First detection of Leishmania donovani in sand flies ...

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Mar 26, 2010 - The area is a rocky and hilly savannah region, with a Sudano-Sahe- lian climate ... pended from the roof of the house, or outdoors suspended 80 cm .... the Old World parasites L. donovani sensu stricto (s.s.) and L. donovani s.l. .... and parasitological prevalence of leishmaniasis in school children in Keana ...
Tropical Medicine and International Health

doi:10.1111/tmi.13123

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First detection of Leishmania donovani in sand flies from Cameroon and its epidemiological implications Aim e Tateng Ngouateu1,2, Oscar David Kirstein3, Omer B eb e Ngouateu2,4, Andreas Kr€ uger5, 6 7 1 3 Esther von Stebut , Marcus Maurer , Vincent Khan Payne , Alon Warburg and Blaise Dondji2,8 1 2 3 4 5 6 7 8

Research Unit of Biology and Applied Ecology, University of Dschang, Dschang, Cameroon Laboratory of the Leishmaniasis Research Project, Mokolo District Hospital, Mokolo, Cameroon Department of Microbiology and Molecular Genetics, Hebrew University of Jerusalem, Jerusalem, Israel Department of Animal Biology and Physiology, University of Yaounde I, Yaounde, Cameroon Tropical Medicine Branch, Bundeswehr-Hospital Hamburg, Hamburg, Germany Department of Dermatology and Venerology, University of Cologne, Cologne, Germany Department of Dermatology and Allergy, Charite – Universit€atsmedizin Berlin, Berlin, Germany Laboratory of Cellular Immunology and Parasitology, Central Washington University, Ellensburg, WA, USA

Summary

objectives A sound knowledge of the vector-host-parasite transmission dynamics is a prerequisite for adequate control measures of vector-borne diseases. To achieve this, an entomological investigation was conducted in the cutaneous leishmaniasis (CL) endemic focus of Mokolo District, northern Cameroon to identify the insect vector(s) of the disease. methods Phlebotomine sand flies were collected in and around Mokolo using New Standard CDC Miniature Light Traps. Individual sand flies were used for morphological species identification, and the remainder of the body for DNA analysis. Sand flies were demonstrated to harbour Leishmania spp. parasites using ITS1 PCR. Mitochondrial vertebrate-specific Cytochrome b -PCR was used to identify blood meals ingested by female sand flies. PCR amplicons were sequenced for Leishmania and blood sources discrimination. results This study revealed the presence of Leishmania donovani complex DNA (n = 1) in Phlebotomus duboscqi and of lizard-borne Leishmania tarentolae-like DNA (n = 3) in Sergentomyia spp. in 79 sand fly specimens from Mokolo district. conclusions The causative agent of CL could not be detected in potential vectors. Instead, we found evidence for visceral leishmaniasis (VL) parasites in Phlebotomus duboscqi as well as enzootic reptile parasites in the Mokolo area. We recommend that an epidemiological survey be carried out in the area to evaluate the prevalence and eventually describe the clinical manifestations of VL in the human population. Political instability in neighbouring countries and the resulting refugee migration are likely explanations for the emergence of VL in Mokolo. keywords Leishmania donovani, Cameroon, sand flies, visceral leishmaniasis

Introduction Leishmaniasis is a vector-borne disease caused by protozoa of the genus Leishmania (Kinetoplastida) and transmitted to the vertebrate host by the bite of infected phlebotomine sand flies (Diptera: Psychodidae-Phlebotominae). About 350 million people are at risk of infection worldwide, and about 2 million new cases are registered every year [1]. Because of little interest of pharmaceutical companies and the public entities of countries concerned, leishmaniasis and in particular, its most devastating form, visceral leishmaniasis (VL), is listed among the neglected tropical diseases [2]. Northern Cameroon and

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neighbouring countries constitute one of the Central/West African leishmaniasis foci. Several studies have confirmed its presence in these countries: VL in Gabon [3], CL in Nigeria [4, 5], CL in Central African Republic [6] and VL in Chad [7]. Two foci of leishmaniasis have been identified in the northern region of Cameroon: CL in Mokolo, where the first cases were reported in 1930 [8] and the causative agent identified as Leishmania major MON-26 [9, 10], and VL in Kousseri [11]. Evidence of cases of leishmaniasis in Cameroon dates back to the 1930s; however, no study has attempted to identify the sand fly species transmitting the causative agent in the area. Previous entomological work from the

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CL focus in Mokolo area recorded five Sergentomyia species and two Phlebotomus species of sand flies [12]. Nevertheless, no parasite isolation was successfully achieved from Phlebotomus (Phlebotomus) duboscqi, a known vector of CL, which has been reported in the endemic region [12]. This study was aimed at detecting Leishmania parasites in phlebotomine sand flies collected in the Mokolo CL focus of northern Cameroon.

Materials and methods Study site The study was conducted from January to December 2013 in Mokolo district, Far North Region of Cameroon (10°440 32.8500 N; 13°480 15.2500 E) (Figure 1). The elevation ranges from 750 m to more than 1000 m asl. The area is a rocky and hilly savannah region, with a Sudano-Sahelian climate, characterised by long dry seasons running from October to May, with median temperatures above 24 °C and low relative humidity during the dry season, which can reach up to 100% during the rainy season (Mokolo Meteorological Station). Collection of sand flies Sand flies were collected using CDC Mini Light Traps (BioQuip, Rancho Dominguez, CA, USA) and New Standard CDC Miniature Light Traps (John W. Hock Co., Gainesville, FL, USA). Three traps per night per site were deployed in five local representative sites in a perimeter of approximately 4 km in and around Mokolo town comprising of two urban, two peri-urban/rural and a sylvatic location. The peri-urban/rural settings were villager’s compounds, and the sylvatic site was the peak of a rocky mountain with agricultural activities at its foothills. Except for the latter, all sites were chosen in view of previous CL cases in the respective communities. In urban areas, traps were deployed either indoors, suspended from the roof of the house, or outdoors suspended 80 cm from the ground in downdraft orientation. Traps were operated overnight for 12 h (from 6:00 PM to 6:00 AM). Collected sand flies were transported to the laboratory, where they were sorted, counted and preserved in 1.5-ml tubes containing 96% EtOH. Identification of sand flies For the purpose of this study, that is the detection of Leishmania parasites in sand flies, we focused on female 2

sand flies after sorting. Sand fly genera were identified based upon external characteristics. All specimens belonging to the genus Phlebotomus and approximately 80% of the Sergentomyia spp. females were selected for species identification under a microscope. Our selection was based on previous reports of Phlebotomus spp but not Sergentomyia spp. as potential insect vectors of human leishmaniasis in the focus [12]. For species identification, head and genitalia of individual sand flies were mounted on microscope slides in Hoyer’s medium. Thoraxes were preserved in 1.5-ml tubes for DNA extraction. Identification of species was based on cibarial and pharyngeal armatures, antennal features and spermathecae of females and external genitalia of males, using different keys [13, 14]. We used an Olympus BX50 microscope at 20–409 magnification. Molecular analysis DNA extraction. Prior to dissection, sand flies were placed in a tea strainer, rinsed under tap water, briefly immersed in dilute dish-washing detergent, rinsed again, immersed in 1% of sodium hypochlorite (NaClO) solution for 1 min and rinsed in double distilled water to remove all external contamination. DNA was extracted from individual sand flies using phenol–chloroform [15]. Leishmania infection in sand flies. Leishmania infections were determined by PCR amplification of Leishmaniaspecific ribosomal Internal Transcribed Spacer 1 (ITS 1) [16]. PCR products were visualised on 1.5% agarose gel. DNA from the successful amplicons was sequenced to identify the Leishmania species. Validation of parasite species was based on ITS1 sharing high sequence homology (i.e. 98% for Leishmania donovani complex and 93– 98% for other species, respectively) with existing entries in GenBank. Identification of sand fly blood meal source. DNA extracted from abdomens of blood-fed female sand flies was PCR-amplified using vertebrate-specific mitochondrial cytochrome b gene (cyt b) [15]. Products were visualised on 1.5% agarose gels. DNA from successful amplicons was sequenced to identify vertebrate blood sources. Validation of blood host species (i.e. Homo sapiens) was based on cyt b sharing high sequence homology with existing entries in GenBank. DNA sequencing analysis. PCR amplicons (from human blood samples and sand flies) were sequenced by The Center for Genomic Technologies at the Hebrew

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N

Chad

Cameroon

Far north region

Mokolo

D - Mokolá

Nigeria CAR

Gabon

Congo

E - Mboua C - Hospital A - Mofolé B - Mountain

0

1

2

3 Km

Figure 1 Map of the Far North Region of Cameroon with sand fly collection sites A–E. Basemap and layers source: Esri, Earthstar Geographics, CNES/Airbus DS, USDA, USGS, AeroGRID, IGN, and the GIS User Community. Map was developed on ArcGISTM 10 (ESRI Inc., Redlands, CA, USA).

University of Jerusalem using automated DNA Sequencing, based on BigDye Terminator cycle sequencing chemistry from Applied Biosystems (ABI), ABI PRISM 3730xl DNA Analyzer and the ABI’s Data collection and Sequence Analysis software. The derived sequences were compared against the GenBank database using NCBI BLASTN (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and default search parameters, optimised for highly similar sequences (megablast) [17]. Furthermore, they were aligned using Clustal Omega (http://www.ebi.ac.uk/ Tools/msa/clustalo/) and analysed in MEGA7 [18]. Simple neighbour-joining (NJ) analyses were computed using the Kimura2-Parameter distance model. Bootstrap of 500 replicates was calculated to test the robustness of the resulting taxon ID trees.

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Results A total of 477 females were dissected and identified. Of these, all Phlebotomus duboscqi (n = 44) were tested by ITS1-PCR for Leishmania DNA. In addition, 35 sand flies of the genus Sergentomyia were tested for Leishmania DNA, 14 of which were blood fed and were therefore, also analysed for blood meal identification. All ITS1-positive samples (n = 15) were sequenced for Leishmania species identification, Results are summarised in Table 1. Sequencing of DNA from ITS1-PCR-positive individuals showed L. donovani complex DNA in one sample of Ph. duboscqi (Figure 2). The specimen was from periurban Mokolo with ‘dense’ human population living 3

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Table 1 Results of ITS1 and cyt b PCR and sequence analysis on Leishmania and blood meal host DNA in sand flies from northern Cameroon ITS1 for Leishmania infection and Leishmania ID

Cyt b for Blood meal ID

PCR Results

PCR Results

Sequenced

Sequencing results

+

U

+

U

Homo sapiens (95%) Homo sapiens (85%) Phlebotomus

Sample #

Sand fly species

52

Phlebotomus duboscqi (BM)

61

Phlebotomus duboscqi (BM)

+

Amplicon not good for sequencing

62 72 37

Phlebotomus duboscqi (BM) Phlebotomus duboscqi Sergentomyia simillima (BM)

+ +

+

U

Leishmania donovani (98% at 184 bp) Amplicon not good for sequencing

+

U

Homo sapiens (98%)

50 55

Sergentomyia antennata (BM) Sergentomyia affinis vorax (BM) Sergentomyia distincta (BM)

+ +

Leishmania tarentolae (99% at 135 bp) Leishmania tarentolae (98% at 163 bp)

+ +

U

Dipteran

+

Leishmania tarentolae (98% at 162 bp)

+

U

No significant similarity found

65

Sequencing results (Blast similarity)

BM, with blood meal; bp, base pairs.

Figure 2 Pairwise sequence alignment, produced by Clustal Omega, displaying ITS1 of Mokolo specimen 72 and Leishmania infantum (GenBank accession no. KU680960).

together with domestic animals. The sequence alignment in Figure 2 indicates three polymorphic positions in the Mokolo specimen, resulting in 98% homology with Leishmania infantum (GenBank accession no. KU680960). However, the BLASTN search produced another 99 matches with L. donovani complex sequences, all of which revealed the same alignments and homologies. In addition, sequencing showed L. tarentolae-like DNA in three samples of Sergentomyia spp. (Sergentomyia antennata sensu lato, Sergentomyia affinis subspecies vorax,

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and Sergentomyia distincta; Figure 3). A further 11 positive PCR results remained unresolved because of low quality of the amplicons or that of the sequence, or no sequence similarity with Leishmania spp. The cyt b PCR for blood meal identification produced amplicons for all 17 blood-fed females. However, only seven were sequenced, three of which presented homology to human DNA (Table 1). The sequences of three other samples were of insect origin, and in these females, the gravid condition had apparently been misinterpreted as blood meals.

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LC031456 Leishmania sp. 220 DB-2015 LC028235 Leishmania sp. LC216356 Leishmania sp. LC216359 Leishmania sp. LC216360 Leishmania sp. LC216361 Leishmania sp. LC216362 Leishmania sp. LC216364 Leishmania sp. LC216366 Leishmania sp. 73

LC216367 Leishmania sp. LC216368 Leishmania sp. LC120689 Leishmania sp. KU680858 Leishmania tarentolae Mokolo Isolate 55

75

LC216369 Leishmania sp. LC216363 Leishmania sp. LC216358 Leishmania sp. LC216357 Leishmania sp. LC028233 Leishmania sp. LC086293 Leishmania tarentolae AJ300480 L. adleri HQ830355 Leishmania sp. 99

HQ830356 Leishmania sp. 79

HQ830357 Leishmania sp. HM130606 Leishmania sp. HM130605 Leishmania sp.

0.02

HM130604 Leishmania sp. HM130602 Leishmania sp. HM130601 Leishmania sp.

Figure 3 Neighbour-Joining taxon ID tree, produced by Mega7, displaying Leishmania tarentolae and affiliates, including Mokolo specimen 55.

Discussion Knowledge of the epidemiology of leishmaniasis is an important measure for the management and planning of disease control. When entomological surveys are combined with epidemiological data, they constitute a major component in the fight against vector-borne diseases. Several recent epidemiological and entomological surveys using molecular techniques have incriminated sand flies as vectors of many human and animal leishmaniases [19–21]. Our study is the first to detect L. donovani sensu lato (s.l.), the causative agent of visceral leishmaniasis, in sand flies from Cameroon. The L. donovani complex comprises the Old World parasites L. donovani sensu stricto (s.s.)

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HM130599 Leishmania sp. HQ830359 Leishmania sp. HQ830360 Leishmania sp.

and L. donovani s.l. including L. infantum [22]. As there is so far no parasitological or clinical confirmation for either of L. donovani s.s. or L. infantum in the Mokolo district, and the ITS1 marker does not allow clear differentiation, we can only speculate about the identity of our isolate. Leishmania donovani s.s. has mainly been reported from India and East Africa [23, 24], while L. infantum MON-1 is primarily found in the Mediterranean basin, but was also reported in the neighbouring Central African Republic and Senegal. Leishmania infantum, although primarily linked to VL, may cause a variety of symptoms including cutaneous manifestations [25]. Similarly, L. donovani s.s. has been implicated in CL in South Asia [26]. It is very likely that some cases of the CL in Mokolo were caused by L. infantum instead of L. major. 5

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However, the distribution of VL in western sub-Saharan Africa is generally poorly known as just a few sporadic outbreaks have been observed [27–29]. In Cameroon, the VL focus described to date is further north of Mokolo, that is Kousseri [11, 30, 31] about 300 km away. The detection of L. donovani s.l. in sand flies from Mokolo is most likely explained by the recent movement of populations from Kousseri and neighbouring Nigeria and Central African Republic due to political unrest in the Sahel region. Conflicts and other causes of migration have been reported to influence the spread of diseases including leishmaniasis [32]. Outbreaks and epidemics of leishmaniasis are reported due to movement of populations bringing infected individuals to susceptible vectors or exposing na€ıve individuals to infected vectors [33]. Recent evidence of the impact of political instabilities on the incidence of leishmaniasis has been reported in Sudan and Middle East [34, 35]. Consequently, with the report of L. donovani s.l. in sand flies from Mokolo, a focus of CL due to L. major [9, 10], it is strongly recommended that studies should be conducted to evaluate the presence of L. donovani s.l. in the human population, followed by control measures. Such attempts would limit the occurrence of VL outbreaks in this CL focus. Such outbreaks could be potentially worsened in human subjects previously exposed to L. major in this focus as described in laboratory animals [36]. Nonetheless, VL as well as CL is serious opportunistic coinfections in HIV patients [10, 37]. Results of our study revealed for the first time the presence of Leishmania tarentolae (or a close relative), a lizard parasite, in three species of Sergentomyia from peri-urban (Se. antennata s.l.) and sylvatic areas (Se. distincta, Se. affinis vorax) in Cameroon. The reason for designating the three isolates as ‘tarentolae-like’ was (i) the high sequence homology with L. (Sauroleishmania) tarentolae of up to 98% (published GenBank accession nos. KU680858 and LC086293) and (ii) the equally high similarity to strain 220 from Portugal (accession no. LC031456) that had been published under the name L. tarentolae-like [38]. The report of lizard parasite in our study is not surprising, as the Mokolo region is a hilly and rocky savannah with climatic conditions favouring the proliferation of many species of lizards. Our data equally confirm the recognised role of Sergentomyia spp. in the transmission of reptile Leishmania [38–40] although Sergentomyia spp. has not been incriminated in the transmission of human Leishmania even experimentally [41]. However, it remains to be investigated whether the Mokolo isolate is indeed identical to L. tarentolae. Our three specimens of L. tarentolae were 100% identical and showed highest 6

similarity to ‘Leishmania spp.’ from Europe, which is yet to be assigned to a Linnean species. Despite earlier lack of evidence of the role of Sergentomyia species in the transmission of human leishmaniasis, some studies have reported the presence of human Leishmania DNA in Sergentomyia specimens [42–44]. The number and diversity of Sergentomyia spp. are 97% and 99% of total sand flies, respectively, [12] increasing their potential to be involved in disease transmission. The confirmation of these percentages in our study in a CL endemic focus, combined with the fact that at least one local species fed on humans as shown in our results, highlights the need of further studies to confirm the role of Sergentomyia in human leishmaniasis transmission. Acknowledgements The authors wish to thank the Kuvin Foundation for funding Aime Tateng Ngouateu’s fellowship at the Kuvin Centre, Hadassah Medical School, Jerusalem, Israel, and Professor Warburg’s Laboratory members for their technical support. The field work was funded by the Volkswagen Foundation, Hannover, Germany. We are also very grateful to the families/communities and their chiefs in the Mokolo area for their assistance and consent to work in their compounds. The work described here formed part of a thesis approved by the University of Dschang (Dschang, Cameroon) for a PhD degree to be awarded to ATA. References 1. WHO Expert Committee on the Control of the Leishmaniases & World Health Organization. Control of the leishmaniases: report of a meeting of the WHO Expert Commitee on the Control of Leishmaniases, Geneva, 22–26 March 2010, Geneva 2010: xiii, 186 pp. 2. Alvar J, Yactayo S, Bern C. Leishmaniasis and poverty. Trends Parasitol 2006: 22: 552–557. 3. Tournier E. Note sur un cas de kala-azar infantile observe au Gabon. Bull Soc Pathol Exot Filiales 1920: 13: 175– 1764. 4. Agwale SM, Dondji B, Okolo C, Duhlinska DD. Clinical and parasitological prevalence of leishmaniasis in school children in Keana, Awe L.G.C. of Plateau State, Nigeria. Mem Inst Oswaldo Cruz 1993: 88: 347. 5. Ikeh E, Ajayi J, Bello C. Epidemiology of cutaneous leishmaniasis in Nigeria: a preliminary communication. Trop Doct 1994: 24: 84–85. 6. Kassa-Kalembo E, Kobangue L, Huerre M, Morvan J. First cases of imported cutaneous leishmaniasis in Bangui Central African Republic: efficacy of metronidazole. Med Trop 2003: 63: 597–600.

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Corresponding Author Blaise Dondji, Laboratory of Cellular Immunology and Parasitology, Central Washington University, 400 E. University Way, Ellensburg, WA 98926, USA. E-mail: [email protected]

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