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Infection rate of Trypanosoma brucei s.l., T. vivax, T. congolense “forest type”, and T. simiae in small wild vertebrates in south Cameroon
Acta Tropica 92 (2004) 139–146
Infection rate of Trypanosoma brucei s.l., T. vivax, T. congolense “forest type”, and T. simiae in small wild vertebrates in south Cameroon F. Njiokoua,b,∗ , G. Simoa,d , S.W. Nkinina , C. Laveissi`erec , S. Herderc a LRT, OCEAC, BP 288 Yaound´ e, Cameroon Universit´e de Yaound´e I, Facult´e des Sciences, BP 812 Yaound´e, Cameroon LRCT/IRD - CIRAD, TA 207/G, Campus international de Baillarguet, 34398 Montpellier cedex 5, France d IMPM, MINREST, Yaound´ e, Cameroon b
c
Received 16 June 2003; received in revised form 10 March 2004; accepted 6 April 2004 Available online 14 August 2004
Abstract In order to identify the infection rate of trypanosome species infecting wild animals in four localities (Bipindi, Campo, Fontem and Nditam) of southern Cameroon, 1,141 wild animals were sampled. These animals belonged to 36 species grouped in 8 orders including 407 primates, 347 artiodactyls, 264 rodents, 54 pangolins, 53 small carnivores, 11 saurians and crocodilians and 5 hyraxes. PCR using specific primers for Trypanosoma vivax, T. brucei s.l., T. congolense “forest type”, and T. simiae showed that 18.7% of the animals were infected by at least one of these trypanosome species. A positive PCR result may not indicate absolutely an active infection because PCR can detect also transient infections. T. vivax (Duttonella) had the highest infection rate (9.5%) and was found in almost all the host orders studied. T. brucei s.l. mostly infected primates, rodents and some duikers (Cephalophus dorsalis and C. monticola). Trypanosomes of the subgenus Nannomonas had a lower infection rate of 5.5% (2.4% for T. simiae and 3.1% for T. congolense “forest type”). They were harboured mainly by primates, ungulates and rodents. Trypanosome infection rates were highest in Nditam (24.5%) and Bipindi (21%). T. brucei s.l. (Trypanozoon) had its maximum infection rate of 10.4% in Bipindi. The “Quantitative Buffy Coat” (QBC® ) and Kit for in vitro isolation techniques were used to identify 48 (6.1%) infected animals. 13 were positive using QBC® , and 42 were positive by KIVI. However, PCR was negative on 16 of these infected animals, probably due to infections with other trypanosome species. This study showed that trypanosomes of the subgenera Duttonella, Nannomonas and Trypanozoon could infect small wild vertebrates as has been shown for large ungulates and carnivores. The presence of T. brucei s.l. in a large range of wild animals strengthens the hypothesis of the existence of a wild animal reservoir of T. b. gambiense in Cameroon. © 2004 Elsevier B.V. All rights reserved. Keywords: Wild animal trypanosomes; PCR; Infection rate; Reservoir; Cameroonian forest ∗ Corresponding author. Tel.: +237 223 22 32; fax: +237 223 00 61. E-mail address: [email protected] (F. Njiokou).
0001-706X/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2004.04.011
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1. Introduction In Africa, trypanosome infections constitute an important cause of morbidity and mortality in domestic and wild animals in areas where tsetse flies are present. Most studies performed on African animal trypanosomiasis (AAT) have been limited to domestic animals because of the difficulties of obtaining blood samples from wild animals. These latter animals may constitute a reservoir host for trypanosomes responsible for animal and human trypanosomiasis (Gibson et al., 1978; Mehlitz et al., 1982; Joshua et al., 1983; Zillmann et al., 1984; Mehlitz, 1986, Asonganyi et al., 1986, 1990). Previous studies have identified most subgenera of salivarian trypanosomes in domestic and wild animals from subsaharan zones (Kupper et al., 1983; Mattioli et al., 1990; Mehlitz, 1986). However, the distribution of these parasites in wild animals is still not well-known since the commonly used diagnostic tools are not highly sensitive and specific. Even with the development of concentration techniques, such as haematocrit centrifuge technique (HTC) (Woo, 1970) and “Quantitative Buffy Coat” (QBC® ) (Bailey and Smith, 1992), the threshold of detection of trypanosomes remains high. Thus, some infected animals are not diagnosed. In addition, parasitological tests do not easily differentiate trypanosome species or sub-species that infect animals. Specific or sub-specific identification of trypanosomes in animals requires isolation, culture and characterisation by costly techniques, such as isoenzyme electrophoresis, restriction fragment length polymorphism and hybridisation. To compensate for the low sensitivity and low specificity of parasitological tests, a diagnostic technique using the polymerase chain reaction (PCR) has been developed. It enables the in vitro amplification of repetitive and specific nuclear DNA sequences of T. brucei s.l., T. vivax, T. simiae and three types (“savannah”, “forest” and “kilifi”) of T. congolense (Moser et al., 1989; Masiga et al., 1992; Artama et al., 1992; McNamara et al., 1994). The use of PCR on blood extracts enables the specific identification (without culture and isolation of parasites) of natural infections of most trypanosomes species and sub-species. In addition, the high sensitivity of this technique helps to considerably reduce the threshold levels of detection to as low as one trypanosome per millilitre of blood (Penchenier et al., 1996).
This study aimed to identify, by PCR, trypanosome species infecting wild animals, and to determine the infection rate of these parasites in wildlife in southern forest area of Cameroon. This identification is important to understand some epidemiological aspects of African trypanosomiasis, notably trypanotolerence in AAT and the existence of human African trypanosomiasis (HAT) animal reservoir.
2. Materials and methods 2.1. Study sites The study was carried in 4 localities in the southern Cameroonian forest characterized by the co-existence of tsetse flies, a large variety of wild fauna and intensive hunting activities by the inhabitants. - Bipindi (3◦ 2 N, 10◦ 22 E) covers several villages that are mainly located along roads (Gr´ebaut et al., 2001). The vegetation is dense equatorial forest interspersed with farmland. This region is surrounded by hills and has a dense network of several fast running streams. These streams pass through cocoa farms offering favourable conditions (such as shade and microclimate) for the presence of tsetse flies. - Campo (2◦ 20 N, 9◦ 52 E) lies along the Atlantic coast and extends along the Ntem river which constitutes the Cameroonian and Equatorial Guinean border. Campo is located in the equatorial rain forest zone. There is a dense network of several rivers, swampy areas and marshes. - Doum´e (4◦ 16 N, 13◦ 25 E) vegetation is mainly characterised by dense equatorial forest with an important network constituted by the river Doum´e and many large ponds. - Nditam locality (5◦ 20 N, 11◦ 9 E) belongs to the Tikar plain. The hydrographic network is dominated by the Mbam river and its affluents whose levels depend on the amount of annual rainfall (about 1600 mm). The vegetation is a mosaic of forests and savannahs, with woodland, grassland and shrubbery. The fauna and flora are highly diversified to the extent that Nditam area is considered as a safari park, where fishing and hunting activities are very frequently practised.
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2.2. Blood collection and parasitological tests Animals that were captured by hunters and trappers and brought to the village were identified using systematic keys (Depierre and Vivien, 1992; Dorst and Dandelot, 1997; Spawls et al., 2002). Cardiac punctures were made on dead animals, while intravenous blood collection were performed on living animals. From living or dead animals whose veins still contained fresh blood, a second blood sample was obtained under sterile conditions in KIVI medium for isolation of parasites (Aerts et al., 1992). Blood collected in EDTA tubes was used for parasitological diagnosis using the QBC® method (Bailey and Smith, 1992) and for PCR. In order not to encourage hunting as some of these animals are protected, no prior contacts were made with hunters before meeting them. Moreover, visits to villages were made unannounced. The follow-up of KIVI was done by microscopical examination of samples in the laboratory. Positive KIVI samples were grown in Cunningham medium (Cunningham, 1977). 2.3. PCR DNA was extracted from blood using the “ReadyAmp® Genomic DNA Purification System” (Promega) kit as described by Penchenier et al. (2000). The supernatant collected was used directly for PCR or stored at −20 ◦ C. Four pairs of specific primers for T. brucei s.l., T. congolense “forest type”, T. vivax and T. simiae (Masiga et al., 1992) were used. PCR amplifications were performed as described by Herder et al. (2002). The amplification was done on 25 l of mixture containing 5 l of DNA extract, 10 mM of Tris–HCl (pH 9), 50 mM of KCl, 3 mM of MgCl2 , 0.8 pM of each primer, 200 M of dNTP (dTTP, dATP, dCTP and dGTP) and 1 unit of Taq DNA polymerase (Appligene–Oncor, USA). One denaturing step at 94 ◦ C for 5 min was programmed before 40 amplification cycles. Each cycle contained a denaturing step at 94 ◦ C for 30 seconds, annealing at 55 ◦ C (T. brucei s.l.) or at 60 ◦ C (T. congolense “forest type”, T. vivax and T. simiae) for 30 s and an extension step at 72 ◦ C for 1 min. A final extension was done at 72 ◦ C for 10 min. The amplified products were analysed on 1.5% agarose gel containing ethidium bromide and visualised by UV light. For each extraction series a neg-
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ative control tube constituted of blood from uninfected animal was incorporated and processed at last. This control tube was used further as negative control during PCR reaction in addition to classical PCR negative control constituted by distilled water. Further more, the experimenter who performed the PCR had no information on the initial parasitological status of the samples. PCR test was repeated on all PCR positive and also on some negative samples randomly selected. 2.4. Statistical analysis The chi-square test (Scherrer, 1984) was used to compare the overall trypanosome infections rate and the prevalence of trypanosome species among animal groups and among localities.
3. Results Wild animals were sampled during 11 field surveys (5 at Bipindi, 2 at Campo, 2 at Doum´e and 2 at Nditam) from 1999 to 2001. One thousand, one hundred and forty one wild animals examined belonged to 36 species grouped into 8 orders as follows: 407 (35.7%) primates, 347 (30.4%) artiodactyls, 264 (23.1%) rodents, 54 (4.7%) pangolins, 53 (4.6%) small carnivores, 11 (1%) saurians and crocodilians and 5 (0.4%) hyraxes (Table 1). Parasitological tests (QBC® and/or KIVI) were done on 789 (69%) of the 1,141 animals examined. These tests revealed 48 (6.1%) infected animals of which 42 were positive with KIVI and 13 with QBC® . Of the 762 blood samples analysed by QBC® , 13 (1.7%) were positive (presence of live trypanosomes with fluorescent nucleus and kinetoplast due to acridine orange). Specific morphological identification was not possible. Of the 234 KIVI performed 42 (18%) were positive. 8 trypanosome strains were isolated: SitaBip 1 from a Sitatunga in Bipindi, CroCamp 1 and CroCamp 2 from crocodiles in Campo, CepCamp3, CepCamp4 and CepCamp5 from blue duikers in Campo, NanDoum1 from a palm civet in Doum´e, and HochNdi1 from a white-nosed monkey in Nditam. Of the 1,141 animals examined, 213 (18.7%) were positive by PCR (Table 1). A typical result of PCR analysis for T. brucei s.l. is shown in Fig. 1. Trypanosome
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Table 1 Trypanosomes infection rates in different animal species and orders Common names
Scientific names
N
Number of positive PCR TB s.l.
TCF
TS
TV
NP
White-eyelid mangabey Crested mangabey Cloaked mangabey De Brazza’s monkey Guereza white colobus Greater white-nosed monkey Mandrill Mona monkey Moustached monkey Dwarf guenon Bosman potto Golden potto
Cercocebus torquatus Cercocebus neglectus Cercocebus albigena Cercopithecus neglectus Colobus guereza Cercopithecus nictitans Mandrilus sphinx Cercopithecus mona Cercopithecus cephus Miopithecus talapoin Perodicticus potto Arctocebus calabarensis
5 2 12 1 14 155 5 46 101 55 8 3
1 – 2 – 1 10 – 1 4 5 2 1
– – – – 1 6 – 2 2 – 1 1
– – 1 – – 3 – 1 – – – –
2 – 2 1 2 22 – 8 11 5 3 –
3 – 5 1 3 33 – 12 15 9 4 1
Water chevrotain Royal antelope Blackstriped duiker Yellow-backed duiker Blue duiker Ogilby’s duiker Peter’s duiker Sitatunga
Primates ST Hyemoschus aquaticus Neotragus pygmaeus Cephalophus dorsalis Cephalophus silvicultor Cephalophus monticola Cephalophus ogilbyi Cephalophus callipygus Tragelaphus spekei
407 1 5 37 3 290 1 5 5
27 (6.6) – – 4 – 14 – – 1
13 (3.2) – – 2 – 16 – – –
5 (1.2) – – 3 – 14 – – –
56 (14) – – 3 – 24 – – 1
86 (21) – – 10 – 62 – – 1
Brush-tailed porcupine Greater cane rat Giant forest squirrel Red-legged sun squirrel Giant rat
Artiodactyls ST Atherurus africanus Thryonomys sp. Protoxerus stangeri Heliosciurus rufobrachium Cricetomys gambianus
347 106 15 1 6 136
19 (5.5) 11 2 – 1 6
18 (5.2) 2 – – 1 1
17 (5) 3 – – – 2
28 (8) 7 – – – 4
73 (21) 20 2 – 2 11
Golden cat Black legged mangoose Dark mangoose Small-spotted genet African civet Two-spotted palm civet
Rodents ST Profelis aurata Bdeogale nigripis Crossarchus obscurus Genetta servalina Viverra civetta Nandinia binotata
264 1 1 8 8 3 32
20 (7.6) – – 1 1 – 3
4 (1.5) – – – – – –
5 (1.9) – – – – – –
11 (4.8) – – – 1 – 3
35 (13.3) – – 1 1 – 5
Long-tailed pangolin Tree pangolin
Carnivores ST Manis tetradactyla Manis tricuspis
53 34 20
5 (9.4) 2 1
0 – –
0 – –
4 (7.5) 2 5
7 (13.2) 4 6
Crocodile Monitor lizard
Pangolins ST Crocodylus niloticus Varanus ornatus
54 3 8
3 (5.6) – –
0 – –
0 – –
7 (13.2) 1 1
10 (18.5) 1 1
Tree dassie
Saurians and crocodilians ST Dendrohyrax arboreus
11 5
0 (0) –
0 (0) –
0 (0) –
2 (18.2) –
2 (18.2) –
Hyraxes ST
5
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
1141
74 (6.5)
35 (3.1)
27 (2.4)
108 (9.5)
213 (18.7)
Total Sample rate of infection (%)
Percentages of infected animals are given in brackets. TB s.l., Trypanosoma brucei s.l.; TCF, Trypanosoma congolense “forest type”; TV, Trypanosoma vivax; TS, Trypanosoma simiae; N, number of animals examined; ST, sub-total; NP, number of wild animals harbouring at least one parasite species (this number is lower than the total number of infections recorded because some animals had multiple infections).
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Fig. 1. Example of agarose electrophoresis obtained after amplification of T. brucei s.l. specific sequences. M, molecular marker; C− , negative control; C+ , positive control; 1–9, wild animal blood samples tested; 1, 2, 4, 5, 6, 8 and 9, negative samples; 3 and 7, positive samples.
infection rates for the subgenera Duttonella (T. vivax) and Trypanozoon (T. brucei s.l.) were 9.5% and 6.5%, respectively, while that of the subgenus Nannomonas was relatively low, 5.5%, of which 56% were T. congolense “forest type” and 44% were T. simiae (Table 1). The infection rates differed significantly (χ2 = 64; P < 0.0001). The observed frequency was T. vivax > T. brucei s.l. > T. congolense “forest type” > T. simiae. As for the wild animal categories, primates, artiodactyls and rodents harboured all four sibling parasites, whereas pangolins and small carnivores were infected by only T. brucei s.l. and T. vivax, and reptiles by only T. vivax. None of the hyraxes examined was infected (Table 2). The overall trypanosome infection rates were not significantly different between the seven animal groups (χ2 = 7.29; P = 0.29), nor between the five
groups with sample size superior to 50 individuals (χ2 = 6.33; P = 0.17). The infection rates in Nditam, Bipindi, Campo and Doum´e were 24.5%, 21%, 11.7% and 10%, respectively. These values differed significantly between the four localities (Table 2): wild animals in Nditam and in Bipindi harboured significantly more trypanosome infections than those in Campo and in Doum´e. The prevalence values of T. simiae and T. vivax did not differ between localities, whereas animals in Bipindi and in Nditam were significantly more infected by T. brucei s.l. and T. congolense “forest type” respectively (Table 2). Of the 213 wild animals that were positive by PCR, 25 (11.7%) had double infections, 3 (1.4%) harboured triple infections and the rest had a single infection. Dou-
Table 2 Trypanosome infection rates in different localities Localities
Bipindi Campo Doum´e Nditam Total χ2 P
N
n (%) TB s. l
TCF
TS
TV
NP
499 154 178 310
52 (10.4) 5 (3.2) 5 (2.8) 12 (3.9)
10 (2) 4 (2.6) 0 (0) 21 (6.8)
12 (2.4) 3 (2) 0 (0) 12 (3.9)
46 (9.2) 9 (5.8) 12 (6.7) 41 (13.2)
103 (21) 18 (11.7) 16 (10) 76 (24.5)
1141
74 (6.5)
35 (3.1)
27 (2.4)
108 (9.5)
213 (18.7)
19.9 0.0002
20.4 0.0001
7.2 0.07 ns
7.4 0.06 ns
17.2 0.0006
TB s.l., Trypanosoma brucei s.l.; TCF, Trypanosoma congolense “forest type”; TV, Trypanosoma vivax; TS, Trypanosoma simiae; N, number of animals tested; n, number of positive animals for specific primers used; (%), percentage of positive PCR; NP, number of animal with at least one positive PCR (this number is lower than the total number of positive PCR as some animals harboured more than one trypanosome species). P, level of significance of the χ2 test; ns, non significant.
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Table 3 Parasitological positive hosts identified or not identified by PCR Hosts
Not identified
Identified
Trypanosome DNAs detected
Blue duiker Greater white-nosed monkey Mona monkey Sitatunga Crocodile Bosman potto Tree pangolin Palm civet
7 5
19 6
TB, TV, TCF, TS TB, TV
1 1 1 0 1 0
1 1 1 3 0 1
TB, TV, TCF TB, TV TV, TB, TV, TCF
Total
16
32
TV
TB, Trypanosoma brucei s.l.; TV, Trypanosoma vivax; TCF, Trypanosoma congolense “forest type”; TS, Trypanosoma simiae.
ble infections included 11 animals infected with T. brucei s.l. and T. vivax, 7 with T. simiae and T. vivax, 4 with T. congolense “forest type” and T. vivax, 2 with T. simiae and T. congolense “forest type” and 1 with T. brucei s.l. and T. congolense “forest type”. Triple infections involved T. brucei s.l., T. vivax and T. simiae, T. brucei s.l., T. vivax and T. congolense “forest type”, and T. brucei s.l., T. simiae and T. congolense “forest type”. Concerning the eight stocks isolated by KIVI, two PCR results using the four couples of primers studied was available: Firstly, PCR detected T. brucei s.l. from the original blood sample of SitaBip1, T. vivax from the original blood samples of NanDoum1, CroCamp2, CepCamp3 and CepCamp4, and none of the four trypanosomes in the original blood samples of CroCamp1, and CepCamp5. Secondly, PCR using the same primers confirmed the presence of T. brucei s.l. in SitaBip1 stocks and none of the four trypanosomes in the other unidentified trypanosome stocks. Of the 48 animals that were positive using parasitological tests, 16 were negative by PCR whereas 32 animals were positive. Trypanosomes could be positively identified using the available primers in all groups of hosts except pangolins (Table 3). PCR was positive in 125 animals negative with parasitological examinations. 4. Discussion The rate of trypanosome infection detected by the parasitological tests (6.1%) was relatively low, prob-
ably due to the low sensitivity of the QBC® as well as several technical constraints, in particular difficulties to get blood samples and inoculate them into KIVI flask under sterile conditions, and the lapse of time between animal slaughtering and performing QBC® (2–6 h). These difficulties could considerably decrease the accuracy of parasitological techniques (Bailey and Smith, 1992; Ancelle et al., 1997). Our PCR results showed that 18.7% of wild animals were infected by at least one of the four trypanosome species. The high infection rate of trypanosomes in wild animals in Nditam and Bipindi could be explained by the fact that Bipindi is currently the HAT focus in Cameroon with the highest sleeping sickness prevalence (Gr´ebaut et al., 2001). Nditam is located near the Adamaoua Province, the main region of livestock location with the highest prevalence of African animal trypanosomiasis in Cameroon. T. vivax (Duttonella) had the highest infection rate of 9.5%, whereas T. congolense “forest type” and T. simiae (both Nannomonas) species had the lowest infection rates of 3.1% and 2.4% respectively. These levels of infection differed from previous study conducted in West African (Komoin-Oka et al., 1994). The difference may be ascribed to the wild animal host species. In west Africa, studies performed on large mammals (Loxodonta africana, Syncerus caffer, Kobus kob, Kobus defassa) revealed mostly T. congolense and very few T. vivax (Komoin-Oka et al., 1994; Truc et al., 1997), whereas the present study detected mostly T. vivax (Duttonella), few T. simiae and T. congolense “forest type” (Nannomonas) in a large variety of small vertebrates. Despite relatively small sample numbers, (pangolins, small carnivores and reptiles) these animals were found to be infected by T. vivax and T. brucei s.l. T. vivax (Duttonella) was very frequent and was often associated with T. brucei s.l. (Trypanozoon) or T. simiae and T. congolense (Nannomonas). Very few mixed infections involved trypanosomes of the subgenera Nannomonas and Trypanozoon. However, some animal hosts found in this study were quite unexpected, when comparing the review by Hoare (1972). For example, T. vivax, a parasite of various domestic and wild ungulates was detected in monitor Lizard and crocodile. T. simiae, a suid parasite, already found in non-suid hosts (camels, cattle and horse) was detected from two monkeys, two duikers and two rodents. These findings may be partially explained because PCR detects also
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transient infections. A PCR positive result indicates the presence of the corresponding parasite DNA and not necessary an active infection (Herder et al., 2002). The positive PCR results may indicate the presence of T. vivax DNA in these animals without active infection, because of the clearance of this parasite by the host immune system. The more probable hypothesis is that the trypanosomes detected have been inoculated by infected tsetse flies, but have failed to establish an infection. Previous results in West Africa showed that 46% of G. p. palpalis blood meals were from humans and 46% from Sitatunga (Laveissi`ere et al., 1985) whereas in south Cameroon, the analysis of 137 tsetse blood meals showed that 48% were from humans, 25% from domestic animals (pig and sheep), 16% from wild animal (Sitatunga and Golden Cat), and 10% not yet identified, probably also from wild animals (Njiokou et al., 2003). Tsetse flies could then carry trypanosomes from one host to another. Morlais et al. (1998) revealed T. vivax in tsetse flies from southern forest of Cameroon. Detection of trypanosome species in non-common hosts needs to be confirmed by their isolation with appropriate medium, culture and characterisation. In this study, T. vivax DNA was detected from original blood samples of NanDoum1, CroCamp2, CepCamp3 and CepCamp4 strains but was absent in these isolated stocks; the explanation could be their degeneration in the KIVI medium which is not suitable for this species, but also because some of the positive animals might not harbour active infections. T. brucei s.l. showed the second highest infection rate and was present in both large and small vertebrates. T. brucei s.l. infection rate was low in three localities: Doum´e (2.8%), Campo (3.2%), and Nditam (3.9%), and high in Bipindi (10.4%). The high level of infection rate of T. brucei s.l. may be due to the presence of T. b. gambiense in a large range of small wild mammals in Bipindi (Herder et al., 2002). Apart from Doum´e, where no trypanosomes of the sub-genus Nannomonas (T. simiae and T. congolense) were detected, all species have been identified in animals from Bipindi, Campo and Nditam. T. vivax was detected in wild animals from all localities. Negative PCR results in 16 infected animals could be due to the presence of other trypanosome species. Previous studies detected other trypanosome species, such as T. congolense “savannah type”, T. con-
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golense “kilifi type” and Megatrypanum (Itard, 1981; Mattioli et al., 1990; Herder et al., 2002). Crocodiles could harbour reptilian trypanosomes such as Trypanosoma grayi not investigated in the present study. This study showed that the four trypanosome species DNAs could be detected in a large range of wild animals by the PCR. However, PCR positive results indicate the presence of the parasite DNA in the vertebrate hosts and not necessarily an active infection. As PCR detects also transient infections, it is therefore useful to confirm new hosts status by the isolation of the parasites in an appropriate medium and characterisation. The use of specific primers for other trypanosome species and sub-species could enable the identification of all trypanosomes infecting animals and may allow a better understanding of the role of wild animals in the epidemiology of African trypanosomiasis.
Acknowledgements This work was supported by Institut de Recherche pour le D´eveloppement (IRD). We thank C. F. Bilong, A. Monkiedje, L. Basco and three anonymous referees for critical reading of the manuscript, and Ernest Mooh and Samuel Bahebegue for technical assistance.
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Cunningham, I., 1977. New culture medium for maintenance of tsetse tissues and growth of trypanosomatids. J. Protozool. 21, 325–329. Depierre, D., Vivien, J., 1992. Mammif`eres sauvages du Cameroun. Presses de l’office nationale des forˆets, boulevard de Constance 77300, Fontainebleau. Dorst, J., Dandelot, P., 1997. Guide des mammif`eres d’Afrique. Delachaux et Niesfl´e, 2`eme edition. Gibson, W.C., Mehlitz, D., Lanham, S., Godfrey, D.G., 1978. The identification of Trypanosoma brucei gambiense in Liberian pigs and dogs by isoenzymes and by resistance to human plasma. Tropenmed. Parasitol. 29, 335–345. Gr´ebaut, P., Bodo, J.M., Assona, A., Foumane Ngane, V., Njiokou, F., Ollivier, G., Soula, G., Laveissi`ere, C., 2001. Recherche des facteurs de risque de la Trypanosomiase Humaine Africaine dans le foyer de Bipindi au Cameroun. Med. Trop. 61, 377– 383. Herder, S., Simo, G., Nkinin, S., Njiokou, F., 2002. Identification of trypanosomes in wild animals from southern Cameroon using the polymerase chain reaction (PCR). Parasite 9, 345–349. Hoare, C.A., 1972. The trypanosomes of mammals. In: A Zoological Monograph. Blackwell Scientific Publications, Oxford and Edinburgh. Itard, J., 1981. Les trypanosomoses animales africaines. In: Pr´ecis de parasitologie v´et´erinaire, Minist`ere de la coop´eration et du d´eveloppement, Paris. Manuels et pr´ecis d’´elevage 10, 303– 469. Joshua, R.A., Magaji, Y., Kayit, Y.S., 1983. Isolation of human serum resistant Trypanozoon from cattle in Nigeria. Tropenmed. Parasit. 34, 201–202. Komoin-Oka, C., Truc, P., Bengaly, Z., Formenty, P., Duvallet, G., Lauginie, F., Raath, J.P., N’Depo, A.E., Leforban, Y., 1994. Etude de la pr´evalence des infections a` trypanosomes chez diff´erentes esp`eces d’animaux sauvages du parc national de la Como´e en Cˆote d’Ivoire: R´esultats pr´eliminaires sur la comparaison de trois m´ethodes de diagnostic. R´ev. Elev. Med. V´et. Pays Trop. 47 (2), 189–194. Kupper, W., Wolters, M., Tscharf, I., 1983. Observations on kob antelope (Kobus kob) in northen Ivory Coast and their epizootiological role in trypanosomiasis transmission. Z. Angew Zool. 70, 227–283. Laveissi`ere, C., Couret, D., Staak, C., Hervouet, J.P., 1985. Glossina palpalis et ses hˆotes en secteur forestier de Cˆote d’Ivoire. Relation avec l’´epid´emiologie de la trypanosomiase humaine. Cah. ORSTOM, S´er. Ent. M´ed. Parasitol. 23, 297–303. Masiga, D.K., Smith, J.A., Hayes, P., Bromidge, J.T., Gibson, W.C., 1992. Sensitive detection of trypanosomes in tsetse flies by DNA amplification. Int. J. Parasitol. 22, 909–918.
Mattioli, R.C., Jean, O., Belem, A.M.G., 1990. Incidence de la trypanosomose sur la faune sauvage d’un ranch de gibier au Burkina Faso. Rev. Elev. Med. V´et. Pays Trop. 44, 165–168. McNamara, J.J., Mohammed, G., Gibson, W.C., 1994. Trypanosoma godfreyi (Nannomonas) sp. nov. from tsetse flies in the Gambia: biological and biochemical characterization. Parasitology 109, 497–509. Mehlitz, D., Zillmann, U., Scott, C.M., Godfrey, D.G., 1982. Epidemiological studies on the animal reservoir of gambiense sleeping sickness. 3: Characterization of Trypanozoon stocks by isoenzymes and sensitivity to human serum. Tropenmed. Parasitol. 33, 113–118. Mehlitz, D., 1986. Le r´eservoir animal de la maladie du sommeil a` Trypanosoma brucei gambiense. Etudes et synth`eses de l’I.E.M.V.T., Ed. Deutsche Gesellschaft f¨ur Technische Zusammenarbeit (GTZ) (Eds.), Eschborn, RFA 18: 156. Morlais, I., Gr´ebaut, P., Bodo, J.M., Djoha, S., Cuny, G., 1998. Characterization of trypanosome infections by polymerase chain reaction (PCR) amplification in wild tsetse flies in Cameroon. Parasitology 116, 547–554. Moser, D.R., Cook, G.A., Ochs, D.E., Bailey, C.P., McKane, M.R., Donelson, J.E., 1989. Detection of Trypanosoma congolense and Trypanosoma brucei subspecies by DNA amplification using the polymerase chain reaction. Parasitology 99, 57–66. Njiokou F., Simo, G., Mbida Mbida, A., Truc, P., Herder, S., 2003. Identification of vertebrate blood meals in tsetse flies by the Heteroduplex PCR based method. 27th International Scientifique Conference for Trypanosomiasis Research and Control, Pretoria. Penchenier, L., Dumas, V., Gr´ebaut, P., Reifemberg, J.M., Cuny, G., 1996. Improvement of blood and fly gut processing for diagnosis of Trypanosomosis. Parasite 4, 387–389. Penchenier, L., Simo, G., Gr´ebaut, P., Nkinin, S., Laveissi`ere, C., Herder, S., 2000. Diagnosis of human trypanosomiasis, due to Trypanosoma brucei gambiense in Central Africa, by Polymerase Chain Reaction. Trans. R. Soc. Trop. Med. Hyg. 94, 392–394. Scherrer, B., 1984. Biostatistique. Ga¨etan Morin (Eds.), Quebec. Spawls, S., Howell, K., Drewes, R., Ashe, J., 2002. A field guide to the reptiles of East Africa. Elsevier (Eds.), London. Truc, P., Formenty, P., Diallo, P.B., Komoin-Oka, C., Lauginie, F., 1997. Identification of trypanosomes isolated by KIVI from wild animals in Cˆote d’Ivoire: diagnostic, taxonomic and epidemiological considerations. Acta Tropica. 67, 1–10. Woo, P.T.K., 1970. The haematocrit centrifuge technique for the diagnosis of african trypanosomiasis. Acta Tropica. 27, 384–386. Zillmann, D., Mehlitz, D., Sachs, R., 1984. Identity of Trypanozoon stocks isolated from man and domestic animal in Liberia. Tropenmed. Parasitol. 35, 105–108.
Infection rate of Trypanosoma brucei s.l., T. vivax, T. congolense “forest type”, and T. simiae in small wild vertebrates in south Cameroon F. Njiokoua,b,∗ , G. Simoa,d , S.W. Nkinina , C. Laveissi`erec , S. Herderc a LRT, OCEAC, BP 288 Yaound´ e, Cameroon Universit´e de Yaound´e I, Facult´e des Sciences, BP 812 Yaound´e, Cameroon LRCT/IRD - CIRAD, TA 207/G, Campus international de Baillarguet, 34398 Montpellier cedex 5, France d IMPM, MINREST, Yaound´ e, Cameroon b
c
Received 16 June 2003; received in revised form 10 March 2004; accepted 6 April 2004 Available online 14 August 2004
Abstract In order to identify the infection rate of trypanosome species infecting wild animals in four localities (Bipindi, Campo, Fontem and Nditam) of southern Cameroon, 1,141 wild animals were sampled. These animals belonged to 36 species grouped in 8 orders including 407 primates, 347 artiodactyls, 264 rodents, 54 pangolins, 53 small carnivores, 11 saurians and crocodilians and 5 hyraxes. PCR using specific primers for Trypanosoma vivax, T. brucei s.l., T. congolense “forest type”, and T. simiae showed that 18.7% of the animals were infected by at least one of these trypanosome species. A positive PCR result may not indicate absolutely an active infection because PCR can detect also transient infections. T. vivax (Duttonella) had the highest infection rate (9.5%) and was found in almost all the host orders studied. T. brucei s.l. mostly infected primates, rodents and some duikers (Cephalophus dorsalis and C. monticola). Trypanosomes of the subgenus Nannomonas had a lower infection rate of 5.5% (2.4% for T. simiae and 3.1% for T. congolense “forest type”). They were harboured mainly by primates, ungulates and rodents. Trypanosome infection rates were highest in Nditam (24.5%) and Bipindi (21%). T. brucei s.l. (Trypanozoon) had its maximum infection rate of 10.4% in Bipindi. The “Quantitative Buffy Coat” (QBC® ) and Kit for in vitro isolation techniques were used to identify 48 (6.1%) infected animals. 13 were positive using QBC® , and 42 were positive by KIVI. However, PCR was negative on 16 of these infected animals, probably due to infections with other trypanosome species. This study showed that trypanosomes of the subgenera Duttonella, Nannomonas and Trypanozoon could infect small wild vertebrates as has been shown for large ungulates and carnivores. The presence of T. brucei s.l. in a large range of wild animals strengthens the hypothesis of the existence of a wild animal reservoir of T. b. gambiense in Cameroon. © 2004 Elsevier B.V. All rights reserved. Keywords: Wild animal trypanosomes; PCR; Infection rate; Reservoir; Cameroonian forest ∗ Corresponding author. Tel.: +237 223 22 32; fax: +237 223 00 61. E-mail address: [email protected] (F. Njiokou).
0001-706X/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2004.04.011
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1. Introduction In Africa, trypanosome infections constitute an important cause of morbidity and mortality in domestic and wild animals in areas where tsetse flies are present. Most studies performed on African animal trypanosomiasis (AAT) have been limited to domestic animals because of the difficulties of obtaining blood samples from wild animals. These latter animals may constitute a reservoir host for trypanosomes responsible for animal and human trypanosomiasis (Gibson et al., 1978; Mehlitz et al., 1982; Joshua et al., 1983; Zillmann et al., 1984; Mehlitz, 1986, Asonganyi et al., 1986, 1990). Previous studies have identified most subgenera of salivarian trypanosomes in domestic and wild animals from subsaharan zones (Kupper et al., 1983; Mattioli et al., 1990; Mehlitz, 1986). However, the distribution of these parasites in wild animals is still not well-known since the commonly used diagnostic tools are not highly sensitive and specific. Even with the development of concentration techniques, such as haematocrit centrifuge technique (HTC) (Woo, 1970) and “Quantitative Buffy Coat” (QBC® ) (Bailey and Smith, 1992), the threshold of detection of trypanosomes remains high. Thus, some infected animals are not diagnosed. In addition, parasitological tests do not easily differentiate trypanosome species or sub-species that infect animals. Specific or sub-specific identification of trypanosomes in animals requires isolation, culture and characterisation by costly techniques, such as isoenzyme electrophoresis, restriction fragment length polymorphism and hybridisation. To compensate for the low sensitivity and low specificity of parasitological tests, a diagnostic technique using the polymerase chain reaction (PCR) has been developed. It enables the in vitro amplification of repetitive and specific nuclear DNA sequences of T. brucei s.l., T. vivax, T. simiae and three types (“savannah”, “forest” and “kilifi”) of T. congolense (Moser et al., 1989; Masiga et al., 1992; Artama et al., 1992; McNamara et al., 1994). The use of PCR on blood extracts enables the specific identification (without culture and isolation of parasites) of natural infections of most trypanosomes species and sub-species. In addition, the high sensitivity of this technique helps to considerably reduce the threshold levels of detection to as low as one trypanosome per millilitre of blood (Penchenier et al., 1996).
This study aimed to identify, by PCR, trypanosome species infecting wild animals, and to determine the infection rate of these parasites in wildlife in southern forest area of Cameroon. This identification is important to understand some epidemiological aspects of African trypanosomiasis, notably trypanotolerence in AAT and the existence of human African trypanosomiasis (HAT) animal reservoir.
2. Materials and methods 2.1. Study sites The study was carried in 4 localities in the southern Cameroonian forest characterized by the co-existence of tsetse flies, a large variety of wild fauna and intensive hunting activities by the inhabitants. - Bipindi (3◦ 2 N, 10◦ 22 E) covers several villages that are mainly located along roads (Gr´ebaut et al., 2001). The vegetation is dense equatorial forest interspersed with farmland. This region is surrounded by hills and has a dense network of several fast running streams. These streams pass through cocoa farms offering favourable conditions (such as shade and microclimate) for the presence of tsetse flies. - Campo (2◦ 20 N, 9◦ 52 E) lies along the Atlantic coast and extends along the Ntem river which constitutes the Cameroonian and Equatorial Guinean border. Campo is located in the equatorial rain forest zone. There is a dense network of several rivers, swampy areas and marshes. - Doum´e (4◦ 16 N, 13◦ 25 E) vegetation is mainly characterised by dense equatorial forest with an important network constituted by the river Doum´e and many large ponds. - Nditam locality (5◦ 20 N, 11◦ 9 E) belongs to the Tikar plain. The hydrographic network is dominated by the Mbam river and its affluents whose levels depend on the amount of annual rainfall (about 1600 mm). The vegetation is a mosaic of forests and savannahs, with woodland, grassland and shrubbery. The fauna and flora are highly diversified to the extent that Nditam area is considered as a safari park, where fishing and hunting activities are very frequently practised.
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2.2. Blood collection and parasitological tests Animals that were captured by hunters and trappers and brought to the village were identified using systematic keys (Depierre and Vivien, 1992; Dorst and Dandelot, 1997; Spawls et al., 2002). Cardiac punctures were made on dead animals, while intravenous blood collection were performed on living animals. From living or dead animals whose veins still contained fresh blood, a second blood sample was obtained under sterile conditions in KIVI medium for isolation of parasites (Aerts et al., 1992). Blood collected in EDTA tubes was used for parasitological diagnosis using the QBC® method (Bailey and Smith, 1992) and for PCR. In order not to encourage hunting as some of these animals are protected, no prior contacts were made with hunters before meeting them. Moreover, visits to villages were made unannounced. The follow-up of KIVI was done by microscopical examination of samples in the laboratory. Positive KIVI samples were grown in Cunningham medium (Cunningham, 1977). 2.3. PCR DNA was extracted from blood using the “ReadyAmp® Genomic DNA Purification System” (Promega) kit as described by Penchenier et al. (2000). The supernatant collected was used directly for PCR or stored at −20 ◦ C. Four pairs of specific primers for T. brucei s.l., T. congolense “forest type”, T. vivax and T. simiae (Masiga et al., 1992) were used. PCR amplifications were performed as described by Herder et al. (2002). The amplification was done on 25 l of mixture containing 5 l of DNA extract, 10 mM of Tris–HCl (pH 9), 50 mM of KCl, 3 mM of MgCl2 , 0.8 pM of each primer, 200 M of dNTP (dTTP, dATP, dCTP and dGTP) and 1 unit of Taq DNA polymerase (Appligene–Oncor, USA). One denaturing step at 94 ◦ C for 5 min was programmed before 40 amplification cycles. Each cycle contained a denaturing step at 94 ◦ C for 30 seconds, annealing at 55 ◦ C (T. brucei s.l.) or at 60 ◦ C (T. congolense “forest type”, T. vivax and T. simiae) for 30 s and an extension step at 72 ◦ C for 1 min. A final extension was done at 72 ◦ C for 10 min. The amplified products were analysed on 1.5% agarose gel containing ethidium bromide and visualised by UV light. For each extraction series a neg-
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ative control tube constituted of blood from uninfected animal was incorporated and processed at last. This control tube was used further as negative control during PCR reaction in addition to classical PCR negative control constituted by distilled water. Further more, the experimenter who performed the PCR had no information on the initial parasitological status of the samples. PCR test was repeated on all PCR positive and also on some negative samples randomly selected. 2.4. Statistical analysis The chi-square test (Scherrer, 1984) was used to compare the overall trypanosome infections rate and the prevalence of trypanosome species among animal groups and among localities.
3. Results Wild animals were sampled during 11 field surveys (5 at Bipindi, 2 at Campo, 2 at Doum´e and 2 at Nditam) from 1999 to 2001. One thousand, one hundred and forty one wild animals examined belonged to 36 species grouped into 8 orders as follows: 407 (35.7%) primates, 347 (30.4%) artiodactyls, 264 (23.1%) rodents, 54 (4.7%) pangolins, 53 (4.6%) small carnivores, 11 (1%) saurians and crocodilians and 5 (0.4%) hyraxes (Table 1). Parasitological tests (QBC® and/or KIVI) were done on 789 (69%) of the 1,141 animals examined. These tests revealed 48 (6.1%) infected animals of which 42 were positive with KIVI and 13 with QBC® . Of the 762 blood samples analysed by QBC® , 13 (1.7%) were positive (presence of live trypanosomes with fluorescent nucleus and kinetoplast due to acridine orange). Specific morphological identification was not possible. Of the 234 KIVI performed 42 (18%) were positive. 8 trypanosome strains were isolated: SitaBip 1 from a Sitatunga in Bipindi, CroCamp 1 and CroCamp 2 from crocodiles in Campo, CepCamp3, CepCamp4 and CepCamp5 from blue duikers in Campo, NanDoum1 from a palm civet in Doum´e, and HochNdi1 from a white-nosed monkey in Nditam. Of the 1,141 animals examined, 213 (18.7%) were positive by PCR (Table 1). A typical result of PCR analysis for T. brucei s.l. is shown in Fig. 1. Trypanosome
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Table 1 Trypanosomes infection rates in different animal species and orders Common names
Scientific names
N
Number of positive PCR TB s.l.
TCF
TS
TV
NP
White-eyelid mangabey Crested mangabey Cloaked mangabey De Brazza’s monkey Guereza white colobus Greater white-nosed monkey Mandrill Mona monkey Moustached monkey Dwarf guenon Bosman potto Golden potto
Cercocebus torquatus Cercocebus neglectus Cercocebus albigena Cercopithecus neglectus Colobus guereza Cercopithecus nictitans Mandrilus sphinx Cercopithecus mona Cercopithecus cephus Miopithecus talapoin Perodicticus potto Arctocebus calabarensis
5 2 12 1 14 155 5 46 101 55 8 3
1 – 2 – 1 10 – 1 4 5 2 1
– – – – 1 6 – 2 2 – 1 1
– – 1 – – 3 – 1 – – – –
2 – 2 1 2 22 – 8 11 5 3 –
3 – 5 1 3 33 – 12 15 9 4 1
Water chevrotain Royal antelope Blackstriped duiker Yellow-backed duiker Blue duiker Ogilby’s duiker Peter’s duiker Sitatunga
Primates ST Hyemoschus aquaticus Neotragus pygmaeus Cephalophus dorsalis Cephalophus silvicultor Cephalophus monticola Cephalophus ogilbyi Cephalophus callipygus Tragelaphus spekei
407 1 5 37 3 290 1 5 5
27 (6.6) – – 4 – 14 – – 1
13 (3.2) – – 2 – 16 – – –
5 (1.2) – – 3 – 14 – – –
56 (14) – – 3 – 24 – – 1
86 (21) – – 10 – 62 – – 1
Brush-tailed porcupine Greater cane rat Giant forest squirrel Red-legged sun squirrel Giant rat
Artiodactyls ST Atherurus africanus Thryonomys sp. Protoxerus stangeri Heliosciurus rufobrachium Cricetomys gambianus
347 106 15 1 6 136
19 (5.5) 11 2 – 1 6
18 (5.2) 2 – – 1 1
17 (5) 3 – – – 2
28 (8) 7 – – – 4
73 (21) 20 2 – 2 11
Golden cat Black legged mangoose Dark mangoose Small-spotted genet African civet Two-spotted palm civet
Rodents ST Profelis aurata Bdeogale nigripis Crossarchus obscurus Genetta servalina Viverra civetta Nandinia binotata
264 1 1 8 8 3 32
20 (7.6) – – 1 1 – 3
4 (1.5) – – – – – –
5 (1.9) – – – – – –
11 (4.8) – – – 1 – 3
35 (13.3) – – 1 1 – 5
Long-tailed pangolin Tree pangolin
Carnivores ST Manis tetradactyla Manis tricuspis
53 34 20
5 (9.4) 2 1
0 – –
0 – –
4 (7.5) 2 5
7 (13.2) 4 6
Crocodile Monitor lizard
Pangolins ST Crocodylus niloticus Varanus ornatus
54 3 8
3 (5.6) – –
0 – –
0 – –
7 (13.2) 1 1
10 (18.5) 1 1
Tree dassie
Saurians and crocodilians ST Dendrohyrax arboreus
11 5
0 (0) –
0 (0) –
0 (0) –
2 (18.2) –
2 (18.2) –
Hyraxes ST
5
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
1141
74 (6.5)
35 (3.1)
27 (2.4)
108 (9.5)
213 (18.7)
Total Sample rate of infection (%)
Percentages of infected animals are given in brackets. TB s.l., Trypanosoma brucei s.l.; TCF, Trypanosoma congolense “forest type”; TV, Trypanosoma vivax; TS, Trypanosoma simiae; N, number of animals examined; ST, sub-total; NP, number of wild animals harbouring at least one parasite species (this number is lower than the total number of infections recorded because some animals had multiple infections).
F. Njiokou et al. / Acta Tropica 92 (2004) 139–146
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Fig. 1. Example of agarose electrophoresis obtained after amplification of T. brucei s.l. specific sequences. M, molecular marker; C− , negative control; C+ , positive control; 1–9, wild animal blood samples tested; 1, 2, 4, 5, 6, 8 and 9, negative samples; 3 and 7, positive samples.
infection rates for the subgenera Duttonella (T. vivax) and Trypanozoon (T. brucei s.l.) were 9.5% and 6.5%, respectively, while that of the subgenus Nannomonas was relatively low, 5.5%, of which 56% were T. congolense “forest type” and 44% were T. simiae (Table 1). The infection rates differed significantly (χ2 = 64; P < 0.0001). The observed frequency was T. vivax > T. brucei s.l. > T. congolense “forest type” > T. simiae. As for the wild animal categories, primates, artiodactyls and rodents harboured all four sibling parasites, whereas pangolins and small carnivores were infected by only T. brucei s.l. and T. vivax, and reptiles by only T. vivax. None of the hyraxes examined was infected (Table 2). The overall trypanosome infection rates were not significantly different between the seven animal groups (χ2 = 7.29; P = 0.29), nor between the five
groups with sample size superior to 50 individuals (χ2 = 6.33; P = 0.17). The infection rates in Nditam, Bipindi, Campo and Doum´e were 24.5%, 21%, 11.7% and 10%, respectively. These values differed significantly between the four localities (Table 2): wild animals in Nditam and in Bipindi harboured significantly more trypanosome infections than those in Campo and in Doum´e. The prevalence values of T. simiae and T. vivax did not differ between localities, whereas animals in Bipindi and in Nditam were significantly more infected by T. brucei s.l. and T. congolense “forest type” respectively (Table 2). Of the 213 wild animals that were positive by PCR, 25 (11.7%) had double infections, 3 (1.4%) harboured triple infections and the rest had a single infection. Dou-
Table 2 Trypanosome infection rates in different localities Localities
Bipindi Campo Doum´e Nditam Total χ2 P
N
n (%) TB s. l
TCF
TS
TV
NP
499 154 178 310
52 (10.4) 5 (3.2) 5 (2.8) 12 (3.9)
10 (2) 4 (2.6) 0 (0) 21 (6.8)
12 (2.4) 3 (2) 0 (0) 12 (3.9)
46 (9.2) 9 (5.8) 12 (6.7) 41 (13.2)
103 (21) 18 (11.7) 16 (10) 76 (24.5)
1141
74 (6.5)
35 (3.1)
27 (2.4)
108 (9.5)
213 (18.7)
19.9 0.0002
20.4 0.0001
7.2 0.07 ns
7.4 0.06 ns
17.2 0.0006
TB s.l., Trypanosoma brucei s.l.; TCF, Trypanosoma congolense “forest type”; TV, Trypanosoma vivax; TS, Trypanosoma simiae; N, number of animals tested; n, number of positive animals for specific primers used; (%), percentage of positive PCR; NP, number of animal with at least one positive PCR (this number is lower than the total number of positive PCR as some animals harboured more than one trypanosome species). P, level of significance of the χ2 test; ns, non significant.
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Table 3 Parasitological positive hosts identified or not identified by PCR Hosts
Not identified
Identified
Trypanosome DNAs detected
Blue duiker Greater white-nosed monkey Mona monkey Sitatunga Crocodile Bosman potto Tree pangolin Palm civet
7 5
19 6
TB, TV, TCF, TS TB, TV
1 1 1 0 1 0
1 1 1 3 0 1
TB, TV, TCF TB, TV TV, TB, TV, TCF
Total
16
32
TV
TB, Trypanosoma brucei s.l.; TV, Trypanosoma vivax; TCF, Trypanosoma congolense “forest type”; TS, Trypanosoma simiae.
ble infections included 11 animals infected with T. brucei s.l. and T. vivax, 7 with T. simiae and T. vivax, 4 with T. congolense “forest type” and T. vivax, 2 with T. simiae and T. congolense “forest type” and 1 with T. brucei s.l. and T. congolense “forest type”. Triple infections involved T. brucei s.l., T. vivax and T. simiae, T. brucei s.l., T. vivax and T. congolense “forest type”, and T. brucei s.l., T. simiae and T. congolense “forest type”. Concerning the eight stocks isolated by KIVI, two PCR results using the four couples of primers studied was available: Firstly, PCR detected T. brucei s.l. from the original blood sample of SitaBip1, T. vivax from the original blood samples of NanDoum1, CroCamp2, CepCamp3 and CepCamp4, and none of the four trypanosomes in the original blood samples of CroCamp1, and CepCamp5. Secondly, PCR using the same primers confirmed the presence of T. brucei s.l. in SitaBip1 stocks and none of the four trypanosomes in the other unidentified trypanosome stocks. Of the 48 animals that were positive using parasitological tests, 16 were negative by PCR whereas 32 animals were positive. Trypanosomes could be positively identified using the available primers in all groups of hosts except pangolins (Table 3). PCR was positive in 125 animals negative with parasitological examinations. 4. Discussion The rate of trypanosome infection detected by the parasitological tests (6.1%) was relatively low, prob-
ably due to the low sensitivity of the QBC® as well as several technical constraints, in particular difficulties to get blood samples and inoculate them into KIVI flask under sterile conditions, and the lapse of time between animal slaughtering and performing QBC® (2–6 h). These difficulties could considerably decrease the accuracy of parasitological techniques (Bailey and Smith, 1992; Ancelle et al., 1997). Our PCR results showed that 18.7% of wild animals were infected by at least one of the four trypanosome species. The high infection rate of trypanosomes in wild animals in Nditam and Bipindi could be explained by the fact that Bipindi is currently the HAT focus in Cameroon with the highest sleeping sickness prevalence (Gr´ebaut et al., 2001). Nditam is located near the Adamaoua Province, the main region of livestock location with the highest prevalence of African animal trypanosomiasis in Cameroon. T. vivax (Duttonella) had the highest infection rate of 9.5%, whereas T. congolense “forest type” and T. simiae (both Nannomonas) species had the lowest infection rates of 3.1% and 2.4% respectively. These levels of infection differed from previous study conducted in West African (Komoin-Oka et al., 1994). The difference may be ascribed to the wild animal host species. In west Africa, studies performed on large mammals (Loxodonta africana, Syncerus caffer, Kobus kob, Kobus defassa) revealed mostly T. congolense and very few T. vivax (Komoin-Oka et al., 1994; Truc et al., 1997), whereas the present study detected mostly T. vivax (Duttonella), few T. simiae and T. congolense “forest type” (Nannomonas) in a large variety of small vertebrates. Despite relatively small sample numbers, (pangolins, small carnivores and reptiles) these animals were found to be infected by T. vivax and T. brucei s.l. T. vivax (Duttonella) was very frequent and was often associated with T. brucei s.l. (Trypanozoon) or T. simiae and T. congolense (Nannomonas). Very few mixed infections involved trypanosomes of the subgenera Nannomonas and Trypanozoon. However, some animal hosts found in this study were quite unexpected, when comparing the review by Hoare (1972). For example, T. vivax, a parasite of various domestic and wild ungulates was detected in monitor Lizard and crocodile. T. simiae, a suid parasite, already found in non-suid hosts (camels, cattle and horse) was detected from two monkeys, two duikers and two rodents. These findings may be partially explained because PCR detects also
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transient infections. A PCR positive result indicates the presence of the corresponding parasite DNA and not necessary an active infection (Herder et al., 2002). The positive PCR results may indicate the presence of T. vivax DNA in these animals without active infection, because of the clearance of this parasite by the host immune system. The more probable hypothesis is that the trypanosomes detected have been inoculated by infected tsetse flies, but have failed to establish an infection. Previous results in West Africa showed that 46% of G. p. palpalis blood meals were from humans and 46% from Sitatunga (Laveissi`ere et al., 1985) whereas in south Cameroon, the analysis of 137 tsetse blood meals showed that 48% were from humans, 25% from domestic animals (pig and sheep), 16% from wild animal (Sitatunga and Golden Cat), and 10% not yet identified, probably also from wild animals (Njiokou et al., 2003). Tsetse flies could then carry trypanosomes from one host to another. Morlais et al. (1998) revealed T. vivax in tsetse flies from southern forest of Cameroon. Detection of trypanosome species in non-common hosts needs to be confirmed by their isolation with appropriate medium, culture and characterisation. In this study, T. vivax DNA was detected from original blood samples of NanDoum1, CroCamp2, CepCamp3 and CepCamp4 strains but was absent in these isolated stocks; the explanation could be their degeneration in the KIVI medium which is not suitable for this species, but also because some of the positive animals might not harbour active infections. T. brucei s.l. showed the second highest infection rate and was present in both large and small vertebrates. T. brucei s.l. infection rate was low in three localities: Doum´e (2.8%), Campo (3.2%), and Nditam (3.9%), and high in Bipindi (10.4%). The high level of infection rate of T. brucei s.l. may be due to the presence of T. b. gambiense in a large range of small wild mammals in Bipindi (Herder et al., 2002). Apart from Doum´e, where no trypanosomes of the sub-genus Nannomonas (T. simiae and T. congolense) were detected, all species have been identified in animals from Bipindi, Campo and Nditam. T. vivax was detected in wild animals from all localities. Negative PCR results in 16 infected animals could be due to the presence of other trypanosome species. Previous studies detected other trypanosome species, such as T. congolense “savannah type”, T. con-
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golense “kilifi type” and Megatrypanum (Itard, 1981; Mattioli et al., 1990; Herder et al., 2002). Crocodiles could harbour reptilian trypanosomes such as Trypanosoma grayi not investigated in the present study. This study showed that the four trypanosome species DNAs could be detected in a large range of wild animals by the PCR. However, PCR positive results indicate the presence of the parasite DNA in the vertebrate hosts and not necessarily an active infection. As PCR detects also transient infections, it is therefore useful to confirm new hosts status by the isolation of the parasites in an appropriate medium and characterisation. The use of specific primers for other trypanosome species and sub-species could enable the identification of all trypanosomes infecting animals and may allow a better understanding of the role of wild animals in the epidemiology of African trypanosomiasis.
Acknowledgements This work was supported by Institut de Recherche pour le D´eveloppement (IRD). We thank C. F. Bilong, A. Monkiedje, L. Basco and three anonymous referees for critical reading of the manuscript, and Ernest Mooh and Samuel Bahebegue for technical assistance.
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