pathology observed in human disease [12–14]. ForL. tropica andL. major, patterns of responses are species specific with different sex effects and largely different host susceptibility genes [6, 15, 16]. So,L. killicki causes in humans cutaneous lesions, which tend to form dry ulcers similar in their aspect to those caused byL. tropica [1, 6, 7]. Nevertheless, their healing requires a longer period of treatment. CL caused byL. killicki is called chronic CL because lesions persist for years, as opposed to CL caused byL. major for which lesions usually resolve with scare after few months only [7].The aims of this work were therefore to develop an animal model that allows us to study basic physiopathological and immunological aspects of the infection caused byL. killicki and to evaluate its suitability for pharmacological purposes.


## 2. Materials and Methods

### 2.1. Parasites

L. major (MHOM/DZ/10/LIPA175/11) andL. killicki (MHOM/DZ/11/LIPA281/11) were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 25 mM glucose, 100 μg/mL streptomycin, and 100 IU/mL penicillin at 25°C. Strain virulence was maintained by a regular passage in susceptible BALB/c mice.


### 2.2. Ethics Statement

All experiments were carried out in compliance with the guidelines of the Federation of European Laboratory Animal Science Associations (FELASA) and approved by the Ethical Committee of the Pasteur Institute in Algiers.


### 2.3. Mouse Infection

A total of 120 BALB/c mice were obtained from the animal breeding stock facility of Pasteur Institute in Algiers. Six-week-old females were kept in conventional conditions with barriers, controlled temperature, and light cycle. Food and water were providedad libitum. Infective promastigotes were isolated at the metacyclic stage from stationary phase cultures (6 days old). Metacyclic promastigotes were isolated on a Ficoll gradient [17], washed once with phosphate buffered saline (PBS) (pH 7.3), and then resuspended in PBS (pH 7.3). 103 metacyclic promastigotes in 10 μL of PBS were injected in the left ear of each mouse [13].


### 2.4.L. killicki Animal Model

To establish anin vivo model of cutaneous lesions caused byL. killicki, we compared the appearance and the induration thickness of the lesion following intradermal inoculation withL. killicki orL. major into the ear dermis of BALB/c mice. To monitor lesion development, mice were divided into three groups: 5 mice for the control noninfected mice, 5 mice infected withL. major, and 5 mice infected withL. killicki. Experiments were conducted during 12 consecutive weeks after infection in mice infected withL. major and during 30 consecutive weeks after infection for mice infected withL. killicki. Lesion development was monitored by measuring the ear thickness using a digital micrometer caliper (Fisherbrand) at weeks 2, 4, 6, 10, and 12 after infection forL. major and at weeks 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 30 after infection forL. killicki. Results are expressed as the difference between thicknesses of the inoculated ear and the noninoculated contralateral ear (internal control).


### 2.5. Detection and Quantification of Parasites in Tissues

To look at the capacity ofL. killicki and ofL. major to disseminate into internal organ, the presence of parasite DNA and of live parasites was investigated at weeks 2, 4, 8, and 12 forL. major infected mice and at weeks 2, 4, 8, 12, 16, and 20 for theL. killicki group. Mice were euthanized, and the retromaxillary draining lymph nodes, spleen, and liver were collected to extract DNA. At the 12th week, an aliquot from each sample was seeded to LIT (Liver Infusion Tryptose) medium and incubated for 4 weeks in order to isolate live parasites. Samples of tissue were homogenized in PBS using potter grinders and 1.5 mL microtubes with single-use blue pellet pestles (Polylabo, France). Homogenates were then aliquoted and stored at −20°C until DNA extraction. Total DNA was purified using the QIAamp DNA mini kit (QIAGEN) according to the manufacturer’s protocol. Parasite DNA was detected after amplification of the ribosomal internal transcribed spacer 1 (ITS1) using primers LITSR and L5.8S previously designed by Schönian et al. [18]. The PCR mix (25 μL) contained 2.5 μL of DNA, 10x buffer, 300 μM MgCl2, 200 μM dNTP, 500 nM of each primer, and 2 U of Taq DNA polymerase. Amplification products were separated on a 1% agarose gel and visualized after staining with ethidium bromide.To investigate parasite proliferation during lesion expansion, we measured the parasite load at the inoculation site. At weeks 4, 8, and 12 forL. major infected mice and at weeks 4, 8, 12, 16, 20, and 30 forL. killicki groups, mice were euthanized. Parasite proliferation within the lesion was monitored by counting the number of amastigotes in Giemsa-stained smears under 100x magnification. The number of infected macrophages and the mean number of amastigotes per macrophage were determined in one hundred randomly selected fields. The results are expressed as the mean number of parasites per 100 macrophages.


### 2.6. Drug Treatment

In a first attempt to investigate the capacity of this newin vivo murine model of infection withL. killicki to be used for the screening of new antileishmanial compounds, we compared the outcome of Glucantime treatment which consisted in injecting drug into lesions at weeks 4 and 8 forL. major andL. killicki, respectively. In all experiments, the treatment was initiated when the infection was well established and when the lesions were obvious 4 and 8 weeks after the inoculation withL. major andL. killicki, respectively. Two days before drug administration, mice were randomly divided into 2 groups of thirty animals. N-Methylglucamine antimoniate (pentavalent antimony: SbV) was diluted in PBS and then administered to mice by injection directly into lesions at a dose of 28 mg per kg of body weight every 5 days for 15 days. Ear thickness was measured weekly during the treatment and after the end of the treatment. The antimony treatment efficiency was monitored by calculating ear thickness and parasite load indexes:Ear thickness index = mean thickness of ear from treated mice/mean thickness of ear from untreated mice.Parasite load index = mean parasite load in untreated mice/mean parasite load in treated mice.


### 2.7. Statistical Analysis

Values are given as the mean ± SEM for groups ofn samples. Analysis of variance (ANOVA) and Student’s t-test were performed using GraphPad Prism Software (GraphPad Software Inc., San Diego, California, USA) and Microsoft Office Excel 2013 was used to determine the significance of differences.


## 2.1. Parasites

L. major (MHOM/DZ/10/LIPA175/11) andL. killicki (MHOM/DZ/11/LIPA281/11) were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 25 mM glucose, 100 μg/mL streptomycin, and 100 IU/mL penicillin at 25°C. Strain virulence was maintained by a regular passage in susceptible BALB/c mice.


## 2.2. Ethics Statement

All experiments were carried out in compliance with the guidelines of the Federation of European Laboratory Animal Science Associations (FELASA) and approved by the Ethical Committee of the Pasteur Institute in Algiers.


## 2.3. Mouse Infection

A total of 120 BALB/c mice were obtained from the animal breeding stock facility of Pasteur Institute in Algiers. Six-week-old females were kept in conventional conditions with barriers, controlled temperature, and light cycle. Food and water were providedad libitum. Infective promastigotes were isolated at the metacyclic stage from stationary phase cultures (6 days old). Metacyclic promastigotes were isolated on a Ficoll gradient [17], washed once with phosphate buffered saline (PBS) (pH 7.3), and then resuspended in PBS (pH 7.3). 103 metacyclic promastigotes in 10 μL of PBS were injected in the left ear of each mouse [13].


## 2.4.L. killicki Animal Model

To establish anin vivo model of cutaneous lesions caused byL. killicki, we compared the appearance and the induration thickness of the lesion following intradermal inoculation withL. killicki orL. major into the ear dermis of BALB/c mice. To monitor lesion development, mice were divided into three groups: 5 mice for the control noninfected mice, 5 mice infected withL. major, and 5 mice infected withL. killicki. Experiments were conducted during 12 consecutive weeks after infection in mice infected withL. major and during 30 consecutive weeks after infection for mice infected withL. killicki. Lesion development was monitored by measuring the ear thickness using a digital micrometer caliper (Fisherbrand) at weeks 2, 4, 6, 10, and 12 after infection forL. major and at weeks 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 30 after infection forL. killicki. Results are expressed as the difference between thicknesses of the inoculated ear and the noninoculated contralateral ear (internal control).


## 2.5. Detection and Quantification of Parasites in Tissues

To look at the capacity ofL. killicki and ofL. major to disseminate into internal organ, the presence of parasite DNA and of live parasites was investigated at weeks 2, 4, 8, and 12 forL. major infected mice and at weeks 2, 4, 8, 12, 16, and 20 for theL. killicki group. Mice were euthanized, and the retromaxillary draining lymph nodes, spleen, and liver were collected to extract DNA. At the 12th week, an aliquot from each sample was seeded to LIT (Liver Infusion Tryptose) medium and incubated for 4 weeks in order to isolate live parasites. Samples of tissue were homogenized in PBS using potter grinders and 1.5 mL microtubes with single-use blue pellet pestles (Polylabo, France). Homogenates were then aliquoted and stored at −20°C until DNA extraction. Total DNA was purified using the QIAamp DNA mini kit (QIAGEN) according to the manufacturer’s protocol. Parasite DNA was detected after amplification of the ribosomal internal transcribed spacer 1 (ITS1) using primers LITSR and L5.8S previously designed by Schönian et al. [18]. The PCR mix (25 μL) contained 2.5 μL of DNA, 10x buffer, 300 μM MgCl2, 200 μM dNTP, 500 nM of each primer, and 2 U of Taq DNA polymerase. Amplification products were separated on a 1% agarose gel and visualized after staining with ethidium bromide.To investigate parasite proliferation during lesion expansion, we measured the parasite load at the inoculation site. At weeks 4, 8, and 12 forL. major infected mice and at weeks 4, 8, 12, 16, 20, and 30 forL. killicki groups, mice were euthanized. Parasite proliferation within the lesion was monitored by counting the number of amastigotes in Giemsa-stained smears under 100x magnification. The number of infected macrophages and the mean number of amastigotes per macrophage were determined in one hundred randomly selected fields. The results are expressed as the mean number of parasites per 100 macrophages.


## 2.6. Drug Treatment

In a first attempt to investigate the capacity of this newin vivo murine model of infection withL. killicki to be used for the screening of new antileishmanial compounds, we compared the outcome of Glucantime treatment which consisted in injecting drug into lesions at weeks 4 and 8 forL. major andL. killicki, respectively. In all experiments, the treatment was initiated when the infection was well established and when the lesions were obvious 4 and 8 weeks after the inoculation withL. major andL. killicki, respectively. Two days before drug administration, mice were randomly divided into 2 groups of thirty animals. N-Methylglucamine antimoniate (pentavalent antimony: SbV) was diluted in PBS and then administered to mice by injection directly into lesions at a dose of 28 mg per kg of body weight every 5 days for 15 days. Ear thickness was measured weekly during the treatment and after the end of the treatment. The antimony treatment efficiency was monitored by calculating ear thickness and parasite load indexes:Ear thickness index = mean thickness of ear from treated mice/mean thickness of ear from untreated mice.Parasite load index = mean parasite load in untreated mice/mean parasite load in treated mice.


## 2.7. Statistical Analysis

Values are given as the mean ± SEM for groups ofn samples. Analysis of variance (ANOVA) and Student’s t-test were performed using GraphPad Prism Software (GraphPad Software Inc., San Diego, California, USA) and Microsoft Office Excel 2013 was used to determine the significance of differences.


## 3. Results

### 3.1. Lesion Appearance and Development

A striking difference in the onset, the type, and the severity of lesions was observed between bothLeishmaniaspecies. Cutaneous redness, which is the first symptom of infection caused by tissue inflammation, was detectable 2 weeks after the infection of mice withL. major but 4 weeks after infection withL. killicki.After 4 weeks,L. major-infected mice exhibited lesions with elevated borders and sharp craters (Figure 1(a)). Mice infected withL. killicki developed a detectable lesion later: the ear thickness increased progressively during the time course of the experiment (Figure 1(b)). Infection never caused lesion ulceration, which was observed inL. major-infected mice. Strikingly, lesion phenotypes induced byL. killicki were clearly distinct from those induced byL. major.Figure 1
Clinical evolution of the ear lesions in BALB/c mice. Lesions’ appearance in mice infected withL. major(4th week after infection) (a) orL. killicki (20th week after infection) (b).
(a)
(b)In mice infected byL. major metacyclic promastigotes, thickening of the ear was observed at the inoculation site as early as 3 weeks after infection. The ear thickness rapidly increased, reached a maximum of 2.4 mm at week 10, and then regressed (Figure 2). ForL. killicki, the ear thickness expanded linearly and more slowly throughout the time course of the experiment to reach 1.4 mm and 4.3 mm at weeks 10 and 30, respectively.Figure 2
Evolution of indurations following infection of BALB/c mice withLeishmania killicki orLeishmania major. The ear thickness is expressed as the difference between the thicknesses of the inoculated ear and the noninoculated contralateral ear. The data represent the mean values of measures ± standard deviations (n=5). Note: 12 weeks (∗) after infection withL. major, ears became necrotic and showed a loss of tissue which has prompted us to interrupt the experiment at this time.


### 3.2. Parasite Burden in the Ear Dermis and Occurrence in Other Organs or Tissues

The presence of parasites in the ear dermis of mice inoculated withL. major was observed as early as the 4th week (Figure 3). Moreover, the parasite burden steadily increased until the 8th week and the maximum was reached at the 10th week (Figures 1 and 3). As from this time, the ear thickness and the lesion size slightly decreased until the ear perforated.Figure 3
Parasite burden measured in ears of BALB/c mice infected withLeishmania killicki orLeishmania major. Following intradermal injection of 103 metacyclic promastigotes, the parasite load was estimated as described in Materials and Methods. Each bar is representative of the mean of five determinations ± standard deviations. Statistical analysis (p∗<0.05 and p∗∗<0.01) was performed using Student’s t-test under GraphPad Prism (n=5 mice/group).The onset of parasite burden inL. killicki-infected mice was different. Parasite expansion began later than forL. major, 8 weeks after infection, reached its maximum at week 16, and was maintained until the end of the experiment at week 30. The maximal parasite burden was significantly higher than forL. major-infected mice (p<0.001). InL. killicki-infected mice, no further parasite expansion was observed after the 12th week, while the induration thickness increased (Figures 1 and 3).Beyond the capacity ofLeishmania to replicate at the inoculation site, the dissemination ofL. killicki andL. major in various tissues or organs was further investigated (Table 1).L. major andL. killicki were both detected in culture at 12 weeks. Indeed, DNA detection evidenced the presence of parasites at the inoculation site from the 2nd week after infection for both species. However, the major difference was the delay observed for the colonization of the organ following infection.L. major colonized draining lymph nodes and spleen more quickly, 2 weeks after infection compared to 4 weeks forL. killicki (Table 1). DNA was detected in liver 4 weeks after infection for both species. After this time point, the PCR remained positive until the 12th week forL. major and the 20th week forL. killicki.Table 1
Detection ofL. major and L. killicki DNA and parasitesin various tissues of BALB/c mice. ND, not determined.
Leishmania
Tissue or organ
PCR after inoculation/LIT culture
2 weeks
4 weeks
8 weeks
12 weeks
16 weeks
20 weeks
L. major
Ear (inoculation site)
+
+
+
+/ND
Draining lymph node
+
+
+
+/+
Spleen
+
+
+
+/+
Liver
−
+
+
+/+
L. killicki
Ear (inoculation site)
+
+
+
+/ND
+
+
Draining lymph node
−
+
+
+/+
+
+
Spleen
−
+
+
+/+
+
+
Liver
−
+
+
+/+
ND
ND


### 3.3. Compared Clinical Evolution ofL. killicki andL. major Lesions and Parasite Load under Antimonial Treatment

The ear thickness was roughly similar inL. major-infected mice when treated with drug or PBS (Figure 4(a)). The reduction in the lesions observed inL. major-infected mice 10 weeks after infection was due to tissue necrosis and loss. These results show that antimony has no or undetectable effect onL. major lesions after the end of the treatment, although at concentration we used in our experiment (Figure 4(c)). Interestingly, the expansion of lesion halted in treatedL. killicki-infected mice submitted to chemotherapy (Figure 4(b)). Accordingly, the ear thickness index increased constantly during the time course of the treatment (Figure 4(c)).Figure 4
Effect of Glucantime treatment on ear induration in mice infected withLeishmania major (a) orLeishmania killicki (b) and on the ear thickness index (c). Grey bars indicate the Glucantime treatment period. The induration thickness is expressed as the difference of thicknesses between infected ears and contralateral noninoculated ear (control). Data are expressed as mean values ± standard deviations (error bars) (n=5).
(a)
(b)
(c)Glucantime treatment slightly affects the parasite load with only 0.12-fold reduction of the mean number ofL. major amastigotes/100 macrophages in lesions (see Figures 5(a) and 5(c)). However, it is more efficient inL. killicki-infected mice, which exhibited twofold reduction inL. killicki amastigotes (Figures 5(b) and 5(c)). Parasite loads were significantly different as early as one week after the beginning of the treatment, that is, at the third injection of Glucantime (p<0.001). Overall, a better concordance between parasite load and ear thickness was observed in mice infected withL. killicki during antimony treatment than in mice infected withL. major.Figure 5
Parasite load in mice infected withLeishmania major (a) orLeishmania killicki (b) and evolution of the parasite load index following antimony treatment (c). The parasite load index was calculated as follows: mean parasite load in untreated mice/mean parasite load in treated mice. Each bar is representative of the mean parasite load determined in 5 mice ± standard deviations (error bars). Statistical analysis (p∗∗∗<0.01) was performed using Student’s t-test under GraphPad Prism (n=5 mice/group). Grey bar indicates the Glucantime treatment period.
(a)
(b)
(c)


## 3.1. Lesion Appearance and Development

A striking difference in the onset, the type, and the severity of lesions was observed between bothLeishmaniaspecies. Cutaneous redness, which is the first symptom of infection caused by tissue inflammation, was detectable 2 weeks after the infection of mice withL. major but 4 weeks after infection withL. killicki.After 4 weeks,L. major-infected mice exhibited lesions with elevated borders and sharp craters (Figure 1(a)). Mice infected withL. killicki developed a detectable lesion later: the ear thickness increased progressively during the time course of the experiment (Figure 1(b)). Infection never caused lesion ulceration, which was observed inL. major-infected mice. Strikingly, lesion phenotypes induced byL. killicki were clearly distinct from those induced byL. major.Figure 1
Clinical evolution of the ear lesions in BALB/c mice. Lesions’ appearance in mice infected withL. major(4th week after infection) (a) orL. killicki (20th week after infection) (b).
(a)
(b)In mice infected byL. major metacyclic promastigotes, thickening of the ear was observed at the inoculation site as early as 3 weeks after infection. The ear thickness rapidly increased, reached a maximum of 2.4 mm at week 10, and then regressed (Figure 2). ForL. killicki, the ear thickness expanded linearly and more slowly throughout the time course of the experiment to reach 1.4 mm and 4.3 mm at weeks 10 and 30, respectively.Figure 2
Evolution of indurations following infection of BALB/c mice withLeishmania killicki orLeishmania major. The ear thickness is expressed as the difference between the thicknesses of the inoculated ear and the noninoculated contralateral ear. The data represent the mean values of measures ± standard deviations (n=5). Note: 12 weeks (∗) after infection withL. major, ears became necrotic and showed a loss of tissue which has prompted us to interrupt the experiment at this time.


## 3.2. Parasite Burden in the Ear Dermis and Occurrence in Other Organs or Tissues

The presence of parasites in the ear dermis of mice inoculated withL. major was observed as early as the 4th week (Figure 3). Moreover, the parasite burden steadily increased until the 8th week and the maximum was reached at the 10th week (Figures 1 and 3). As from this time, the ear thickness and the lesion size slightly decreased until the ear perforated.Figure 3
Parasite burden measured in ears of BALB/c mice infected withLeishmania killicki orLeishmania major. Following intradermal injection of 103 metacyclic promastigotes, the parasite load was estimated as described in Materials and Methods. Each bar is representative of the mean of five determinations ± standard deviations. Statistical analysis (p∗<0.05 and p∗∗<0.01) was performed using Student’s t-test under GraphPad Prism (n=5 mice/group).The onset of parasite burden inL. killicki-infected mice was different. Parasite expansion began later than forL. major, 8 weeks after infection, reached its maximum at week 16, and was maintained until the end of the experiment at week 30. The maximal parasite burden was significantly higher than forL. major-infected mice (p<0.001). InL. killicki-infected mice, no further parasite expansion was observed after the 12th week, while the induration thickness increased (Figures 1 and 3).Beyond the capacity ofLeishmania to replicate at the inoculation site, the dissemination ofL. killicki andL. major in various tissues or organs was further investigated (Table 1).L. major andL. killicki were both detected in culture at 12 weeks. Indeed, DNA detection evidenced the presence of parasites at the inoculation site from the 2nd week after infection for both species. However, the major difference was the delay observed for the colonization of the organ following infection.L. major colonized draining lymph nodes and spleen more quickly, 2 weeks after infection compared to 4 weeks forL. killicki (Table 1). DNA was detected in liver 4 weeks after infection for both species. After this time point, the PCR remained positive until the 12th week forL. major and the 20th week forL. killicki.Table 1
Detection ofL. major and L. killicki DNA and parasitesin various tissues of BALB/c mice. ND, not determined.
Leishmania
Tissue or organ
PCR after inoculation/LIT culture
2 weeks
4 weeks
8 weeks
12 weeks
16 weeks
20 weeks
L. major
Ear (inoculation site)
+
+
+
+/ND
Draining lymph node
+
+
+
+/+
Spleen
+
+
+
+/+
Liver
−
+
+
+/+
L. killicki
Ear (inoculation site)
+
+
+
+/ND
+
+
Draining lymph node
−
+
+
+/+
+
+
Spleen
−
+
+
+/+
+
+
Liver
−
+
+
+/+
ND
ND


## 3.3. Compared Clinical Evolution ofL. killicki andL. major Lesions and Parasite Load under Antimonial Treatment

The ear thickness was roughly similar inL. major-infected mice when treated with drug or PBS (Figure 4(a)). The reduction in the lesions observed inL. major-infected mice 10 weeks after infection was due to tissue necrosis and loss. These results show that antimony has no or undetectable effect onL. major lesions after the end of the treatment, although at concentration we used in our experiment (Figure 4(c)). Interestingly, the expansion of lesion halted in treatedL. killicki-infected mice submitted to chemotherapy (Figure 4(b)). Accordingly, the ear thickness index increased constantly during the time course of the treatment (Figure 4(c)).Figure 4
Effect of Glucantime treatment on ear induration in mice infected withLeishmania major (a) orLeishmania killicki (b) and on the ear thickness index (c). Grey bars indicate the Glucantime treatment period. The induration thickness is expressed as the difference of thicknesses between infected ears and contralateral noninoculated ear (control). Data are expressed as mean values ± standard deviations (error bars) (n=5).
(a)
(b)
(c)Glucantime treatment slightly affects the parasite load with only 0.12-fold reduction of the mean number ofL. major amastigotes/100 macrophages in lesions (see Figures 5(a) and 5(c)). However, it is more efficient inL. killicki-infected mice, which exhibited twofold reduction inL. killicki amastigotes (Figures 5(b) and 5(c)). Parasite loads were significantly different as early as one week after the beginning of the treatment, that is, at the third injection of Glucantime (p<0.001). Overall, a better concordance between parasite load and ear thickness was observed in mice infected withL. killicki during antimony treatment than in mice infected withL. major.Figure 5
Parasite load in mice infected withLeishmania major (a) orLeishmania killicki (b) and evolution of the parasite load index following antimony treatment (c). The parasite load index was calculated as follows: mean parasite load in untreated mice/mean parasite load in treated mice. Each bar is representative of the mean parasite load determined in 5 mice ± standard deviations (error bars). Statistical analysis (p∗∗∗<0.01) was performed using Student’s t-test under GraphPad Prism (n=5 mice/group). Grey bar indicates the Glucantime treatment period.
(a)
(b)
(c)


## 4. Discussion

In this work we seek for the first time to establish a CL animal model for the emergingLeishmania parasite in Algeria:L. killicki. In an attempt to reproduce the natural biology ofLeishmania transmission, Belkaid et al. [19]  established a dermal model of infection in which low numbers ofL. major promastigotes were injected into the ear. Based on this methodology, we established a dermal model of infection usingL. killicki, which involved a cutaneous lesion in the ear dermis of mice, similar to those observed in patients withL. killicki infection, that is, localized lesions that do not heal spontaneously. The appearance of lesions induced byL. killicki is different from those produced byL. major orL. tropica sensu stricto [6, 16]. Experimental infection of BALB/c mice withL. tropica produced lesions that developed up to 3 months after infection and then regressed [6, 16]. In mice infected withL. killicki, we never observed regression of the ear thickness but a continuous extension of the lesion. Indeed, in humans,L. killicki induces chronic lesions that persist up to one year [2]. In the mouse, we observed thatL. killicki lesions do not spontaneously heal but persist and develop all along the experiment. Further studies will be required to understand the underlying immunological determinants allowing the long-term persistence ofL. killicki in lesions. In the old world, onlyL. infantum andL. donovani are known to cause visceral forms of leishmaniosis (VL). Nevertheless, these two species can also be the causative agents of some forms of CL [20, 21]. In the same way,L. tropica asL. major cause CL in humans but different studies reported the isolation and characterization ofL. tropica in patients with VL [8, 22–24]. In mice,L. tropica orL. major are also known to cause visceral infections; that is, they are detected in spleen and liver but the rapidity of the visceral dissemination, the symptoms observed, and the parasite load differ betweenL. tropica andL. major [25–27]. We observed thatL. killicki had also the capacity to disseminate and to persist in internal organs of mice. Nevertheless, the ability ofL. killicki to cause VL in humans remains unknown and has not been reported to date.L. killicki is considered as an emerging pathogen resistant to pentavalent antimonial treatment [1, 2, 7, 10]. Therefore, it would be interesting to evaluate the suitability of thisL. killicki infection model in experimental pharmacology. This infection model could test the leishmanicidal activity of known drugs and be predictive of their clinical efficacy. Currently, animal models of infection optimized to test antileishmanial compounds are available forL. major andL. amazonensis [28, 29]. Human infections caused byL. tropicacomplex are considered to be refractory to most classical treatments, including antimonial containing drugs, unlikeL. major infections [22, 30, 31]. In our study, we have observed that antimony treatment does not affect the outcome of lesions induced byL. major in contrast toL. killicki. In fact, the outcome of the experiment cannot be further followed in mice infected withL. majorbecause of lesions’ necrosis. The treatment of mice infected withL. killicki resulted in the reduction of lesions size and of the thickness which were associated with a drastic diminution of the parasite load at the inoculation site.


## 5. Conclusions

Our observations support the notion that thisL. killicki model of infection has several practical advantages over theL. major model. First, the drug regimen can be evaluated over a longer time period (up to 30 weeks) as compared toL. major, where tissue loss and the appearance of ulcerative lesions 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 the leishmanicidal activity of new molecules in a simple way. The continuous emergence of antimony resistance inLeishmania spp. in various parts of the world necessitates the development of new alternative antileishmanial drugs [32–34]. To this end, this study supports the notion that this newL. killicki experimental model might be useful for screening and validating new compoundsin vivo.



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