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Natural infection of Ctenodactylus gundi by Leishmania major in Tunisia
Accepted Manuscript Title: Natural infection of Ctenodactylus gundi by Leishmania major in Tunisia Authors: Wissem Ghawar, Jih`ene Bettaieb, Sadok Salem, Mohammed-Ali Snoussi, Kaouther Jaouadi, Rihab Yazidi, Afif Ben-Salah PII: DOI: Reference:
S0001-706X(17)30015-3 https://doi.org/10.1016/j.actatropica.2017.09.022 ACTROP 4450
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
6-1-2017 6-5-2017 25-9-2017
Please cite this article as: Ghawar, Wissem, Bettaieb, Jih`ene, Salem, Sadok, Snoussi, Mohammed-Ali, Jaouadi, Kaouther, Yazidi, Rihab, Ben-Salah, Afif, Natural infection of Ctenodactylus gundi by Leishmania major in Tunisia.Acta Tropica https://doi.org/10.1016/j.actatropica.2017.09.022 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Natural infection of Ctenodactylus gundi by Leishmania major in Tunisia.
Wissem Ghawara,b*, Jihène Bettaieba,b,c, Sadok Salema,b, Mohammed-Ali Snoussia,b, Kaouther Jaouadia,b, Rihab Yazidia,b, and Afif Ben-Salaha,b,c,d.
a
Department of Medical Epidemiology, Institut Pasteur de Tunis, 13, Place Pasteur, BP 74
1002, Tunis-Belvédère, Tunisia. b
Laboratory of Transmission, Control and Immunobiology of Infections (LR11IPT02),
Institut Pasteur de Tunis, 13, Place Pasteur, BP 74 1002, Tunis-Belvédère, Tunisia. c
Faculty of Medicine of Tunis, University Tunis El Manar, Tunis, Tunisia.
d
Department of Family and Community Medicine, College of Medicine and Medical
Sciences (CMMS), Arabian Gulf University (AGU), Manama, Bahraïn.
*Corresponding Author: PhD. WissemGhawar, Department of Medical Epidemiology, Pasteur Institute of Tunis, 13, Place Pasteur, BP 74 1002, Tunis-Belvédère, Tunisia; email: [email protected]; Telephone: (+216) 71 843 755; Fax: (+216) 71 791 833.
Email addresses for all authors: Wissem Ghawar ([email protected]), Jihène Bettaieb ([email protected]), Sadok Salem ([email protected]), Mohammed-Ali Snoussi ([email protected]), Kaouther Jaouadi ([email protected]), Rihab Yazidi ([email protected]) and Afif Ben-Salah ([email protected]).
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Graphical abstract
Summary: The study demonstrated the detection of Leishmania parasites in Ctenodactylus gundi from two endemic area of cutaneous leishmaniasis in Tunisia.
Pictogram:
Highlights
Confirmed Leishmania infection in Ctenodactylus gundi.
We detect Leishmania with clinical, parasitological and molecular tools.
The molecular methods used allow identifying the species involved as Leishmania major and Leishmania tropica.
Ctenodactylus gundi is a potential reservoir of Leishmania major in Tunisia.
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Abstract Incriminating new rodent species, as reservoir hosts of Leishmania parasites is crucial for understanding the transmission cycle of cutaneous leishmaniasis in Tunisia. Ctenodactylus (C.) gundi was previously described as extremely abundant in all Tunisian Leishmania (L.) tropica foci in south Tunisia besides its presence in L. major endemic area. The aim of this study was to detect Leishmania species parasites among C. gundi in two endemic regions in Tunisia: Sidi Bouzid and Tataouine. Total DNA was isolated from the spleens and the livers of 92 C. gundi. Leishmaniasis clinical manifestations were detected among 11 rodents (12%). Leishmania parasites were detected in 30 (32.6%) rodents using direct exam method. Leishmania DNA was detected in 40 (43.5%) C. gundi by combining results among spleens and livers using ITS1-PCR. Positive samples were confirmed to be L. major except for only one specimen which was L. tropica. These results demonstrated, for the first time, the high natural infection rate of C. gundi with L. major parasites in Tunisia. Hence, C. gundi should be considered as potential reservoir host of Leishmania parasites causing cutaneous leishmaniasis in Tunisia.
Keywords: Ctenodactylus gundi, Leishmania major, Leishmania tropica, rodent reservoir, cutaneous leishmaniasis, Tunisia.
1. Introduction Cutaneous leishmaniasis (CL), caused by three species of Leishmania parasites: Leishmania (L.) major, L. tropica (Wright, 1903) and L. infantum (Nicolle, 1908), represent the most important and endemic disease in Tunisia (Ben-Ismail and Ben Rachid, 1989). Leishmania major with the single zymodeme MON-25 is the principal agent causing zoonotic CL in Tunisia with more than 90% of the registered cases of leishmaniasis (Aoun and
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Bouratbine, 2014). Since 2002, zoonotic CL caused by L. major was endemic into the whole center and southern parts of the country, and occurs in 15 out of the 24 governorates (Salah et al., 2007). The cyclic epidemics of the disease depend on climatic factors (Toumi et al., 2012) with an annual incidence ranges from 2000 to 10000 cases (Bettaieb et al., 2014). This incidence presents a close spatial association with the abundance of Phlebotomus papatasi, the proven vector (Chelbi et al., 2009). However, L. major has been recently detected from other species of phlebotomine sand flies in Tunisia: Sergentomyia minuta (Jaouadi et al., 2015) and Sergentomyia clydei (Ayari et al., 2016). Wild rodents such as Psammomys (P.) obesus, Meriones (M.) shawi and Meriones libycus were incriminated as the reservoir hosts of L. major parasites (Ben-Ismail et al., 1987a; Ben-Ismail et al., 1987b; Ben-Ismail et al., 1989; Fichet-Calvet et al., 2003; Ghawar et al., 2011b). However, these parasites were recently detected in other small mammals such as Mustela nivalis and Atelerix algirus; but their role as reservoir hosts was not confirmed (Chemkhi et al., 2015; Ghawar et al., 2011a). Sidi Bouzid governorate, in central Tunisia, is the most important foci of this disease with 2530% of the Zoonotic CL reported cases annually (Salah et al., 2007). Chronic CL caused by L. killicki (belonging to the L. tropica complex) (Chaara et al., 2015; Pratlong et al., 2009) and transmitted by Phlebotomus sergenti (Tabbabi et al., 2011a) was usually recognized as an anthroponotic infection. This disease was firstly described in Tataouine governorate, in south Tunisia and form mixed zones with the zoonotic CL (Aoun et al., 2015). Recently, L. tropica parasites were detected, using molecular tools among small samples of wild animals such as: Ctenodactylus (C.) gundi, Rock Hyraxes (Procavia capensis), Golden Jackals (Canis aureus) and Red Foxes (Vulpes vulpes) around the Old World (Bousslimi et al., 2012; Jaouadi et al., 2011; Talmi-Frank et al., 2010a; Talmi-Frank et al., 2010b). The implication of these animals in the sylvatic cycle of L. tropica may support the presence of a zoonotic transmission cycle and could influence disease emergence. In
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Tunisia, the North African rock-dwelling rodent: C. gundi habits in the mountainous area of Tataouine (the original focus of L. tropica) as well as in all emerging Tunisian foci of CL caused by L. tropica (Ben-Ismail and Ben Rachid, 1989; Bouratbine et al., 2005; Haouas et al., 2005). Thus supports the implication of this rodent species as possible reservoir in the L. tropica life cycle in Tunisia. The main purpose of the present study was to investigate the different species involved in the natural Leishmania infection of C. gundi collected from two CL endemic regions in Tunisia: Sidi Bouzid and Tataouine.
2. Materials and methods 2.1. Study site and rodent trapping Rodents were trapped from three study sites in Sidi Bouzid and Tataouine regions, in central and south Tunisia, respectively. Ctenodactylus gundi were trapped in Khbina (average altitude 231 m; N35 1215.96 E9 42.3824) in Sidi Bouzid Governorate; Guermessa (average altitude 297 m; N32 5931.56 E10 15.138) and Mdhila (average altitude 374 m; N33 0238.46 E10 22.1422) in Tataouine Governorate. Ctenodactylus gundi were abundant in the rockymountains of the three study sites in addition to the presence of P. obesus and M. shawi as previously described for Khbina (Sidi Bouzid) (Ghawar et al., 2011b). Trappings were done by placing pincer traps recovered by sands, in areas where rodents have been spotted by vision or by the presence of their wastes. Three expeditions in which we have realized eight field trips (one week for each one) were realized: two in March 2010 in which rodent from Sidi Bouzid were collected; two between May and July 2011, and four between April and May 2013 in which rodents from Tataouine were captured with an equal alternation between the two study sites. 2.2. Clinical manifestations of Leishmania
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Each captured rodent was identified, sexed, and searched for cutaneous lesions in the different parts of the body. Clinical signs of Leishmania infection were assessed through skin examination. Signs included depilation, hyper-pigmentation of the higher edge of the ear, the presence of small nodules and/or partial destruction of organs. 2.3. Detection of Leishmania parasites by parasitological methods . Each captured rodent was anesthetized by Ether, weighted to the nearest 0.1 g on a pan balance, and then sacrificed by cardiac exsanguination without producing any stress to the animal. External measurements (the head, the body, the tail, the hind feet, and the ear) were recorded. Samples of heart, liver, kidney, spleen, lung and brain were taken, then frozen, and stored in liquid nitrogen for molecular analysis. Both ears of each rodent caught were removed and macerated together in physiological saline. A subsample of the produced solution was smeared in a slide, stained by May-Grunwald-Giemsa, and observed at 1000X for direct examination of Leishmania amastigotes. The rest of the solution was placed in Novy-MacNeal-Nicolle (NNN) medium for parasites culture (Ghawar et al., 2011b). 2.4. DNA extraction and parasite detection by PCR-Restriction Fragment Length Polymorphism (RFLP) DNA from each rodent spleen and liver was extracted using the QIAamp® DNA Mini Kit (QIAgen, Germany). The presence of the Leishmania DNA was tested by targeting the ribosomal internal transcribed spacer 1 (ITS1) using the primers LITSR and L5.8S followed by a Restriction Fragment Length Polymorphism (RFLP) analysis. ITS1-PCR products were digested by HaeIII restriction enzyme using previously described protocol (Schonian et al., 2003). The following references strains were used as positive controls: L. major MON-25 MHOM/TN2009/S600, L. killicki MON-8 MHOM/TN/2011/MX and L. infantum MON-1 MHOM/TN/94/LV50.
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2.5. Data analysis Chi2 as well as Fisher exact tests permitted to compare association of categorical variables. STATA software version11 was used to carry out all statistical analysis (Stata Corporation). 2.6. Ethical statement All animal experimentations comply with institutional, national and international guidelines. The study and rodents handling protocols were approved by the ethics committee at Pasteur Institute of Tunis (5P50AI074178-04).
3. Results A total of 92 C. gundi were collected. Twenty of them (21.7%) were captured from Khbina in Sidi Bouzid governorate in 2010 and 72 (78.3%) were captured from Tataouine governorate in 2011 and 2013 subdivided into two sites: 21 (22.8%) from Guermessa and 51 (55.4%) from Mdhila. Among the captured rodents, we found 45 (48.9%) males and 47 (51.1%) females. 3.1. Clinical manifestations The clinical examination of all captured rodents showed various aspects of skin lesions. When we consider any clinical sign among the whole study sample (n = 92), disease was detected among 11 rodents (12%). Depilation, small nodules and hyper-pigmentation were the clinical signs observed in both ears (72.72%; n = 8) and the back (36.36%; n = 4) (Figure 1) with a specimen showing a combined infection in these parts of body.
3.2. Infection prevalence Leishmania infection prevalence among C. gundi was 32.6% (n = 30) using the direct examination of ears homogenates (Figure 2) and 43.5% (n = 40) by combining result for both
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tested organs using the ITS1-PCR method. Unfortunately, we were unable to isolate Leishmania strains from these rodents by culture method due to contamination. The overall infection rate was 57.6% (n = 53), by combining the results of Giemsa stained smears and ITS1-PCR.
Table 1 details the distribution of the infection rate and the identified positive samples among C. gundi according to the used diagnostic method by sampling site location.
The proportion of C. gundi with leishmaniasis lesions among those who showed a positive sample by any diagnostic method (direct exam and/or ITS1-PCR) was 13.2% (7/53). Thus, asymptomatic infection among positive samples was 86.8% (46/53). Moreover, five specimens were positives by the two used diagnostic methods and showing a clinical manifestation. Relationship between positivity rates determined by each diagnostic method and the clinical exam is presented in Figure 3.
Additionally, Leishmania infection, determined by the molecular method, varied among C. gundi according to the tested organs. In fact, Leishmania DNA were detected in 25 versus 17 out of 92 C. gundi, yielding a positivity rate of 27.2% versus 18.5% (p = NS) among spleens and livers, respectively; with only two positive rodent samples showing infection in both organs. Furthermore, RFLP analysis allowed to identify 29 (72.5%) from the 40 positives samples by ITS1-PCR (Figure 4): 15/25 (60%) among spleens identified as L. major and 14/17 (82.5%) among livers were identified as L. major in the exception of one sample identified as L. tropica.
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4. Discussion The present study is to our knowledge the first one in North Africa assessing the importance of Leishmania infection and its clinical manifestation among C. gundi using a large sample size captured in two endemic areas of CL. Our finding represents a first report confirming the high rate and the symptomatic natural Leishmania infection of C. gundi in North Africa. In our study, the prevalence reached 32% (30 out of 92) by direct examination and 43.5% (40 out of 92) by ITS1-PCR method which agrees with rates previously described on a small sample size (six and thirteen) of this species of rodents (Bousslimi et al., 2012; Jaouadi et al., 2011). Moreover, infection prevalence determined by the direct exam in C. gundi was much higher than those described among P. obesus (ranging from 13% to 29%) and M. shawi (ranging from 10% to 16%) in Tunisia (Ben-Ismail et al., 1987a; Ben-Ismail et al., 1989; Fichet-Calvet et al., 2003; Ghawar et al., 2011b). Unfortunately, no study recorder the Leishmania infection rate using molecular tools among the proven rodent reservoir hosts in Tunisia. The overall prevalence among C. gundi (57.6%) showed in this study, using parasitological and molecular tools, was higher when compared to those observed for the classic rodent reservoir hosts of L. major in Tunisia: P. obesus and M. shawi, using parasitological and serological tools (Ben-Ismail et al., 1989; Ben-Ismail R., 1987a; Fichet-Calvet et al., 2003; Ghawar et al., 2011b). It is well known that some Leishmania species causing CL could migrate to visceral organs of reservoir hosts (Davami et al., 2014; Mohebali et al., 2004; Parhizkari et al., 2011). Our findings confirm this fact, and more interestingly, Leishmania infection by ITS1-PCR method varied among C. gundi according to the target organ. It was 27.2% versus 18.5% among spleens and livers, respectively, with a low spleen-liver co-infection. In agreement with
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previous studies (Bensoussan et al., 2006; Bousslimi et al., 2012; Faiman et al., 2013), we were unable to identify 13 ITS1-PCR positive samples out of 42 using the RFLP analysis. This can be explained by the fact that PCR-ITS method can provide DNA amplification of other Trypanosoma species which are widely frequent in rodents and other mammals reservoir hosts of Leishmania parasites (Desquesnes et al., 2013; Rjeibi et al., 2015). On the other hand, RFLP analysis allowed us to identify 15/25 (60%) versus 13/17 (76.5%) positive samples as L. major among spleens and livers, respectively. While, only one sample was identified as L. tropica among livers. Disagreement between this result and those previously described for this species of rodents with only one detection of L. major and few detection of L. tropica (Bousslimi et al., 2012; Jaouadi et al., 2011) might be explained either by the sampling site location or the inability of the RFLP analysis to identify all positive samples. Furthermore, the C. gundi infected with L. tropica was surprisingly trapped in the well known L. major endemic area of Sidi Bouzid. This finding supports the fact that the geographical distribution of L. tropica parasites in Tunisia can be much wider than described. CL due to L. major may cause cutaneous lesions among established rodent reservoir hosts of Leishmania parasites (Ben-Ismail et al., 1987a; Ghawar et al., 2011b). Preferred locations of Leishmania lesions among rodent reservoir hosts were ears and tails (Ghawar et al., 2011b). This is not valid for C. gundi, where lesions were found only in ears and backs, possibly because C. gundi has a very short tail (Macdonald, 1984). Interestingly, C. gundi showed a high rate (86.8%) of asymptomatic leishmaniasis infection compared with 40% for the rodent reservoir hosts (P. obesus and M. shawi) of L. major in Tunisia (Ghawar et al., 2011b). Early dissemination of Leishmania parasites to the spleen was also reported in asymptomatic animals (Jaouadi et al., 2011; Schilling and Glaichenhaus, 2001).
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Our findings can be explained by the fact that L. major is endemic in the two studies regions. Sidi Bouzid governorate is an important endemic focus of CL with an average incidence rate of 669.7 cases/100000 inhabitants (Salah et al., 2007). However, Tataouine governorate is a mixed focus of CL with an average incidence of 50–150 cases per year with dominance of the zoonotic versus the chronic CL (Aoun et al., 2015; Bousslimi et al., 2010). Prevalence of this disease has a close temporal association with the abundance of Phlebotomus papatasi (Chelbi et al., 2007). In addition, C. gundi appears numerous and widespread with a geographical range from the center to the south in Tunisia (Honigs and Greven, 2003). This species was considered to be the most frequent mammal in the southern part of the country (Kock and Schomber, 1961). They live in small family groups. Each group spends a lot of time in their main shelter and all members of the family stay there overnight. Besides, they have temporary shelters used during their movements in the day (Gouat, 1989; Gouat and Gouat, 1983). These ecological parameters of C. gundi were in agreement with the criteria presumed by Ashford (Ashford, 1996) to define a reservoir host. But according to the WHO (WHO, 1990), reservoir hosts should be able to maintain the parasites and present a strong host-fly contact. In this context, we know that C. gundi defecate and urinate in their main shelter (Honigs and Greven, 2003) which present a favorable biotope for the sandflies. In fact, as previously described, a variety of sandflies vectors of Leishmania parasites causing CL were present in C. gundi biotope (Tabbabi et al., 2011b). Actually, during a study realized in Tataouine, ten species of sandflies were recorded in this rodent habitat. Proven vectors of the Zoonotic CL were predominant with a relative abundance of 0.23 and 0.09 for Sergentomiya minuta and Phlebotomus papatasi, respectively; and a relative abundance of 0.05 for P. sergenti, the proven vector for the Chronic CL (Tabbabi et al., 2011b).
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All these parameters implicit the putative role of C. gundi as reservoir host of two Leishmania species parasites in Tunisia but the study of the close interaction between this rodent and the proven sandflies vectors remains a priority to confirm that.
5. Conclusions C. gundi was found to be naturally infected by two species of Leishmania parasites (L. major and L. tropica) with the predominance of L. major, in endemic areas for both human leishmaniasis. Thus allow to suggest that C. gundi could play a major role in their epidemiological cycles and should be considered as possible reservoir host of Leishmania parasites causing cutaneous leishmaniasis in Tunisia.
Competing interests The authors declare that they have no competing interests. Funding This work was supported by the Laboratory of Transmission, Control and Immunobiology of Infections (LR11IPT02), Institut Pasteur de Tunis; and the Ministry of Higher Education and Research of Tunisia.
Acknowledgements We are grateful to the staff of the Regional Directorate of Public Health of Tataouine and Sidi Bouzid for their support to the achievement of the field work.
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Psammomys obesus and Meriones shawi: reservoirs of zoonotic cutaneous leishmaniasis in Sidi Bouzid (central Tunisia). Vector Borne Zoonotic Dis 11, 1561-1568. Gouat, P., 1989. b. Communal gestion of space and defense of territory by the gundi, Ctenodactylus gundi. Mammalia 53, 477. Gouat, P., Gouat, J., 1983. L’habitat du goundi (Ctenodactylus gundi) dans le massif de l’Aures (Algérie). Mammalia 47, 507-518. Haouas, N., Chargui, N., Chaker, E., Ben Said, M., Babba, H., Belhadj, S., Kallel, K., Pratlong, F., Dedet, J.P., Mezhoud, H., Azaiez, R., 2005. Anthroponotic cutaneous leishmaniasis in Tunisia: presence of Leishmania killicki outside its original focus of Tataouine. Trans R Soc Trop Med Hyg 99, 499-501. Honigs, S., Greven, H., 2003. Biology of the gundi, Ctenodactylus gundi (Rodentia: Ctenodactylidae), and its occurrence in Tunisia. Kaupia 12, 43-55. Jaouadi, K., Ghawar, W., Salem, S., Gharbi, M., Bettaieb, J., Yazidi, R., Harrabi, M., Hamarsheh, O., Ben Salah, A., 2015. First report of naturally infected Sergentomyia minuta with Leishmania major in Tunisia. Parasit Vectors 8, 649. Jaouadi, K., Haouas, N., Chaara, D., Gorcii, M., Chargui, N., Augot, D., Pratlong, F., Dedet, J.P., Ettlijani, S., Mezhoud, H., Babba, H., 2011. First detection of Leishmania killicki (Kinetoplastida, Trypanosomatidae) in Ctenodactylus gundi (Rodentia, Ctenodactylidae), a possible reservoir of human cutaneous leishmaniasis in Tunisia. Parasit Vectors 4, 159. Kock, D., Schomber, H., 1961. Beitrag zur Kenntnis der Lebens-und Verhaltensweise des Gundi, Ctenodactylus gundi (Rothmann, 1776). Säugetierkundliche Mitteilungen 92, 165166. Macdonald, D., 1984. The encyclopedia of mammals. New York, Facts on File Publications. Coverage of all mammalian orders and families, enhanced with fine photographs and color artwork.
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Mohebali, M., Javadian, E., Yaghoobi-Ershadi, M.R., Akhavan, A.A., Hajjaran, H., Abaei, M.R., 2004. Characterization of Leishmania infection in rodents from endemic areas of the Islamic Republic of Iran. East Mediterr Health J 10, 591-599. Parhizkari, M., Motazedian, M.H., Asqari, Q., Mehrabani, D., 2011. The PCR-based detection of Leishmania major in Mus musculus and other rodents caught in southern Iran: a guide to sample selection. Ann Trop Med Parasitol 105, 319-323. Pratlong, F., Dereure, J., Ravel, C., Lami, P., Balard, Y., Serres, G., Lanotte, G., Rioux, J.A., Dedet, J.P., 2009. Geographical distribution and epidemiological features of Old World cutaneous leishmaniasis foci, based on the isoenzyme analysis of 1048 strains. Trop Med Int Health 14, 1071-1085. Rjeibi, M.R., Ben Hamida, T., Dalgatova, Z., Mahjoub, T., Rejeb, A., Dridi, W., Gharbi, M., 2015. First report of surra (Trypanosoma evansi infection) in a Tunisian dog. Parasite 22, 3. Salah, A.B., Kamarianakis, Y., Chlif, S., Alaya, N.B., Prastacos, P., 2007. Zoonotic cutaneous leishmaniasis in central Tunisia: spatio temporal dynamics. Int J Epidemiol 36, 991-1000. Schilling, S., Glaichenhaus, N., 2001. T cells that react to the immunodominant Leishmania major LACK antigen prevent early dissemination of the parasite in susceptible BALB/c mice. Infect Immun 69, 1212-1214. Schonian, G., Nasereddin, A., Dinse, N., Schweynoch, C., Schallig, H.D., Presber, W., Jaffe, C.L., 2003. PCR diagnosis and characterization of Leishmania in local and imported clinical samples. Diagn Microbiol Infect Dis 47, 349-358. Tabbabi, A., Bousslimi, N., Rhim, A., Aoun, K., Bouratbine, A., 2011a. First report on natural infection of Phlebotomus sergenti with Leishmania promastigotes in the cutaneous leishmaniasis focus in southeastern Tunisia. Am J Trop Med Hyg 85, 646-647.
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Tabbabi, A., Ghrab, J., Aoun, K., Ready, P.D., Bouratbine, A., 2011b. Habitats of the sandfly vectors of Leishmania tropica and L. major in a mixed focus of cutaneous leishmaniasis in southeast Tunisia. Acta Trop 119, 131-137. Talmi-Frank, D., Jaffe, C.L., Nasereddin, A., Warburg, A., King, R., Svobodova, M., Peleg, O., Baneth, G., 2010a. Leishmania tropica in rock hyraxes (Procavia capensis) in a focus of human cutaneous leishmaniasis. Am J Trop Med Hyg 82, 814-818. Talmi-Frank, D., Kedem-Vaanunu, N., King, R., Bar-Gal, G.K., Edery, N., Jaffe, C.L., Baneth, G., 2010b. Leishmania tropica infection in golden jackals and red foxes, Israel. Emerg Infect Dis 16, 1973-1975. Toumi, A., Chlif, S., Bettaieb, J., Ben Alaya, N., Boukthir, A., Ahmadi, Z.E., Ben Salah, A., 2012. Temporal dynamics and impact of climate factors on the incidence of zoonotic cutaneous leishmaniasis in central Tunisia. PLoS Negl Trop Dis 6, e1633. WHO, 1990. Control of leishmaniasis. Report of WHO Expert Committee, Geneva, Switzerland.
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Figure 1. Clinical manifestation as hyper-pigmentation observed among Ctenodactylus gundi on the ear (a) and on the back (b).
Figure 2. Leishmania amastigotes in a Giemsa stained smears from the ear of Ctenodactylus gundi under oil immersion (X1000).
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Figure 3. Infected Ctenodactylus gundi by ITS1-PCR and direct exam, and their relation to the clinical manifestation.
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Figure 4. RFLP products of the amplified ITS1 fragment from the spleens and the livers of Ctenodactylus gundi. M1 and M2: 100 bp and 1 Kb size markers (Invitrogen®); Lm, Lt and Li: respectively L. major (two fragments of 132-bp and 206-bp), L. tropica (three fragments of 188-bp, 57-bp and 26-bp respectively) and L. infantum (three fragments of 187-bp, 72-bp and 55-bp respectively) references strains. Positive samples from the spleens: G8, G12, G14, G19 and G21 (G12 not identified by RFLP). Positive samples from the livers: G23, G24, G25, G26, G33 (G23 not identified by RFLP). A: positive sample by ITS1-PCR.
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Table 1. Prevalence of Leishmania infection among Ctenodactylus gundi according to the diagnostic method by capture sites. Governorates
Sidi Bouzid
Tataouine
Khbina
Guermessa
Mdhila
Total
P
(20)
(21)
(51)
(92)
value
Clinical exam (n, %)
1 (9.1)
6 (54.5)
4 (36.4)
11 (12)
NS
Direct exam (n, %)
3 (10)
6 (20)
21 (70)
30 (32.6)
NS
Total ITS1-PCR
6 (30)
8 (38.1)
26 (51)
40 (43.5)
NS
ITS1-PCR among spleens (n, %)
2 (8)
7 (28)
16 (64)
25 (27.2)
NS
ITS1-PCR among livers (n, %)
4 (23.5)
1 (5.9)
12 (70.6)
17 (18.5)
NS
Delegations (n)
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S0001-706X(17)30015-3 https://doi.org/10.1016/j.actatropica.2017.09.022 ACTROP 4450
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
6-1-2017 6-5-2017 25-9-2017
Please cite this article as: Ghawar, Wissem, Bettaieb, Jih`ene, Salem, Sadok, Snoussi, Mohammed-Ali, Jaouadi, Kaouther, Yazidi, Rihab, Ben-Salah, Afif, Natural infection of Ctenodactylus gundi by Leishmania major in Tunisia.Acta Tropica https://doi.org/10.1016/j.actatropica.2017.09.022 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Natural infection of Ctenodactylus gundi by Leishmania major in Tunisia.
Wissem Ghawara,b*, Jihène Bettaieba,b,c, Sadok Salema,b, Mohammed-Ali Snoussia,b, Kaouther Jaouadia,b, Rihab Yazidia,b, and Afif Ben-Salaha,b,c,d.
a
Department of Medical Epidemiology, Institut Pasteur de Tunis, 13, Place Pasteur, BP 74
1002, Tunis-Belvédère, Tunisia. b
Laboratory of Transmission, Control and Immunobiology of Infections (LR11IPT02),
Institut Pasteur de Tunis, 13, Place Pasteur, BP 74 1002, Tunis-Belvédère, Tunisia. c
Faculty of Medicine of Tunis, University Tunis El Manar, Tunis, Tunisia.
d
Department of Family and Community Medicine, College of Medicine and Medical
Sciences (CMMS), Arabian Gulf University (AGU), Manama, Bahraïn.
*Corresponding Author: PhD. WissemGhawar, Department of Medical Epidemiology, Pasteur Institute of Tunis, 13, Place Pasteur, BP 74 1002, Tunis-Belvédère, Tunisia; email: [email protected]; Telephone: (+216) 71 843 755; Fax: (+216) 71 791 833.
Email addresses for all authors: Wissem Ghawar ([email protected]), Jihène Bettaieb ([email protected]), Sadok Salem ([email protected]), Mohammed-Ali Snoussi ([email protected]), Kaouther Jaouadi ([email protected]), Rihab Yazidi ([email protected]) and Afif Ben-Salah ([email protected]).
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Graphical abstract
Summary: The study demonstrated the detection of Leishmania parasites in Ctenodactylus gundi from two endemic area of cutaneous leishmaniasis in Tunisia.
Pictogram:
Highlights
Confirmed Leishmania infection in Ctenodactylus gundi.
We detect Leishmania with clinical, parasitological and molecular tools.
The molecular methods used allow identifying the species involved as Leishmania major and Leishmania tropica.
Ctenodactylus gundi is a potential reservoir of Leishmania major in Tunisia.
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Abstract Incriminating new rodent species, as reservoir hosts of Leishmania parasites is crucial for understanding the transmission cycle of cutaneous leishmaniasis in Tunisia. Ctenodactylus (C.) gundi was previously described as extremely abundant in all Tunisian Leishmania (L.) tropica foci in south Tunisia besides its presence in L. major endemic area. The aim of this study was to detect Leishmania species parasites among C. gundi in two endemic regions in Tunisia: Sidi Bouzid and Tataouine. Total DNA was isolated from the spleens and the livers of 92 C. gundi. Leishmaniasis clinical manifestations were detected among 11 rodents (12%). Leishmania parasites were detected in 30 (32.6%) rodents using direct exam method. Leishmania DNA was detected in 40 (43.5%) C. gundi by combining results among spleens and livers using ITS1-PCR. Positive samples were confirmed to be L. major except for only one specimen which was L. tropica. These results demonstrated, for the first time, the high natural infection rate of C. gundi with L. major parasites in Tunisia. Hence, C. gundi should be considered as potential reservoir host of Leishmania parasites causing cutaneous leishmaniasis in Tunisia.
Keywords: Ctenodactylus gundi, Leishmania major, Leishmania tropica, rodent reservoir, cutaneous leishmaniasis, Tunisia.
1. Introduction Cutaneous leishmaniasis (CL), caused by three species of Leishmania parasites: Leishmania (L.) major, L. tropica (Wright, 1903) and L. infantum (Nicolle, 1908), represent the most important and endemic disease in Tunisia (Ben-Ismail and Ben Rachid, 1989). Leishmania major with the single zymodeme MON-25 is the principal agent causing zoonotic CL in Tunisia with more than 90% of the registered cases of leishmaniasis (Aoun and
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Bouratbine, 2014). Since 2002, zoonotic CL caused by L. major was endemic into the whole center and southern parts of the country, and occurs in 15 out of the 24 governorates (Salah et al., 2007). The cyclic epidemics of the disease depend on climatic factors (Toumi et al., 2012) with an annual incidence ranges from 2000 to 10000 cases (Bettaieb et al., 2014). This incidence presents a close spatial association with the abundance of Phlebotomus papatasi, the proven vector (Chelbi et al., 2009). However, L. major has been recently detected from other species of phlebotomine sand flies in Tunisia: Sergentomyia minuta (Jaouadi et al., 2015) and Sergentomyia clydei (Ayari et al., 2016). Wild rodents such as Psammomys (P.) obesus, Meriones (M.) shawi and Meriones libycus were incriminated as the reservoir hosts of L. major parasites (Ben-Ismail et al., 1987a; Ben-Ismail et al., 1987b; Ben-Ismail et al., 1989; Fichet-Calvet et al., 2003; Ghawar et al., 2011b). However, these parasites were recently detected in other small mammals such as Mustela nivalis and Atelerix algirus; but their role as reservoir hosts was not confirmed (Chemkhi et al., 2015; Ghawar et al., 2011a). Sidi Bouzid governorate, in central Tunisia, is the most important foci of this disease with 2530% of the Zoonotic CL reported cases annually (Salah et al., 2007). Chronic CL caused by L. killicki (belonging to the L. tropica complex) (Chaara et al., 2015; Pratlong et al., 2009) and transmitted by Phlebotomus sergenti (Tabbabi et al., 2011a) was usually recognized as an anthroponotic infection. This disease was firstly described in Tataouine governorate, in south Tunisia and form mixed zones with the zoonotic CL (Aoun et al., 2015). Recently, L. tropica parasites were detected, using molecular tools among small samples of wild animals such as: Ctenodactylus (C.) gundi, Rock Hyraxes (Procavia capensis), Golden Jackals (Canis aureus) and Red Foxes (Vulpes vulpes) around the Old World (Bousslimi et al., 2012; Jaouadi et al., 2011; Talmi-Frank et al., 2010a; Talmi-Frank et al., 2010b). The implication of these animals in the sylvatic cycle of L. tropica may support the presence of a zoonotic transmission cycle and could influence disease emergence. In
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Tunisia, the North African rock-dwelling rodent: C. gundi habits in the mountainous area of Tataouine (the original focus of L. tropica) as well as in all emerging Tunisian foci of CL caused by L. tropica (Ben-Ismail and Ben Rachid, 1989; Bouratbine et al., 2005; Haouas et al., 2005). Thus supports the implication of this rodent species as possible reservoir in the L. tropica life cycle in Tunisia. The main purpose of the present study was to investigate the different species involved in the natural Leishmania infection of C. gundi collected from two CL endemic regions in Tunisia: Sidi Bouzid and Tataouine.
2. Materials and methods 2.1. Study site and rodent trapping Rodents were trapped from three study sites in Sidi Bouzid and Tataouine regions, in central and south Tunisia, respectively. Ctenodactylus gundi were trapped in Khbina (average altitude 231 m; N35 1215.96 E9 42.3824) in Sidi Bouzid Governorate; Guermessa (average altitude 297 m; N32 5931.56 E10 15.138) and Mdhila (average altitude 374 m; N33 0238.46 E10 22.1422) in Tataouine Governorate. Ctenodactylus gundi were abundant in the rockymountains of the three study sites in addition to the presence of P. obesus and M. shawi as previously described for Khbina (Sidi Bouzid) (Ghawar et al., 2011b). Trappings were done by placing pincer traps recovered by sands, in areas where rodents have been spotted by vision or by the presence of their wastes. Three expeditions in which we have realized eight field trips (one week for each one) were realized: two in March 2010 in which rodent from Sidi Bouzid were collected; two between May and July 2011, and four between April and May 2013 in which rodents from Tataouine were captured with an equal alternation between the two study sites. 2.2. Clinical manifestations of Leishmania
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Each captured rodent was identified, sexed, and searched for cutaneous lesions in the different parts of the body. Clinical signs of Leishmania infection were assessed through skin examination. Signs included depilation, hyper-pigmentation of the higher edge of the ear, the presence of small nodules and/or partial destruction of organs. 2.3. Detection of Leishmania parasites by parasitological methods . Each captured rodent was anesthetized by Ether, weighted to the nearest 0.1 g on a pan balance, and then sacrificed by cardiac exsanguination without producing any stress to the animal. External measurements (the head, the body, the tail, the hind feet, and the ear) were recorded. Samples of heart, liver, kidney, spleen, lung and brain were taken, then frozen, and stored in liquid nitrogen for molecular analysis. Both ears of each rodent caught were removed and macerated together in physiological saline. A subsample of the produced solution was smeared in a slide, stained by May-Grunwald-Giemsa, and observed at 1000X for direct examination of Leishmania amastigotes. The rest of the solution was placed in Novy-MacNeal-Nicolle (NNN) medium for parasites culture (Ghawar et al., 2011b). 2.4. DNA extraction and parasite detection by PCR-Restriction Fragment Length Polymorphism (RFLP) DNA from each rodent spleen and liver was extracted using the QIAamp® DNA Mini Kit (QIAgen, Germany). The presence of the Leishmania DNA was tested by targeting the ribosomal internal transcribed spacer 1 (ITS1) using the primers LITSR and L5.8S followed by a Restriction Fragment Length Polymorphism (RFLP) analysis. ITS1-PCR products were digested by HaeIII restriction enzyme using previously described protocol (Schonian et al., 2003). The following references strains were used as positive controls: L. major MON-25 MHOM/TN2009/S600, L. killicki MON-8 MHOM/TN/2011/MX and L. infantum MON-1 MHOM/TN/94/LV50.
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2.5. Data analysis Chi2 as well as Fisher exact tests permitted to compare association of categorical variables. STATA software version11 was used to carry out all statistical analysis (Stata Corporation). 2.6. Ethical statement All animal experimentations comply with institutional, national and international guidelines. The study and rodents handling protocols were approved by the ethics committee at Pasteur Institute of Tunis (5P50AI074178-04).
3. Results A total of 92 C. gundi were collected. Twenty of them (21.7%) were captured from Khbina in Sidi Bouzid governorate in 2010 and 72 (78.3%) were captured from Tataouine governorate in 2011 and 2013 subdivided into two sites: 21 (22.8%) from Guermessa and 51 (55.4%) from Mdhila. Among the captured rodents, we found 45 (48.9%) males and 47 (51.1%) females. 3.1. Clinical manifestations The clinical examination of all captured rodents showed various aspects of skin lesions. When we consider any clinical sign among the whole study sample (n = 92), disease was detected among 11 rodents (12%). Depilation, small nodules and hyper-pigmentation were the clinical signs observed in both ears (72.72%; n = 8) and the back (36.36%; n = 4) (Figure 1) with a specimen showing a combined infection in these parts of body.
3.2. Infection prevalence Leishmania infection prevalence among C. gundi was 32.6% (n = 30) using the direct examination of ears homogenates (Figure 2) and 43.5% (n = 40) by combining result for both
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tested organs using the ITS1-PCR method. Unfortunately, we were unable to isolate Leishmania strains from these rodents by culture method due to contamination. The overall infection rate was 57.6% (n = 53), by combining the results of Giemsa stained smears and ITS1-PCR.
Table 1 details the distribution of the infection rate and the identified positive samples among C. gundi according to the used diagnostic method by sampling site location.
The proportion of C. gundi with leishmaniasis lesions among those who showed a positive sample by any diagnostic method (direct exam and/or ITS1-PCR) was 13.2% (7/53). Thus, asymptomatic infection among positive samples was 86.8% (46/53). Moreover, five specimens were positives by the two used diagnostic methods and showing a clinical manifestation. Relationship between positivity rates determined by each diagnostic method and the clinical exam is presented in Figure 3.
Additionally, Leishmania infection, determined by the molecular method, varied among C. gundi according to the tested organs. In fact, Leishmania DNA were detected in 25 versus 17 out of 92 C. gundi, yielding a positivity rate of 27.2% versus 18.5% (p = NS) among spleens and livers, respectively; with only two positive rodent samples showing infection in both organs. Furthermore, RFLP analysis allowed to identify 29 (72.5%) from the 40 positives samples by ITS1-PCR (Figure 4): 15/25 (60%) among spleens identified as L. major and 14/17 (82.5%) among livers were identified as L. major in the exception of one sample identified as L. tropica.
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4. Discussion The present study is to our knowledge the first one in North Africa assessing the importance of Leishmania infection and its clinical manifestation among C. gundi using a large sample size captured in two endemic areas of CL. Our finding represents a first report confirming the high rate and the symptomatic natural Leishmania infection of C. gundi in North Africa. In our study, the prevalence reached 32% (30 out of 92) by direct examination and 43.5% (40 out of 92) by ITS1-PCR method which agrees with rates previously described on a small sample size (six and thirteen) of this species of rodents (Bousslimi et al., 2012; Jaouadi et al., 2011). Moreover, infection prevalence determined by the direct exam in C. gundi was much higher than those described among P. obesus (ranging from 13% to 29%) and M. shawi (ranging from 10% to 16%) in Tunisia (Ben-Ismail et al., 1987a; Ben-Ismail et al., 1989; Fichet-Calvet et al., 2003; Ghawar et al., 2011b). Unfortunately, no study recorder the Leishmania infection rate using molecular tools among the proven rodent reservoir hosts in Tunisia. The overall prevalence among C. gundi (57.6%) showed in this study, using parasitological and molecular tools, was higher when compared to those observed for the classic rodent reservoir hosts of L. major in Tunisia: P. obesus and M. shawi, using parasitological and serological tools (Ben-Ismail et al., 1989; Ben-Ismail R., 1987a; Fichet-Calvet et al., 2003; Ghawar et al., 2011b). It is well known that some Leishmania species causing CL could migrate to visceral organs of reservoir hosts (Davami et al., 2014; Mohebali et al., 2004; Parhizkari et al., 2011). Our findings confirm this fact, and more interestingly, Leishmania infection by ITS1-PCR method varied among C. gundi according to the target organ. It was 27.2% versus 18.5% among spleens and livers, respectively, with a low spleen-liver co-infection. In agreement with
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previous studies (Bensoussan et al., 2006; Bousslimi et al., 2012; Faiman et al., 2013), we were unable to identify 13 ITS1-PCR positive samples out of 42 using the RFLP analysis. This can be explained by the fact that PCR-ITS method can provide DNA amplification of other Trypanosoma species which are widely frequent in rodents and other mammals reservoir hosts of Leishmania parasites (Desquesnes et al., 2013; Rjeibi et al., 2015). On the other hand, RFLP analysis allowed us to identify 15/25 (60%) versus 13/17 (76.5%) positive samples as L. major among spleens and livers, respectively. While, only one sample was identified as L. tropica among livers. Disagreement between this result and those previously described for this species of rodents with only one detection of L. major and few detection of L. tropica (Bousslimi et al., 2012; Jaouadi et al., 2011) might be explained either by the sampling site location or the inability of the RFLP analysis to identify all positive samples. Furthermore, the C. gundi infected with L. tropica was surprisingly trapped in the well known L. major endemic area of Sidi Bouzid. This finding supports the fact that the geographical distribution of L. tropica parasites in Tunisia can be much wider than described. CL due to L. major may cause cutaneous lesions among established rodent reservoir hosts of Leishmania parasites (Ben-Ismail et al., 1987a; Ghawar et al., 2011b). Preferred locations of Leishmania lesions among rodent reservoir hosts were ears and tails (Ghawar et al., 2011b). This is not valid for C. gundi, where lesions were found only in ears and backs, possibly because C. gundi has a very short tail (Macdonald, 1984). Interestingly, C. gundi showed a high rate (86.8%) of asymptomatic leishmaniasis infection compared with 40% for the rodent reservoir hosts (P. obesus and M. shawi) of L. major in Tunisia (Ghawar et al., 2011b). Early dissemination of Leishmania parasites to the spleen was also reported in asymptomatic animals (Jaouadi et al., 2011; Schilling and Glaichenhaus, 2001).
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Our findings can be explained by the fact that L. major is endemic in the two studies regions. Sidi Bouzid governorate is an important endemic focus of CL with an average incidence rate of 669.7 cases/100000 inhabitants (Salah et al., 2007). However, Tataouine governorate is a mixed focus of CL with an average incidence of 50–150 cases per year with dominance of the zoonotic versus the chronic CL (Aoun et al., 2015; Bousslimi et al., 2010). Prevalence of this disease has a close temporal association with the abundance of Phlebotomus papatasi (Chelbi et al., 2007). In addition, C. gundi appears numerous and widespread with a geographical range from the center to the south in Tunisia (Honigs and Greven, 2003). This species was considered to be the most frequent mammal in the southern part of the country (Kock and Schomber, 1961). They live in small family groups. Each group spends a lot of time in their main shelter and all members of the family stay there overnight. Besides, they have temporary shelters used during their movements in the day (Gouat, 1989; Gouat and Gouat, 1983). These ecological parameters of C. gundi were in agreement with the criteria presumed by Ashford (Ashford, 1996) to define a reservoir host. But according to the WHO (WHO, 1990), reservoir hosts should be able to maintain the parasites and present a strong host-fly contact. In this context, we know that C. gundi defecate and urinate in their main shelter (Honigs and Greven, 2003) which present a favorable biotope for the sandflies. In fact, as previously described, a variety of sandflies vectors of Leishmania parasites causing CL were present in C. gundi biotope (Tabbabi et al., 2011b). Actually, during a study realized in Tataouine, ten species of sandflies were recorded in this rodent habitat. Proven vectors of the Zoonotic CL were predominant with a relative abundance of 0.23 and 0.09 for Sergentomiya minuta and Phlebotomus papatasi, respectively; and a relative abundance of 0.05 for P. sergenti, the proven vector for the Chronic CL (Tabbabi et al., 2011b).
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All these parameters implicit the putative role of C. gundi as reservoir host of two Leishmania species parasites in Tunisia but the study of the close interaction between this rodent and the proven sandflies vectors remains a priority to confirm that.
5. Conclusions C. gundi was found to be naturally infected by two species of Leishmania parasites (L. major and L. tropica) with the predominance of L. major, in endemic areas for both human leishmaniasis. Thus allow to suggest that C. gundi could play a major role in their epidemiological cycles and should be considered as possible reservoir host of Leishmania parasites causing cutaneous leishmaniasis in Tunisia.
Competing interests The authors declare that they have no competing interests. Funding This work was supported by the Laboratory of Transmission, Control and Immunobiology of Infections (LR11IPT02), Institut Pasteur de Tunis; and the Ministry of Higher Education and Research of Tunisia.
Acknowledgements We are grateful to the staff of the Regional Directorate of Public Health of Tataouine and Sidi Bouzid for their support to the achievement of the field work.
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Bensoussan, E., Nasereddin, A., Jonas, F., Schnur, L.F., Jaffe, C.L., 2006. Comparison of PCR assays for diagnosis of cutaneous leishmaniasis. J Clin Microbiol 44, 1435-1439. Bettaieb, J., Toumi, A., Chlif, S., Chelghaf, B., Boukthir, A., Gharbi, A., Ben Salah, A., 2014. Prevalence and determinants of Leishmania major infection in emerging and old foci in Tunisia. Parasit Vectors 7, 386. Bouratbine, A., Aoun, K., Ghrab, J., Harrat, Z., Ezzedini, M.S., Etlijani, S., 2005. Spread of Leishmania killicki to Central and South-West Tunisia. Parasite 12, 59-63. Bousslimi, N., Aoun, K., Ben-Abda, I., Ben-Alaya-Bouafif, N., Raouane, M., Bouratbine, A., 2010. Epidemiologic and clinical features of cutaneous leishmaniasis in southeastern Tunisia. Am J Trop Med Hyg 83, 1034-1039. Bousslimi, N., Ben-Ayed, S., Ben-Abda, I., Aoun, K., Bouratbine, A., 2012. Natural infection of North African gundi (Ctenodactylus gundi) by Leishmania tropica in the focus of cutaneous leishmaniasis, Southeast Tunisia. Am J Trop Med Hyg 86, 962-965. Chaara, D., Ravel, C., Banuls, A., Haouas, N., Lami, P., Talignani, L., El Baidouri, F., Jaouadi, K., Harrat, Z., Dedet, J.P., Babba, H., Pratlong, F., 2015. Evolutionary history of Leishmania killicki (synonymous Leishmania tropica) and taxonomic implications. Parasit Vectors 8, 198. Chelbi, I., Derbali, M., Al-Ahmadi, Z., Zaafouri, B., El Fahem, A., Zhioua, E., 2007. Phenology of Phlebotomus papatasi (Diptera: Psychodidae) relative to the seasonal prevalence of zoonotic cutaneous leishmaniasis in central Tunisia. J Med Entomol 44, 385388. Chelbi, I., Kaabi, B., Bejaoui, M., Derbali, M., Zhioua, E., 2009. Spatial correlation between Phlebotomus papatasi Scopoli (Diptera: Psychodidae) and incidence of zoonotic cutaneous leishmaniasis in Tunisia. J Med Entomol 46, 400-402.
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Chemkhi, J., Souguir, H., Ali, I.B., Driss, M., Guizani, I., Guerbouj, S., 2015. Natural infection of Algerian hedgehog, Atelerix algirus (Lereboullet 1842) with Leishmania parasites in Tunisia. Acta Trop 150, 42-51. Davami, M.H., Motazedian, M.H., Kalantari, M., Asgari, Q., Mohammadpour, I., SotoodehJahromi, A., Solhjoo, K., Pourahmad, M., 2014. Molecular Survey on Detection of Leishmania Infection in Rodent Reservoirs in Jahrom District, Southern Iran. Journal of arthropod-borne diseases 8, 139-146. Desquesnes, M., Holzmuller, P., Lai, D.H., Dargantes, A., Lun, Z.R., Jittaplapong, S., 2013. Trypanosoma evansi and surra: a review and perspectives on origin, history, distribution, taxonomy, morphology, hosts, and pathogenic effects. BioMed research international 2013, 194176. Faiman, R., Abbasi, I., Jaffe, C., Motro, Y., Nasereddin, A., Schnur, L.F., Torem, M., Pratlong, F., Dedet, J.P., Warburg, A., 2013. A newly emerged cutaneous leishmaniasis focus in northern Israel and two new reservoir hosts of Leishmania major. PLoS Negl Trop Dis 7, e2058. Fichet-Calvet, E., Jomaa, I., Ben Ismail, R., Ashford, R.W., 2003. Leishmania major infection in the fat sand rat Psammomys obesus in Tunisia: interaction of host and parasite populations. Ann Trop Med Parasitol 97, 593-603. Ghawar, W., Snoussi, M.A., Hamida, N.B., Boukthir, A., Yazidi, R., Chaabane, S., Chemkhi, J., Zaatour, A., Salah, A.B., 2011a. First report of natural infection of least weasel (Mustela nivalis Linnaeus, 1776) with Leishmania major in Tunisia. Vector Borne Zoonotic Dis 11, 1507-1509. Ghawar, W., Toumi, A., Snoussi, M.A., Chlif, S., Zaatour, A., Boukthir, A., Hamida, N.B., Chemkhi, J., Diouani, M.F., Ben-Salah, A., 2011b. Leishmania major infection among
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Mohebali, M., Javadian, E., Yaghoobi-Ershadi, M.R., Akhavan, A.A., Hajjaran, H., Abaei, M.R., 2004. Characterization of Leishmania infection in rodents from endemic areas of the Islamic Republic of Iran. East Mediterr Health J 10, 591-599. Parhizkari, M., Motazedian, M.H., Asqari, Q., Mehrabani, D., 2011. The PCR-based detection of Leishmania major in Mus musculus and other rodents caught in southern Iran: a guide to sample selection. Ann Trop Med Parasitol 105, 319-323. Pratlong, F., Dereure, J., Ravel, C., Lami, P., Balard, Y., Serres, G., Lanotte, G., Rioux, J.A., Dedet, J.P., 2009. Geographical distribution and epidemiological features of Old World cutaneous leishmaniasis foci, based on the isoenzyme analysis of 1048 strains. Trop Med Int Health 14, 1071-1085. Rjeibi, M.R., Ben Hamida, T., Dalgatova, Z., Mahjoub, T., Rejeb, A., Dridi, W., Gharbi, M., 2015. First report of surra (Trypanosoma evansi infection) in a Tunisian dog. Parasite 22, 3. Salah, A.B., Kamarianakis, Y., Chlif, S., Alaya, N.B., Prastacos, P., 2007. Zoonotic cutaneous leishmaniasis in central Tunisia: spatio temporal dynamics. Int J Epidemiol 36, 991-1000. Schilling, S., Glaichenhaus, N., 2001. T cells that react to the immunodominant Leishmania major LACK antigen prevent early dissemination of the parasite in susceptible BALB/c mice. Infect Immun 69, 1212-1214. Schonian, G., Nasereddin, A., Dinse, N., Schweynoch, C., Schallig, H.D., Presber, W., Jaffe, C.L., 2003. PCR diagnosis and characterization of Leishmania in local and imported clinical samples. Diagn Microbiol Infect Dis 47, 349-358. Tabbabi, A., Bousslimi, N., Rhim, A., Aoun, K., Bouratbine, A., 2011a. First report on natural infection of Phlebotomus sergenti with Leishmania promastigotes in the cutaneous leishmaniasis focus in southeastern Tunisia. Am J Trop Med Hyg 85, 646-647.
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Tabbabi, A., Ghrab, J., Aoun, K., Ready, P.D., Bouratbine, A., 2011b. Habitats of the sandfly vectors of Leishmania tropica and L. major in a mixed focus of cutaneous leishmaniasis in southeast Tunisia. Acta Trop 119, 131-137. Talmi-Frank, D., Jaffe, C.L., Nasereddin, A., Warburg, A., King, R., Svobodova, M., Peleg, O., Baneth, G., 2010a. Leishmania tropica in rock hyraxes (Procavia capensis) in a focus of human cutaneous leishmaniasis. Am J Trop Med Hyg 82, 814-818. Talmi-Frank, D., Kedem-Vaanunu, N., King, R., Bar-Gal, G.K., Edery, N., Jaffe, C.L., Baneth, G., 2010b. Leishmania tropica infection in golden jackals and red foxes, Israel. Emerg Infect Dis 16, 1973-1975. Toumi, A., Chlif, S., Bettaieb, J., Ben Alaya, N., Boukthir, A., Ahmadi, Z.E., Ben Salah, A., 2012. Temporal dynamics and impact of climate factors on the incidence of zoonotic cutaneous leishmaniasis in central Tunisia. PLoS Negl Trop Dis 6, e1633. WHO, 1990. Control of leishmaniasis. Report of WHO Expert Committee, Geneva, Switzerland.
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Figure 1. Clinical manifestation as hyper-pigmentation observed among Ctenodactylus gundi on the ear (a) and on the back (b).
Figure 2. Leishmania amastigotes in a Giemsa stained smears from the ear of Ctenodactylus gundi under oil immersion (X1000).
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Figure 3. Infected Ctenodactylus gundi by ITS1-PCR and direct exam, and their relation to the clinical manifestation.
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Figure 4. RFLP products of the amplified ITS1 fragment from the spleens and the livers of Ctenodactylus gundi. M1 and M2: 100 bp and 1 Kb size markers (Invitrogen®); Lm, Lt and Li: respectively L. major (two fragments of 132-bp and 206-bp), L. tropica (three fragments of 188-bp, 57-bp and 26-bp respectively) and L. infantum (three fragments of 187-bp, 72-bp and 55-bp respectively) references strains. Positive samples from the spleens: G8, G12, G14, G19 and G21 (G12 not identified by RFLP). Positive samples from the livers: G23, G24, G25, G26, G33 (G23 not identified by RFLP). A: positive sample by ITS1-PCR.
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Table 1. Prevalence of Leishmania infection among Ctenodactylus gundi according to the diagnostic method by capture sites. Governorates
Sidi Bouzid
Tataouine
Khbina
Guermessa
Mdhila
Total
P
(20)
(21)
(51)
(92)
value
Clinical exam (n, %)
1 (9.1)
6 (54.5)
4 (36.4)
11 (12)
NS
Direct exam (n, %)
3 (10)
6 (20)
21 (70)
30 (32.6)
NS
Total ITS1-PCR
6 (30)
8 (38.1)
26 (51)
40 (43.5)
NS
ITS1-PCR among spleens (n, %)
2 (8)
7 (28)
16 (64)
25 (27.2)
NS
ITS1-PCR among livers (n, %)
4 (23.5)
1 (5.9)
12 (70.6)
17 (18.5)
NS
Delegations (n)
22