Infection, Genetics and Evolution 10 (2010) 115–121 Contents lists available at ScienceDirect Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid Tripartite interactions between tsetse flies, Sodalis glossinidius and trypanosomes—An epidemiological approach in two historical human African trypanosomiasis foci in Cameroon Oumarou Farikou a,b, Flobert Njiokou a, Jean A. Mbida Mbida a, Guy R. Njitchouang a, Hugues Nana Djeunga a, Tazoacha Asonganyi c, Pere P. Simarro d,Ge´rard Cuny b, Anne Geiger b,* a University of Yaounde´ I, Faculty of Science, BP 812, Yaounde´, Cameroon b UMR 177, IRD-CIRAD, CIRAD TA A-17/G, Campus International de Baillarguet, 34398 Montpellier Cedex 5, France c Faculty of Medicine and Biomedical Sciences, University of Yaounde´ I, Cameroon d World Health Organization, Control of Neglected Tropical Diseases, Geneva, Switzerland ARTICLE INFO ABSTRACT Article history: Epidemiological surveys were conducted in two historical human African trypanosomiasis foci in South Received 3 August 2009 Cameroon, Bipindi and Campo. In each focus, three sampling areas were defined. In Bipindi, only Glossina Received in revised form 14 October 2009 palpalis was identified, whereas four species were identified in Campo, G. palpalis being highly Accepted 20 October 2009 predominant (93%). For further analyses, 75 flies were randomly chosen among the flies trapped in each Available online 29 October 2009 of the six villages. Large and statistically significant differences were recorded between both (1) the prevalence of Sodalis glossinidius (tsetse symbiont) and the prevalence of trypanosome infection of the Keywords: major fly species G. p. palpalis and (2) the respective prevalence of symbiont and infection between the Glossina two foci. Despite these differences, the rate of infected flies harbouring the symbiont was very similar Trypanosome species Sodalis glossinidius (75%) in both foci, suggesting that symbionts favour fly infection by trypanosomes. This hypothesis was Vector competence statistically tested and assessed, showing that S. glossinidius is potentially an efficient target for Historical foci controlling tsetse fly vectorial competence and consequently sleeping sickness. Cameroon ß 2009 Elsevier B.V. All rights reserved. Human African trypanosomiasis Nagana 1. Introduction brucei gambiense, responsible for the chronic form of the disease in West and Central Africa, T. brucei rhodesiense, responsible for the Tsetse flies are medically and agriculturally important vectors acute form in East Africa, and T. brucei brucei, pathogenic for that transmit African trypanosomes, the causative agents of animals only. The endemic characteristics of the HAT foci in West sleeping sickness in humans, and nagana in animals. Sleeping and Central Africa differ greatly from each other; some remain sickness, fatal if untreated, still affects a wide range of people in active for years at a low endemic level while others show periodic sub-Saharan Africa (World Health Organization, 2006), where epidemic outbreaks (Penchenier et al., 1999). The Bipindi and more than 60 million people are at risk. Nagana is estimated to cost Campo foci (southern Cameroon), where our study was conducted, African agriculture US$ 4.5 billion per year (Reinhardt, 2002). are weakly endemic. Nevertheless, several new cases of HAT are The drugs currently used are not satisfactory: some are toxic detected in the two foci every year (V. Ebo’o Eyenga, pers. comm.). and all are difficult to administer (Barrett, 2006). Furthermore, In addition, T. brucei gambiense has been detected in animals drug resistance is increasing (Matovu et al., 2001). Therefore, the (Njiokou et al., 2006) living in the Bipindi and Campo areas, investigation of novel drugs and/or novel disease control strategies suggesting their potential role as a reservoir of parasites favouring must be pursued. their maintenance and their circulation at a low level. This could The causative agent of the human African trypanosomiasis explain the periodic outbreaks of sleeping sickness in these foci (HAT) is a trypanosome belonging to the Trypanosoma brucei (Njiokou et al., 2006). Besides, trypanosomes belonging to the species complex, classically subdivided into three subspecies: T. subgenera Duttonella, Nannomonas and Trypanozoon have also been detected in wild animals in south Cameroon (Njiokou et al., 2004). * Corresponding author. Tel.: +33 04 67 59 39 25; fax: +33 04 67 59 38 94. The biological process leading to transmission of the trypano- E-mail address: [email protected] (A. Geiger). somes from one mammalian host to another is complex. Prior to 1567-1348/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2009.10.008 116 O. Farikou et al. / Infection, Genetics and Evolution 10 (2010) 115–121 being transmitted, the parasite must first establish in the tsetse ten cases of sleeping sickness were detected (V. Ebo’o Eyenga, fly midgut following an infective blood meal and then mature pers. comm.). either in the salivary glands or in the mouthparts, depending on the trypanosome species (Vickerman et al., 1988; Van Den 2.2. Sampling and dissection Abbeele et al., 1999). The factors involved in the establishment are still largely unknown, and only a small proportion of flies Five entomological surveys were conducted in July 2007 and develop mature infection and transmit the parasite (Maudlin and February 2008 in Bipindi (in the Memel, Ebiminbang and Lambi/ Welburn, 1994). Many factors, including midgut lectins and the Bidjouka villages) and in November 2007, March 2008 and October insect immune system, have been suggested to affect the success 2008 in Campo (in the Mabiogo, Akak and Campo Beach/Ipono or failure of the parasite’s development (Hao et al., 2001). villages). The geographical position of the sampling sites was Besides the parasite, tsetse flies may harbour three different determined using the global positioning system (GPS). Tsetse flies symbiotic micro-organisms (Aksoy, 2000). Among them, Sodalis were captured using pyramidal traps (Lancien, 1981) planted in glossinidius (Cheng and Aksoy, 1999; Dale and Maudlin, 1999)is suitable tsetse fly biotopes. Each trap remained for 4 consecutive suspected of favouring the establishment of the parasite in the days and flies were harvested twice a day. insect midgut through a complex biochemical mechanism The different Glossina species were first identified and then (Maudlin and Ellis, 1985; Welburn and Maudlin, 1999; Dale sorted into teneral (young flies that had never taken a blood meal) and Welburn, 2001). and nonteneral flies, according to morphological criteria (Grebaut In previous studies, no exclusive relationship was found et al., 2004). The nonteneral flies were dissected in a drop of sterile between the presence of S. glossinidius and the ability of the fly 0.9% saline solution, and their midgut was examined under the to acquire Trypanosoma congolense (Geiger et al., 2005a). We have light microscope (magnification Â100) for the presence of also shown that Glossina palpalis gambiensis (palpalis group) and trypanosomes. Trypanosome-infected and -noninfected organs Glossina morsitans morsitans (morsitans group) harbour genetically were then separately transferred into microfuge tubes containing distinct populations of S. glossinidius (Geiger et al., 2005b), ethanol (958) for further symbiont and trypanosome PCR suggesting that fly vector competence might be linked to given identification. The instruments used were carefully cleaned after genotypes of S. glossinidius rather than to a mere presence/absence the dissection of each fly to prevent contamination. During field of the symbiont. We also demonstrated that the ability of T. brucei manipulations, the microfuge tubes were maintained at room gambiense and T. brucei brucei to establish in the G. palpalis temperature; thereafter, in the laboratory, they were stored at gambiensis insect midgut is statistically linked to the presence of S. À20 8C until use. glossinidius-specific genotypes (Geiger et al., 2007). Because of these puzzling results and to gain clearer insight into 2.3. DNA extraction and PCR amplification the mechanisms involved, including the possibility that S. glossinidius controls the transmission of the parasites under Frozen samples were thawed and air-dried. DNA was extracted natural field conditions, a large tsetse fly sampling campaign from organs using the classical protocol (Maniatis et al., 1982). was conducted in two historical sleeping sickness foci, Bipindi and Tissues were homogenised with a pestle in a cethyl trimethyl Campo in the south of Cameroon. For each fly, we have determined ammonium bromide (CTAB) buffer (CTAB 2%; 0.1 M Tris, pH 8; the species, the presence/absence of S. glossinidius, and have 0.02 M EDTA pH 8; 1.4 M NaCl) and incubated at 60 8C for 30 min. identified the trypanosome species and subspecies. The prevalence The DNA was extracted from the lysis mixture with chloroform/ of each of them was calculated for the population of flies. isoamylic alcohol (24/1; V/V) and precipitated by adding Prevalence was also calculated separately within each sampling isopropanol (V/V). After centrifugation (10,000 Â g, 15 min), the area (six villages). Finally, all the data were statistically analysed in pellet was rinsed with 70% ethanol, air-dried, and re-suspended in order to identify possible relationships between them. The results distilled sterile water. The DNA samples were stored at À20 8C of this study are presented herein. until PCR amplification. The primers used to amplify DNA are presented in Table 1. 2. Materials and methods Specific trypanosome primers (Moser et al., 1989; Masiga et al., 1992; Majiwa et al., 1994; Herder et al., 2002) were tested on fly 2.1. Sampling area organs. To detect possible dissection and/or extraction contam- ination, controls were done both on noninfected midguts and on This study was conducted in 2007 and 2008 in two HAT foci the salivary glands of flies showing T. congolense midgut infection. (Bipindi and Campo) situated in the Ocean Division of the South S. glossinidius primers (Darby et al., 2005) were tested on the Province of Cameroon. The Campo focus (28200N, 98520E) presents midgut.