http://orcid.org/0000-0001-8427-0769
Figures
Abstract
Background
Important control efforts have led to a significant reduction of the prevalence of human African trypanosomiasis (HAT) in Côte d’Ivoire, but the disease is still present in several foci. The existence of an animal reservoir of Trypanosoma brucei gambiense may explain disease persistence in these foci where animal breeding is an important source of income but where the prevalence of animal African trypanosomiasis (AAT) is unknown. The aim of this study was to identify the trypanosome species circulating in domestic animals in both Bonon and Sinfra HAT endemic foci.
Methodology/Principal findings
552 domestic animals (goats, pigs, cattle and sheep) were included. Blood samples were tested for trypanosomes by microscopic observation, species-specific PCR for T. brucei sl, T. congolense, T. vivax and subspecies-specific PCR for T. b. gambiense and T. b. gambiense immune trypanolysis (TL). Infection rates varied significantly between animal species and were by far the highest in pigs (30%). T. brucei s.l was the most prevalent trypanosome species (13.7%) followed by T. congolense. No T. b. gambiense was identified by PCR while high TL positivity rates were observed using T. b. gambiense specific variants (up to 27.6% for pigs in the Bonon focus).
Conclusion
This study shows that domestic animals are highly infected by trypanosomes in the studied foci. This was particularly true for pigs, possibly due to a higher exposure of these animals to tsetse flies. Whereas T. brucei s.l. was the most prevalent species, discordant results were obtained between PCR and TL regarding T. b. gambiense identification. It is therefore crucial to develop better tools to study the epidemiological role of potential animal reservoir for T. b. gambiense. Our study illustrates the importance of “one health” approaches to reach HAT elimination and contribute to AAT control in the studied foci.
Author summary
In Africa, significant efforts to control human African trypanosomiasis (HAT) over the past three decades have drastically reduced the prevalence of the disease and elimination seems today an achievable goal. However, potential animal reservoirs of Trypanosoma brucei gambiense may compromise this ambitious objective. In the Bonon and Sinfra HAT endemic foci in Côte d’Ivoire, no recent data are available about the prevalence of animal African trypanosomiasis (AAT). The aim of this study was to identify trypanosomes circulating in domestic animals in these two HAT foci using serological, parasitological and molecular tools. We showed that T. brucei s.l. and T. congolense were the most prevalent trypanosome species and that pigs and cattle were the most infected animals. Discordant results were observed between the T. b. gambiense specific molecular and serological tools and the presence of an animal reservoir for T. b. gambiense remains unclear. Nevertheless, improved control strategies can be proposed based on this study to reach HAT elimination and contribute to AAT control in the study areas.
Citation: N’Djetchi MK, Ilboudo H, Koffi M, Kaboré J, Kaboré JW, Kaba D, et al. (2017) The study of trypanosome species circulating in domestic animals in two human African trypanosomiasis foci of Côte d'Ivoire identifies pigs and cattle as potential reservoirs of Trypanosoma brucei gambiense. PLoS Negl Trop Dis 11(10): e0005993. https://doi.org/10.1371/journal.pntd.0005993
Editor: Marleen Boelaert, Institute of Tropical Medicine, BELGIUM
Received: April 16, 2017; Accepted: September 25, 2017; Published: October 18, 2017
Copyright: © 2017 N’Djetchi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This study was funded by Programme d’Appui à l’Enseignement supérieur/Union Economique et Monétaire Ouest Africaine (http://www.uemoa.int/) to MK. This study also received support from a Word Health Organization (WHO) - Institut de Recherche pour le Développement (IRD) collaborative project to VJ. MK, DK, HI, and JK are supported by the “Jeunes Equipes AIRD” JEAI program (ERHATryp project). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Human African trypanosomiasis (HAT) or sleeping sickness is a vector borne parasitic disease caused by Trypanosoma brucei gambiense (T. b. gambiense) in West and Central Africa and T. b. rhodesiense in East Africa. T. b. gambiense is responsible for 98% of all HAT cases reported in the last decade and remains an important public health concern in sub-Saharan Africa [1]. However, with less than 3000 cases reported in 2015 [2], HAT elimination seems an achievable goal [3]. A similar situation occurred in the 1960s but, after an early sense of victory, the disease reemerged. Gambiense HAT is generally considered as an anthroponotic disease, but the absence of animal reservoirs has never been demonstrated. The existence of an animal reservoir for T. b. gambiense could be one of the factors that causes reemergence of the disease after successful control campaigns [4].
In Côte d’Ivoire, significant efforts to control the disease over the past three decades have been made and drastically reduced the prevalence of HAT [5]. The last epidemic was contained in the Sinfra and Bonon foci at the early2000s [6–8]. Despite continuous control efforts, few HAT cases are still passively diagnosed from these two foci as well as from historical foci of the Western-Centre part of the country [7–9]. Transmission persistence may be due to the existence of a residual chronic human and/or animal reservoir of T. b. gambiense in these areas where tsetse flies are still present [10–13].
Several studies have highlighted the importance of wild and domestic animals in the transmission cycle of T. b. rhodesiense [14,15], but this is still under debate for T. b. gambiense. Noteworthy, the presence of T. b. gambiense in domestic and wild animals have been reported in Cameroun [16] and Equatorial Guinea [17–19]. In Côte d’Ivoire, such studies are scarce and the last report dates from the early 2000s in which the authors investigated the presence of trypanosomes in pigs in the Bonon HAT focus. High prevalence of T. brucei s.l. was observed but the presence of T. b. gambiense in this animal species remained unclear [20]. It is crucial to increase the efforts in studying the existence of an animal reservoir of T. b. gambiense as this could compromise HAT elimination.
No data are available regarding the prevalence of T. b. brucei, T. congolense and T. vivax animal African trypanosomiasis (AAT), in the Bonon and Sinfra foci, despite that animal breeding represents an increasing source of food and income in these areas with high human population densities.
In the context of the one health approach that was recently suggested for HAT and AAT control [21], the aim of the present study was to characterize trypanosomes circulating in domestic animals in the HAT foci of Bonon and Sinfra in Côte d’Ivoire. We used Trypanosoma species-specific PCR assays, microsatellite genotyping and immune trypanolysis (TL) with three variant antigenic types (VAT) of which two are specific for T. b. gambiense [22]. We show that T. brucei s.l. and T. congolense were the most prevalent trypanosome species in the two foci and that pigs and cattle were the most infected animals, with T. brucei s.l. and T. congolense respectively. Discordant results were observed between the T. b. gambiense specific PCR and TL tests and the existence of an animal reservoir of T. b. gambiense thus remains unclear.
Materials and methods
Study area
The study was carried out in September/October 2013 in the Sinfra and Bonon areas, which are located in the western-central part of Côte d’Ivoire (Fig 1). In recent decades, the mesophyle forest has been progressively replaced by cash crops (mainly cocoa and coffee, but also bananas, cassava, rice and yam) leading to a favorable environmental context for HAT development in these areas [23]. The evolution of HAT prevalence in Côte d’Ivoire is well documented since the 1950s. The number of cases diagnosed from 2000 to 2010 (Fig 1A) shows that these two foci were the most endemic during this period. Control efforts conducted from 1995 till present could largely contain the epidemic [7,11] but few cases are still passively diagnosed each year [7]. Based on the last 10 HAT cases who were diagnosed from 2011 to 2013, we identified 8 and 10 study sites in the Sinfra and Bonon foci, respectively (Fig 1B). These 18 study sites (less than 10 km from the last detected HAT cases) are expected to be those where transmission is still active and where we had the highest chance to detect T. b. gambiense in domestic animals.
A. Localization of the Bonon and Sinfra foci which reported the highest number of HAT cases diagnosed from 2000 to 2010 in Côte d’Ivoire. B. Localization of the last HAT cases diagnosed from 2011 to 2013 and the sites of domestic animals sampling in the Bonon and Sinfra foci. This figure was created by the mapping service of our team based at Institut Pierre Richet (Bouaké, Côte d’Ivoire) specifically for this manuscript.
We focused our study on cattle (Zebu), goats, sheep and pigs since they are the most common domestic animals in the study areas. They are mainly bred in the periphery of villages or along the small rivers crossing the villages, where tsetse flies are often abundant [10,12,24–26]. Generally, sheep, goats and cattle freely graze during the day and are kept in enclosures at night while pigs freely roam day and night.
Sample collection and parasitological investigation in the field
For each animal, 5ml of blood was taken from the jugular vein. Parasitological diagnosis was performed in the field by microscopic examination using the buffy coat technique (BCT) [27]. BCT was considered positive when trypanosomes could be visually detected regardless of the species. In addition, 1 ml of plasma and 1 ml of blood were aliquoted and immediately frozen at -20°C during transport and subsequently at -80°C in the lab for PCR and immune trypanolysis testing.
PCR analysis
DNA from 500 μL of blood was extracted using the DNeasy Tissue kit (Qiagen, Valencia, CA, USA) as described previously [28] and subjected to diagnostic PCR assays using Trypanosoma species specific primers for T. brucei s.l. (TBR1-2) [29], T. congolense forest type (TCF1-2) [30], T. congolense savannah type (TCS1-2) [31], T. vivax (TVW1-2) [30]. Positives samples for T. brucei s.l were tested for T. b. gambiense using primers targeting the TgsGP gene [32]. All PCR reactions were carried out using 5 μl of DNA template in a reaction volume of 50 μL 1xPCR reaction buffer comprising 0.2 mM of dNTP, 0.2 μM of each primer and 2.5 U of Taq polymerase. A positive control was added to the corresponding PCR and distilled water was used as negative control. The PCR products were visualized by electrophoresis in a 2% agarose gel stained with GelRed and illuminated with UV light.
Microsatellite genotyping
Samples positive in the T. brucei s.l. specific TBR PCR were further characterized by seven microsatellite markers Ch1/18, Ch1/D2/7 [33], M6C8 [34], Micbg5, Micbg6, Misatg4, Misatg9 [35], as previously described [36]. Reference stocks of T. b. gambiense (n = 18), T. b. gambiense group 2 (n = 3), T. b. brucei (n = 1) and T. b. rhodesiense (n = 1) were included. A neighbor-joining tree was computed under multiple sequence alignment (MSA) [37] with Mega 5 [38] on a Cavalli-Sforza and Edward's chord distance matrix [39] as recommended by Takezaki et al. [40].
Trypanolysis test
Plasma samples were analyzed with the immune trypanolysis test (TL) using cloned populations of T. b. gambiense variant antigen type (VATs) LiTat 1.3, LiTat 1.5 and LiTat 1.6 as previously described [22,41]. LiTat 1.3 and LiTat 1.5 VATs are reported to be specific for T. b. gambiense, while LiTat 1.6 VAT can both be expressed in T. b. gambiense and T. b. brucei [22].
Statistical analysis
All statistical analyses were done with JMP11 (SAS Institute). Proportions of positive animals for BCT, PCR and TL were compared regarding foci and host species using the Chi-square analysis.
Ethics statement
Sample collection was conducted within the framework of epidemiological surveillance activities supervised by the HAT National Elimination Program (HAT NEP). No ethical statement is required by local authorities. Any veterinarian may carry out blood sampling on domestic animals, with the authorization of the owner, as it is performed during prophylaxis or diagnostic campaign. No samples other than those for routine screening and diagnostic procedures were collected. Breeders gave their consent for animal sampling after explaining the objectives of the study. For animal care, venous sampling was performed by a veterinary of the Laboratoire National d’Appui au Développement Rural (Ministry of Agriculture). A deworming treatment (Bolumisol, Laprovet) was provided free to all animals sampled and those positive with BCT were treated for trypanosomiasis.
Results
In total, 552 animals were sampled of which 251 from Bonon and 301 from Sinfra as presented in Table 1.
Parasitology
Out of the 552 animals sampled, 57 trypanosome infections (10.3%) were detected by BCT (Table 2). Highest prevalence was observed in pigs (p<0.0001) with a prevalence of almost 30%. No significant differences were observed between the two foci in the infection rates (Fig 2A).
Proportion of BCT (2A), T. brucei s.l. TBR-PCR (2B), T. congolense forest type TCF-PCR (2C) and T. vivax TVW-PCR (2D) positive results on the total sample collection for each host in the two foci. A significant difference between Bonon and Sinfra is indicated by a star.
Molecular diagnosis
No animals were positive with the TCS specific primers. In total, 109 trypanosome infections (19.7%) were detected with at least one PCR (TBR, TCF or TVW) with the highest prevalence observed in pigs (41.6%, Table 2). T. bucei s.l. was the most prevalent trypanosome species (13.8%) followed by T. congolense forest type (8.5%) and T. vivax (2.4%). No T. vivax infection was detected in pigs. Mixed infections with positive results in at least two different PCR assays were observed in 29 animals. Most were mixed infections with T. bucei s.l. / T. congolense forest type and mainly observed in pigs (59%).
TBR, TCF and TVW PCR results for the different hosts in both foci are presented in Fig 2B, 2C and 2D. Pigs and cattle showed higher TBR and TCF-PCR positivity rates compared to sheep and goats. No significant differences were observed between the two foci except for TBR PCR positivity rates in cattle. The highest PCR positivity rate was observed in pigs in Sinfra (43.3%). All 76 DNA samples positive in the TBR PCR were further analyzed with the T. b. gambiense specific TgsGP PCR and all were negative.
Microsatellite genotyping
Among the 76 DNA samples positive in the TBR PCR, only 10 showed amplification in the microsatellite genotyping assays. The NJTree presented in Fig 3 presents a classical shape (e.g. [42])) with one monophyletic lineage that gathers all T. b. gambiense reference stocks and rake for other subspecies. No trypanosome genotypes from the domestic animals sampled in Bonon and Sinfra foci are members of this T. b. gambiense lineage.
Neighbor-joining tree (NJTree), based on Cavalli-Sforza and Ewards Chord distance, of the amplified microsatellite genotypes. Reference stocks are in bold. The unique monophyletic lineage corresponds to Trypanosoma brucei gambiense and is indicated above the corresponding branch. The presence of several missing genotypes prohibited the use of bootstraps. Bo = Bonon, Si = Sinfra, Tbg = Trypanosoma brucei gambiense, Tbg2 = Trypanosoma brucei gambiense group 2, Tbb = Trypanosoma brucei brucei, Tbrh = Trypanosoma brucei rhodesiense.
Trypanolysis
Trypanolysis results for the three VAT types per host in the two foci are presented in Fig 4. No TL positive results were observed in goats and sheep with LiTat 1.3 and 1.5 VAT in both foci and only two goats and two sheep were positive with the LiTat 1.6 VAT. The LiTat 1.6 TL assay showed high positivity rates (more than 35%) in pigs in both foci and in cattle (more than 40%) in the Bonon focus, confirming the PCR-TBR results. We observed the same pattern, but with lower positivity rates, in the T. b. gambiense-specific LiTat 1.3 and 1.5 TL assays (0% for both 1.3 and 1.5 in cattle in Sinfra, 17.6 and 5.9% for 1.3 and 1.5 respectively in cattle in Bonon and between 15.8 and 27.6% for both 1.3 and 1.5 in pigs in the two foci). The difference observed in cattle between Bonon and Sinfra is significant for Litat 1.3.
Proportion of the LiTat 1.6 (4A), LiTat 1.3 (4B) and LiTat 1.5 (4C) TL positive results on the total sample collection for each host in the two foci. A significant difference between Bonon and Sinfra is indicated with a star.
The distribution of LiTat 1.3 and/or LiTat 1.5 TL positive results in cattle (7 individuals = 8%) and pigs (39 individuals = 28.5%) is given in Table 3. Out of the seven cattle which tested positive to either LiTat 1.3 or LiTat 1.5, only one was positive to both variants (13.3%). This proportion was much higher in pigs with 21 of 39 (53.9%) positive animals tested positive to both variants. From these 21 pigs, 15 (10.9%) were also positive to LiTat 1.6 and thus positive to the three VAT types. Animals testing positive to LiTat 1.6 only were also observed for all domestic animal species but with higher proportion in cattle (13.8%) and pigs (22.6%).Nineteen of the 46 samples (43.3%) testing positive to Litat1.3 and/or 1.5 were negative in all species specific PCR assays (TBR, TCF and TVW). Positivity to Litat1.3 and/or 1.5 was the highest in animals testing positive to the TBR PCR (30.6%), it was zero in animals positive only to TVW PCR, but positive in 4/23 (17.4%) animals that were uniquely positive to TCF PCR (Fig 5).
PCR profiles are given as follow: PCR TBR result/PCR TCF result/PCR TVW result. n = number of animals with the corresponding profile. Numbers on the top are the numbers of animal positives with Litat 1.3 and/or 1.5 TL or with LiTat 1.6 only.
Discussion
In this study, we showed that domestic animals are important carriers of trypanosomes in the Sinfra and Bonon HAT foci. T. brucei s.l. infections were highest in pigs in Sinfra and both in pigs and cattle in Bonon. The overall prevalence recorded by PCR was approximately twice the one observed with the parasitological BCT technique. This was expected due to the known higher sensitivity of PCR [28,43]. Infection rates were higher in pigs and cattle than in sheep and goats. This may be related to differences in the host-vector contacts associated with different breeding practices and/or to differential host susceptibility to trypanosome infection. Sheep rather graze in the vicinity of the houses where they are partly nourished by the villagers, limiting the contact with tsetse flies. Goats are browsing freely across the vegetation in the periphery of villages and may be more exposed to tsetse flies. Noteworthy, low trypanosome infection rates have already been described for goats and this was attributed partly to the fact that goats are known to express individual defensive behavior against the bites of tsetse flies [43,44]. Cattle forage across long distances to reach pastures and are thus potentially more exposed to tsetse flies. However, in the Sinfra focus, the areas crossed by cattle herds are particularly anthropized with lower tsetse fly infestations. In contrast, in Bonon, the forest was more recently exploited and cattle are still in contact with tsetse populations [10]. This probably explains why cattle infection rates in Bonon are much higher than in Sinfra. Pigs roam freely in the more humid and shady areas around the villages or along the small rivers and are highly exposed to tsetse flies. Moreover, pigs have already been described as a preferential feeding host for Glossina palpalis palpalis [26,45], the only tsetse species present in the studied areas [26].
In the Sinfra and Bonon areas, the forest was progressively replaced by cocoa, coffee, banana plantations and food crops offering potential favorable conditions to the introduction of T. congolense savannah type from the north of the country where prevalence of this species is high [46]. However we did not detect this trypanosome which is considered as the most pathogenic congolense type [47], in the framework of this study. The prevalence of T. vivax was also low with only 13 infections, mainly detected in sheep. Noteworthy, no T. vivax infections were detected in pigs, confirming that this host is generally refractory to this trypanosome species [48]. T. congolense forest type was found in all host animals as previously observed in other forest areas [49] and mainly detected in pigs. T. brucei s.l. was the predominant species detected in our study areas, which is in line with studies conducted in other HAT forest foci from Cameroon and Equatorial Guinea [17,50].
Among the T. brucei s.l. infections, T. b. gambiense could not be detected by the TgsGP PCR nor by microsatellite genotyping. At first sight, this could indicate that the human pathogenic trypanosome is not circulating in domestic animals in the HAT foci of Bonon and Sinfra. Similar results were reported in northwest Uganda where authors concluded an apparent absence of domestic animal reservoir for T. b. gambiense [51]. However, we observed high positivity rates (up to 28%) with both the T. b. gambiense specific LiTat 1.3 and LiTat 1.5 TL assays. Positivity to more than one T. b. gambiense variant is an indicator that the host was infected for a sufficient amount of time to allow VSG switching. Together, the high prevalence of LiTat 1.3 and/or 1.5 positivity observed in pigs and the fact that more than half of the animal tested positive to both variants, are thus pointing out that pigs could be potential reservoirs of T. b. gambiense in the study area.
Since very few HAT cases have been identified in these foci in the last years (Fig 1), the TL results may suggest an active circulation of T. b. gambiense in pigs and/or cattle in the Sinfra and Bonon foci, but with little contact with humans. In most studies describing the presence of T. b. gambiense in wild and domestic animals, prevalence in animals is often higher than in humans [18,19,50,52]. This may be explained by a higher nutritional preference of tsetse flies for animals and by the fact that human are less exposed to tsetse flies in these areas. This is consistent with previous observations describing the protective role of pigs living at the periphery of villages since pigs are the preferential host of tsetse flies [24–26,53].
The TL results obtained in our study are consistent with recent population genetics data which showed that natural populations of T. b. gambiense are more important than those evidenced by the classical medical survey, suggesting hidden reservoirs of these parasites [42,54]. In the same way, a recent modeling study with animal and human data from a Cameroon focus showed that transmission of T. b. gambiense could not be maintained by humans as the only reservoir [55]. The existence of a domestic animal reservoir for T. b. gambiense could be responsible for the sporadic cases diagnosed in most of the Ivorian forest foci, sometimes a long time after the last epidemic HAT episode [7].
We observed a high discordance between the T. b. gambiense PCR and TL results. The T. b. gambiense specific TgsGP PCR targets a single copy gene [56] and may not be sensitive enough to exclude presence of T. b. gambiense in case of negative result, given the low parasitaemia generally observed in T. b. gambiense infections [57,58]. In addition, the microsatellite genotyping assays are known to lack sensitivity [59]. It is thus possible that T. b. gambiense parasites could have been missed by these two methods. In addition, the skin could be an important anatomical reservoir of T. b. gambiense as was recently demonstrated in experimentally infected mice [60–62]. We can thus not exclude that animals with negative PCR results on blood have trypanosomes in other body compartments such as the dermis. It is also possible that the presence of LiTat 1.3 and/or LiTat 1.5 specific antibodies indicates a previous transient infection. In addition, cross reactions of Litat 1.3 and 1.5 with other trypanosome species cannot be excluded. Bromidge et al. showed in 1993 that a PCR targeting the LiTat 1.3 gene showed a positive result in two stocks described as T. brucei brucei isolated from pigs in Côte d’Ivoire [63]. We thus cannot exclude that T. b. brucei strains circulating in our study areas are expressing the LiTat 1.3 VAT resulting in TL positive results.
In the context of HAT elimination, it will be crucial to improve the sensitivity of the T. b. gambiense PCR in order to detect this species in biological samples of animals, tsetse flies and human. Further studies are also needed to validate the specificity of LiTat 1.3 and 1.5 TL assays in animals. Controlled infection experiments in domestic animals will be needed to more fully evaluate the specificity and sensitivity of the currently available tools and evaluate their usefulness in research on the role of animals in the transmission of T. b. gambiense.
Our data suggest the existence of a potential domestic animal reservoir for T. b. gambiense HAT and provides indications for areas where the transmission may occur in the Sinfra and Bonon foci: the small wet and shady areas around the villages and the forest relics. Vector control using tiny targets [64,65] which are particularly favorable to disrupt the contact between domestic animals and tsetse flies may have a considerable impact on tsetse fly densities and trypanosome transmission in these areas. The concomitant treatment of pigs in the Sinfra focus and both pigs and cattle in the Bonon focus would furthermore help clearing out the potential reservoir of T. b. gambiense but also would contribute to animal trypanosomiasis control. This clearly illustrates the usefulness of applying a one health strategy for both HAT and AAT control.
However, these control measures have to be adapted to the study area and epidemiological context. In the endemic focus of Boffa [66] and in the historical focus of Loos Islands [67] in Guinea, domestic animals seem not to play a role in the epidemiology of HAT. In Cameroun [49,50,68], Congo [69], and Equatorial Guinea [17–19], the presence of human and animal infecting trypanosomes in domestic and/or wild animals could be evidenced, but important differences between study areas were highlighted regarding prevalence in hosts and their geographical distribution. We thus suggest, in low prevalence foci where HAT elimination seems reachable, to conduct animal surveys to define the most appropriate control measures to be implemented.
Conclusion
We have investigated the distribution of animal trypanosomes and the possible existence of a domestic animal reservoir of T. b. gambiense in two hypo-endemic HAT foci in Côte d’Ivoire. Our results show that T. brucei s.l. and T. congolense forest type circulate in the study areas and mainly infect pigs and cattle. Discordant results were obtained on the presence of T. b. gambiense, between PCR and TL methods. PCR did not detect T. b. gambiense while high seroprevalence was observed in TL. In the context of HAT elimination, it will be crucial to further investigate this discordance and to develop better tools and strategies to fully characterize the epidemiological role of an animal reservoir for T. b. gambiense.
Acknowledgments
We acknowledge all the technicians from the HAT teams of CIRDES (Bobo-Dioulasso), IPR (Bouaké), UJLoG (Daloa), CSU of Bonon, General Hospital of Sinfra and National Elimination Program (Abidjan). The authors also thank Saule Ibrah for helpful advice in writing the manuscript.
References
- 1. Franco JR, Simarro PP, Diarra A, Jannin JG (2014) Epidemiology of human African trypanosomiasis. Clin Epidemiol 6: 257–275. pmid:25125985
- 2.
WHO (2016) Lowest caseload recorded as the world prepares to defeat sleeping sickness. http://wwwwhoint/neglected_diseases/news/HAT_lowest_caseload_recorded/en/.
- 3. Franco JR, Simarro PP, Diarra A, Ruiz-Postigo JA, Jannin JG (2014) The journey towards elimination of gambiense human African trypanosomiasis: not far, nor easy. Parasitology 141: 748–760. pmid:24709291
- 4. Molyneux DH (1973) Animal reservoirs and Gambian trypanosomiasis. Ann Soc Belg Med Trop 53: 605–618. pmid:4204667
- 5. Simarro PP, Cecchi G, Franco JR, Paone M, Diarra A, et al. (2015) Monitoring the Progress towards the Elimination of Gambiense Human African Trypanosomiasis. PLoS Negl Trop Dis 9: e0003785. pmid:26056823
- 6. Dje NN, Miezan TW, N'Guessan P, Brika P, Doua F, et al. (2002) [Geographic distribution of trypanosomiasis treated in Ivory Coast from 1993 to 2000]. Bull Soc Pathol Exot 95: 359–361. pmid:12696376
- 7. Kambire R, Lingue K, Courtin F, Sidibe I, Kiendrebeogo D, et al. (2012) [Human African trypanosomiasis in Cote d'Ivoire and Burkina Faso: optimization of epidemiologic surveillance strategies]. Parasite 19: 389–396. pmid:23193524
- 8. Simarro PP, Franco JR, Diarra A, Ruiz Postigo JA, Jannin J (2013) Diversity of human African trypanosomiasis epidemiological settings requires fine-tuning control strategies to facilitate disease elimination. Research and Reports in Tropical Medicine 4: 1–6.
- 9. Koffi M, N'Djetchi M, Ilboudo H, Kaba D, Coulibaly B, et al. (2016) A targeted door-to-door strategy for sleeping sickness detection in low-prevalence settings in Cote d'Ivoire. Parasite 23: 51. pmid:27849517
- 10. Courtin F, Jamonneau V, Oke E, Coulibaly B, Oswald Y, et al. (2005) Towards understanding the presence/absence of Human African Trypanosomosis in a focus of Cote d'Ivoire: a spatial analysis of the pathogenic system. Int J Health Geogr 4: 27. pmid:16269078
- 11. Kaba D, Dje NN, Courtin F, Oke E, Koffi M, et al. (2006) [The impact of war on the evolution of sleeping sickness in west-central Cote d'Ivoire]. Trop Med Int Health 11: 136–143. pmid:16451337
- 12.
Laveissière C, Sané B, Garcia A (2003) Lutte contre la Maladie du Sommeil et Soins de Santé Primaires. Didactiques IRD ed, Paris: 243p.
- 13. Ravel S, de Meeus T, Dujardin JP, Zeze DG, Gooding RH, et al. (2007) The tsetse fly Glossina palpalis palpalis is composed of several genetically differentiated small populations in the sleeping sickness focus of Bonon, Cote d'Ivoire. Infect Genet Evol 7: 116–125. pmid:16890499
- 14. Ruiz JP, Nyingilili HS, Mbata GH, Malele II (2015) The role of domestic animals in the epidemiology of human African trypanosomiasis in Ngorongoro conservation area, Tanzania. Parasit Vectors 8: 510. pmid:26444416
- 15. Welburn SC, Coleman PG, Maudlin I, Fevre EM, Odiit M, et al. (2006) Crisis, what crisis? Control of Rhodesian sleeping sickness. Trends Parasitol 22: 123–128. pmid:16458071
- 16. Njiokou F, Nimpaye H, Simo G, Njitchouang GR, Asonganyi T, et al. (2010) Domestic animals as potential reservoir hosts of Trypanosoma brucei gambiense in sleeping sickness foci in Cameroon. Parasite 17: 61–66. pmid:20387740
- 17. Cordon-Obras C, Berzosa P, Ndong-Mabale N, Bobuakasi L, Buatiche JN, et al. (2009) Trypanosoma brucei gambiense in domestic livestock of Kogo and Mbini foci (Equatorial Guinea). Trop Med Int Health 14: 535–541. pmid:19320872
- 18. Cordon-Obras C, Garcia-Estebanez C, Ndong-Mabale N, Abaga S, Ndongo-Asumu P, et al. (2010) Screening of Trypanosoma brucei gambiense in domestic livestock and tsetse flies from an insular endemic focus (Luba, Equatorial Guinea). PLoS Negl Trop Dis 4: e704. pmid:20544031
- 19. Cordon-Obras C, Rodriguez YF, Fernandez-Martinez A, Cano J, Ndong-Mabale N, et al. (2015) Molecular evidence of a Trypanosoma brucei gambiense sylvatic cycle in the human african trypanosomiasis foci of Equatorial Guinea. Front Microbiol 6: 765. pmid:26257727
- 20. Jamonneau V, Ravel S, Koffi M, Kaba D, Zeze DG, et al. (2004) Mixed infections of trypanosomes in tsetse and pigs and their epidemiological significance in a sleeping sickness focus of Cote d'Ivoire. Parasitology 129: 693–702. pmid:15648692
- 21. Simo G, Rayaisse JB (2015) Challenges facing the elimination of sleeping sickness in west and central Africa: sustainable control of animal trypanosomiasis as an indispensable approach to achieve the goal. Parasit Vectors 8: 640. pmid:26671582
- 22. Van Meirvenne N, Magnus E, Buscher P (1995) Evaluation of variant specific trypanolysis tests for serodiagnosis of human infections with Trypanosoma brucei gambiense. Acta Trop 60: 189–199. pmid:8907397
- 23. Kiendrebeogo D, Kambire R, Jamonneau V, Lingue K, Solano P, et al. (2012) [History of an epidemiological route between Ivory Coast and Burkina Faso: the case of the Koudougou sleeping sickness foci]. Parasite 19: 397–406. pmid:23193525
- 24. Gouteux JP (1985) [Ecology of tsetse flies in the preforested area of the Ivory Coast. Relation to human trypanosomiasis and possibilities for control]. Ann Parasitol Hum Comp 60: 329–347. pmid:2998259
- 25. Laveissière C, Couret D, Staak C, Hervouët JP (1985) Glossina palpalis et ses hôtes en secteur forestier de Côte d'Ivoire: relations avec l'épidémiologie de la trypanosomiase humaine. Cahiers ORSTOMSérie Entomologie Médicale et Parasitologie 23: 297–303.
- 26. Sane B, Laveissiere C, Meda HA (2000) [Diversity of feeding behavior of Glossina palpalis palpalis in the forest belt of the Ivory Coast: relation to the prevalence of human African trypanosomiasis]. Trop Med Int Health 5: 73–78. pmid:10672209
- 27. Murray M, Murray PK, McIntyre WI (1977) An improved parasitological technique for the diagnosis of African trypanosomiasis. Trans R Soc Trop Med Hyg 71: 325–326. pmid:563634
- 28. Koffi M, Solano P, Denizot M, Courtin D, Garcia A, et al. (2006) Aparasitemic serological suspects in Trypanosoma brucei gambiense human African trypanosomiasis: a potential human reservoir of parasites? Acta Trop 98: 183–188. pmid:16723098
- 29. Moser DR, Cook GA, Ochs DE, Bailey CP, McKane MR, et al. (1989) Detection of Trypanosoma congolense and Trypanosoma brucei subspecies by DNA amplification using the polymerase chain reaction. Parasitology 99 Pt 1: 57–66.
- 30. Masiga DK, Smyth AJ, Hayes P, Bromidge TJ, Gibson WC (1992) Sensitive detection of trypanosomes in tsetse flies by DNA amplification. Int J Parasitol 22: 909–918. pmid:1459784
- 31. Majiwa PA, Thatthi R, Moloo SK, Nyeko JH, Otieno LH, et al. (1994) Detection of trypanosome infections in the saliva of tsetse flies and buffy-coat samples from antigenaemic but aparasitaemic cattle. Parasitology 108 (Pt 3): 313–322.
- 32. Radwanska M, Claes F, Magez S, Magnus E, Perez-Morga D, et al. (2002) Novel primer sequences for polymerase chain reaction-based detection of Trypanosoma brucei gambiense. Am J Trop Med Hyg 67: 289–295. pmid:12408669
- 33. MacLeod A, Tweedie A, McLellan S, Taylor S, Hall N, et al. (2005) The genetic map and comparative analysis with the physical map of Trypanosoma brucei. Nucleic Acids Res 33: 6688–6693. pmid:16314301
- 34. Biteau N, Bringaud F, Gibson W, Truc P, Baltz T (2000) Characterization of Trypanozoon isolates using a repeated coding sequence and microsatellite markers. Mol Biochem Parasitol 105: 185–201. pmid:10693742
- 35. Koffi M, Solano P, Barnabe C, de Meeus T, Bucheton B, et al. (2007) Genetic characterisation of Trypanosoma brucei s.l. using microsatellite typing: new perspectives for the molecular epidemiology of human African trypanosomiasis. Infect Genet Evol 7: 675–684. pmid:17704009
- 36. Kabore J, Koffi M, Bucheton B, Macleod A, Duffy C, et al. (2011) First evidence that parasite infecting apparent aparasitemic serological suspects in human African trypanosomiasis are Trypanosoma brucei gambiense and are similar to those found in patients. Infect Genet Evol 11: 1250–1255. pmid:21530681
- 37. Dieringer D, Schlotterer C (2003) Microsatellite analyser (MSA): a platform independent analysis tool for large microsatellite data sets. Mol Ecol Notes 3: 167–169.
- 38. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24: 1596–1599. pmid:17488738
- 39. Cavalli-Sforza LL, Edwards AW (1967) Phylogenetic analysis. Models and estimation procedures. Am J Hum Genet 19: 233–257. pmid:6026583
- 40. Takezaki N, Nei M (1996) Genetic distances and reconstruction of phylogenetic trees from microsatellite DNA. Genetics 144: 389–399. pmid:8878702
- 41. Jamonneau V, Bucheton B, Kabore J, Ilboudo H, Camara O, et al. (2010) Revisiting the immune trypanolysis test to optimise epidemiological surveillance and control of sleeping sickness in West Africa. PLoS Negl Trop Dis 4: e917. pmid:21200417
- 42. Koffi M, De Meeus T, Bucheton B, Solano P, Camara M, et al. (2009) Population genetics of Trypanosoma brucei gambiense, the agent of sleeping sickness in Western Africa. Proc Natl Acad Sci U S A 106: 209–214. pmid:19106297
- 43. Simukoko H, Marcotty T, Phiri I, Geysen D, Vercruysse J, et al. (2007) The comparative role of cattle, goats and pigs in the epidemiology of livestock trypanosomiasis on the plateau of eastern Zambia. Vet Parasitol 147: 231–238. pmid:17493757
- 44. Ravel S, Mediannikov O, Bossard G, Desquesnes M, Cuny G, et al. (2015) A study on African animal trypanosomosis in four areas of Senegal. Folia Parasitol (Praha) 62.
- 45. Simo G, Njiokou F, Mbida Mbida JA, Njitchouang GR, Herder S, et al. (2008) Tsetse fly host preference from sleeping sickness foci in Cameroon: epidemiological implications. Infect Genet Evol 8: 34–39. pmid:17977803
- 46. Koffi M, Sokouri M, Kouadio IK, N'Guetta SP (2014) Molecular characterization of trypanosomes isolated from naturally infected cattle in the "Pays Lobi" of Côte d’ivoire. J Appl Biosci 83: 7570–7578.
- 47. Bengaly Z, Sidibe I, Ganaba R, Desquesnes M, Boly H, et al. (2002) Comparative pathogenicity of three genetically distinct types of Trypanosoma congolense in cattle: clinical observations and haematological changes. Vet Parasitol 108: 1–19. pmid:12191895
- 48.
Hoare CA (1972) The trypanosomes of mammals. A Zoological Monograph. Blackwell Scientific Publications, Oxford: 749.
- 49. Nimpaye H, Njiokou F, Njine T, Njitchouang GR, Cuny G, et al. (2011) Trypanosoma vivax, T. congolense "forest type" and T. simiae: prevalence in domestic animals of sleeping sickness foci of Cameroon. Parasite 18: 171–179. pmid:21678793
- 50. Simo G, Asonganyi T, Nkinin SW, Njiokou F, Herder S (2006) High prevalence of Trypanosoma brucei gambiense group 1 in pigs from the Fontem sleeping sickness focus in Cameroon. Vet Parasitol 139: 57–66. pmid:16567049
- 51. Balyeidhusa AS, Kironde FA, Enyaru JC (2012) Apparent lack of a domestic animal reservoir in Gambiense sleeping sickness in northwest Uganda. Vet Parasitol 187: 157–167. pmid:22245071
- 52. Njiokou F, Laveissiere C, Simo G, Nkinin S, Grebaut P, et al. (2006) Wild fauna as a probable animal reservoir for Trypanosoma brucei gambiense in Cameroon. Infect Genet Evol 6: 147–153. pmid:16236560
- 53. Mehlitz D, Zillman U, Sachs R (1985) The domestic pigs as carriers of Trypanosoma brucei gambiense in West Africa. Tropenmed Parasitol 36, 18.
- 54. Koffi M, De Meeus T, Sere M, Bucheton B, Simo G, et al. (2015) Population Genetics and Reproductive Strategies of African Trypanosomes: Revisiting Available Published Data. PLoS Negl Trop Dis 9: e0003985. pmid:26491968
- 55. Funk S, Nishiura H, Heesterbeek H, Edmunds WJ, Checchi F (2013) Identifying transmission cycles at the human-animal interface: the role of animal reservoirs in maintaining gambiense human african trypanosomiasis. PLoS Comput Biol 9: e1002855. pmid:23341760
- 56. Berberof M, Perez-Morga D, Pays E (2001) A receptor-like flagellar pocket glycoprotein specific to Trypanosoma brucei gambiense. Mol Biochem Parasitol 113: 127–138. pmid:11254961
- 57. Deborggraeve S, Buscher P (2010) Molecular diagnostics for sleeping sickness: what is the benefit for the patient? Lancet Infect Dis 10: 433–439. pmid:20510283
- 58. Deborggraeve S, Buscher P (2012) Recent progress in molecular diagnosis of sleeping sickness. Expert Rev Mol Diagn 12: 719–730. pmid:23153239
- 59. Kabore J, De Meeus T, Macleod A, Ilboudo H, Capewell P, et al. (2013) A protocol to improve genotyping of problematic microsatellite loci of Trypanosoma brucei gambiense from body fluids. Infect Genet Evol 20: 171–176. pmid:23954418
- 60. Caljon G, Van Reet N, De Trez C, Vermeersch M, Perez-Morga D, et al. (2016) The Dermis as a Delivery Site of Trypanosoma brucei for Tsetse Flies. PLoS Pathog 12: e1005744. pmid:27441553
- 61. Capewell P, Cren-Travaille C, Marchesi F, Johnston P, Clucas C, et al. (2016) The skin is a significant but overlooked anatomical reservoir for vector-borne African trypanosomes. Elife 5.
- 62. Trindade S, Rijo-Ferreira F, Carvalho T, Pinto-Neves D, Guegan F, et al. (2016) Trypanosoma brucei Parasites Occupy and Functionally Adapt to the Adipose Tissue in Mice. Cell Host Microbe 19: 837–848. pmid:27237364
- 63. Bromidge T, Gibson W, Hudson K, Dukes P (1993) Identification of Trypanosoma brucei gambiense by PCR amplification of variant surface glycoprotein genes. Acta Trop 53: 107–119. pmid:8098897
- 64. Lehane M, Alfaroukh I, Bucheton B, Camara M, Harris A, et al. (2016) Tsetse Control and the Elimination of Gambian Sleeping Sickness. PLoS Negl Trop Dis 10: e0004437. pmid:27128795
- 65. Rayaisse JB, Esterhuizen J, Tirados I, Kaba D, Salou E, et al. (2011) Towards an optimal design of target for tsetse control: comparisons of novel targets for the control of Palpalis group tsetse in West Africa. PLoS Negl Trop Dis 5: e1332. pmid:21949896
- 66. Kagbadouno MS, Camara M, Rouamba J, Rayaisse JB, Traore IS, et al. (2012) Epidemiology of sleeping sickness in Boffa (Guinea): where are the trypanosomes? PLoS Negl Trop Dis 6: e1949. pmid:23272259
- 67. Kagbadouno M, Camara M, Bouyer J, Hervouet JP, Courtin F, et al. (2009) Tsetse elimination: its interest and feasibility in the historical sleeping sickness focus of Loos islands, Guinea. Parasite 16: 29–35. pmid:19353949
- 68. Njitchouang GR, Njiokou F, Nana-Djeunga H, Asonganyi T, Fewou-Moundipa P, et al. (2011) A new transmission risk index for human African trypanosomiasis and its application in the identification of sites of high transmission of sleeping sickness in the Fontem focus of southwest Cameroon. Med Vet Entomol 25: 289–296. pmid:21198712
- 69. Noireau F, Paindavoine P, Lemesre JL, Toudic A, Pays E, et al. (1989) The epidemiological importance of the animal reservoir of Trypanosoma brucei gambiense in the Congo. 2. Characterization of the Trypanosoma brucei complex. Trop Med Parasitol 40: 9–11. pmid:2740734