Infection with Trypanozoon spp (Tz) (T. b. brucei, T. b. gambiense, T. b. rhodensiense, T. evansi), Tc (T. congolense savannah; T. congolense kilifi; T. congolense forest); Tsg (T. simiae; T. simiae tsavo; T. godfreyi) and Tv (T. vivax) was screened in 6860 adult tsetse. The results indicate that 1736 (25.30%) adults were infected with one or more Trypanosoma taxa (Tables 2, 3 and 4), The Trypanosoma prevalence varied significantly between tsetse taxa (X2 = 750.18, df = 9, P << 0.001) and between countries (X2 = 2038.1, df = 14, P << 0.001). The Permanova analysis as well indicated significant differences between countries (P = 0.009) and taxa (P =0.041) (Table 5) As all taxa were not collected from all countries, the interaction between taxa and countries was only analyzed where a taxon was collected from several countries.
Regardless of tsetse taxon, in west African countries the average Trypanosoma prevalence was 20% (n = 3733), with the highest prevalence recorded in Ghana (61%) and the lowest recorded in Guinea (2.2%). The prevalence in Burkina Faso, Mali and Senegal was 21.9, 6.9 and 14.2% respectively (Figure 1, and Table 2). In east, central and southern African countries, the Trypanosoma infection prevalence was a bit higher than in west African countries with an averaged infection of 31.5% (n = 3127), with the highest prevalence (53.6%) in Zimbabwe and lowest prevalence (2.9%) in DRC. No Trypanosoma infection was detected in Eswatini (Figure 1 and Table 2). Regardless of the country, Trypanosoma prevalence varied from one taxon to another, and G. m. morsitans showed the highest Trypanosoma prevalence (41%) followed by G. pallidipes (38.5%) and the lowest prevalence was detected in G. brevipalpis (9.71%) in east, central and southern Africa. In west Africa, G. medicorum showed the highest Trypanosoma prevalence (39.5%) and the lowest prevalence was detected in G. p. palpalis (2.8%) (Table 3).
Some tsetse taxa were collected from several countries as presented in Figure 2 and Table 4. The highest Trypanosoma prevalence was recorded in G. tachinoides in Ghana (61%). This was followed by high prevalence in G. m. morsitans collected from Zimbabwe (53.9%), Tanzania (53%) and Zambia (48,4%). G. pallidipes from Zimbabwe, Kenya, Zambia and Tanzania also showed high Trypanosoma prevalence of 52.7%, 50.9%, 45.2% and 37.3%, respectively. The lowest Trypanosoma prevalence was found in G. p. gambiensis from Guinea (2.2%). Based on the Trypanosoma prevalence presented in Figure 2 and Table 4, the tested samples can be categorized as: (i) tsetse samples with high prevalence (> 35%) detected in G. tachinoides from Ghana; G. medicorum from Burkina Faso, G. pallidipes from Kenya, Zambia, and Zimbabwe, G. m. morsitans from Tanzania, Zambia, and Zimbabwe; (ii) tsetse samples with medium prevalence (10-35%) detected in G. austeni from South Africa, G. f. fuscipes from Kenya and Uganda, G. m. submorsitans from Burkina Faso, G. p. gambiense from Burkina Faso and Senegal and G. tachinoides from Burkina Faso; (iii) tsetse samples with low prevalence (< 10%) detected in the rest of the samples listed in Table 4 except the G. austeni collected from Eswatini. Despite the difference in Trypanosoma prevalence for each tsetse species, the differences were significant only in G. p. gambiensis (X2= 26.71, df = 4, P < 0.001) and G. tachinoides, (X2= 9.38, df = 1,2, P = 0.002). In contrast, no significant difference was detected between countries for G. austeni (X2= 1.47, df = 4, P = 0.688), G. brevipalpis (X2= 0.34, df = 2, P = 0.559), G. f. fuscipes (X2= 0.15, df = 2, P = 0.702), G. m. morsitans (X2= 1.04, df= 3, P = 0.593) and G. pallidipes (X2= 4.983, df= 1,6, P = 0.418) (Table 4). No Trypanosoma infection was recorded in G. austeni from Eswatini. The best glm model (lowest AICc) selected for the overall Trypanosoma prevalence retained the countries as variables that fitted the data well (AICc = 1521.35) (Supplementary file 1).
Prevalence of different Trypanosoma taxa and mixed infections
The above-mentioned prevalence of Trypanosoma infection was comprised of several different Trypanosoma species and sub-species. Based on the size of the amplified fragment by PCR, the Trypanosoma infection was categorized into four groups: (i) the Tc group including the different forms of Trypanosoma congolense; (ii) Tv group including Trypanosoma vivax infections; (iii) Trypanozoon (Tz) group including T. b. brucei, T. b. gambiense, T. b. rhodesiense, and infections; and (iv) Tsg group including the infections with T. simiae, T. simiae tsavo and T. godfreyi. The screening results revealed that tsetse flies could be infected with single or multiple (double or triple) taxa of Trypanosoma, and the proportion of the infections with the different Trypanosoma taxa and the mixed infection varied with country (X2 = 63.56, df = 14, P <0.001) and species (X2 = 21.86, df = 9, P <0.001) (Supplementary file 1).
The prevalence of the different Trypanosoma species with respect to the above-mentioned groups, indicate that infections with the Tsg group was the highest regardless of countries or tsetse species with an average of 7.06%. The infection rate was higher (14.13%) in east, central and southern African countries than in west Africa (1.13%). Tv infection averaged at 6.75% but with higher prevalence in west African countries (10.37%) than in east, central and southern Africa (2.43%). The prevalence of Tc infection was lower than Tv and Tsg group with an average of 4.78% with higher prevalence in central and southern Africa (8.38%) than in west Africa (1.77%). The Tz group had the lowest prevalence with an average of 2.29%. Like Tv infection, the Tz prevalence was higher in west Africa (3.16%) than central and southern Africa (1.25 %).
The prevalence of infection by a single Trypanosoma group varied significantly from one country to another and from one tsetse species to another. For Tc, Tv, Tz and Tsg the infection prevalence varied significantly with country (X2 = 47.74, df = 14, P < 0.001, X2 = 27.40, df = 14, P = 0.01705, X2 = 106.11, df = 14, P = 0. 001 and, X2 = 44.74, df = 14, P = 0. 001 respectively). Regardless of tsetse species, the highest infection rate for Tc, Tv, Tz and Tsg was found in Tanzania (14.20%), Ghana (14.10%), Ghana (19.66%) and Zimbabwe (39.81%), respectively (Supplementary Table 3). Similarly, the prevalence of Tc, Tz and Tsg varied significantly with tsetse species (X2 = 40.364, df = 1,9, P << 0.001, X2 = 58.253, df = 1,9, P << 0. 001 and X2 = 34.871, df = 1,9, P << 0. 001, respectively), however no significant difference was found in Tv prevalence between tsetse species (X2 = 5.475, df = 1,9, P = 0.07868). Regardless of the country, the highest infection rate of Tc, Tv, Tz and Tsg was found in G. pallidipes (10.68%), G. tachinoides (12.92%), G. medicorum (13.64%) and G. m. morsitans (22.76%), respectively (Supplementary Table 4). No Tc infection was found in samples of G. austeni collected from Eswatini and Tanzania, G. brevipalpis from Mozambique, G. p. palpalis from Democratic Republic of the Congo (DRC) and G. p. gambiensis from Guinea. In addition, no Tv infection was detected in G. austeni collected from Eswatini and Mozambique, G. m. morsitans from Kenya and Zambia, G. pallidipes from Uganda and Zimbabwe. For Tz, G. austeni collected from Eswatini and Mozambique, G. brevipalpis from Mozambique, G. f. fuscipes from Kenya, G. m. morsitans from Kenya and Zambia, G. p. palpalis from DRC and G. p. gambiensis from Guinea did not show any infection (Figure 2 and Supplementary Table 5).
Mixed infections of Trypanosoma groups (double or triple) are rare events with an average prevalence between 0.09 and 1.71% regardless of country or tsetse species. However, double infections seem to be more frequent in some countries than others (X2 = 35.01, df = 14, P = 0.00) for Tv-Tz and in some tsetse species than others (X2 = 21.20, df = 9, P = 0.012) for Tv-Tz (Supplementary file 1). The highest prevalence of the mixed infections Tv-Tz and Tc-Tz were observed in Ghana with 12.39% and 10.68%, respectively, regardless of tsetse species. Although the average Tc-Tsg prevalence was higher than that of Tv-Tz and Tc-Tz, the highest mixed infection with it was found in Zambia with 9.05%. Regardless of the country, the highest mixed infection of Tc-Tsg detected per tsetse species was ~5% in G. m. morsitans and G. pallidipes. The mixed infection of Tsg with either Tv or Tz or both was lower than 2% regardless of the country or tsetse species. Taking into account both the country and tsetse species, the highest mixed infection of Tc-Tsg (12.5%) was detected in G. m. morsitans in Zambia. However, the highest prevalence of Tc-Tz (10.68%) and Tv-Tz (12.39%) was detected in G. tachinoides from Ghana. Although the average prevalence of Tv-Tsg was low (0.54%), a relative high infection rate of 6.17% was found in G. m. morsitans from Tanzania.
A triple infection of Trypanosoma groups (Tc-Tv-Tz) was only detected in G. medicorum from Burkina Faso (1.30%) and G. tachinoides from Ghana (1.71%) (Figure 2 and Supplementary Table 5, Supplementary file 1).
Prevalence of Sodalis infection
The prevalence of Sodalis infection based on the PCR results varied significantly with country (X2 = 108.02, df = 1, 14, P << 0.001) and tsetse species (X2 = 69.60, df = 9, P < 0.001). The best glm model (lowest AICc) selected for the overall Sodalis prevalence retained the countries, the species and their interaction (where possible) as variables that fitted the data well (AICc = 1296.12). Similar to the prevalence of Trypanosoma, the average Sodalis prevalence in east, central and southern Africa (24.6%) was higher than in west Africa (2.70%). Regardless of tsetse species, the highest prevalence of Sodalis infection was found in Tanzania (67.1%) followed by Uganda (43.3%), Kenya (28.5%) and Ethiopia (20.48%) (Table 2). The highest prevalence of Sodalis infection in west Africa was found in Guinea (28.6%). No Sodalis infection was found in Ghana, Mali, Senegal or Eswatini. Regardless of the country, the highest Sodalis prevalence per tsetse species was detected in G. m. morsitans (42.27%) followed by G. pallidipes (30.74%). No Sodalis infection was detected in G. tachinoides. The prevalence of Sodalis infection changed when both the countries and tsetse species are taken into consideration. Based on the Sodalis prevalence the tsetse samples can be categorized into four groups: (i) samples with high prevalence (> 50%) (ii) samples with medium prevalence (between <10% and >50%) (iii) samples with low prevalence (between >0% and 10%) and (iv) samples with no Sodalis infection as shown in Figure 2 and Table 5. The samples showing high Sodalis prevalence includes G. m. morsitans from Kenya (63.5%) and Tanzania (76.5%) and G. pallidipes from Tanzania (74.6%) and Uganda (75%), however the samples with no Sodalis infection includes G. austeni from Eswatini, G. p. gambiensis from Mali and Senegal and G. tachinoides from Burkina Faso and Ghana indicating that there is 95% confidence that the infection rate is less than or equal to 10%, 0.82%, 0.55%, 1.28% and 0.36%, respectively.
Interactions between Sodalis and Trypanosoma infections
Prevalence of co-infections of Sodalis with Trypanosoma
The screening results indicated that the single infection rate was 9.3% (n = 638) and 21.9% (n = 1503) for Sodalis and Trypanosoma, respectively, over all taxa and countries (Figure 3A). No Sodalis infection was found in G. tachinoides, and therefore was excluded from the analysis. A Cochran–Mantel–Haenszel test for repeated tests of independence showed that infection with Sodalis and Trypanosoma did deviate from independence across all taxa (χ2MH = 41.73, df = 1, P < 0.001) and individual Chi squared tests for independence for each taxon showed significant deviation from independence at the Bonferroni corrected α = 0.00833 in G. pallidipes (P < 0.001) and G. p. gambiensis (P < 0.001) (Supplementary Table 6). The prevalence of coinfection of Sodalis and Trypanosoma in wild tsetse populations varied with tsetse taxon and location. No coinfection was found in many taxa and many locations. The co-infection was found only in G. f. fuscipes (2.73%), G. m. morsitans (15.72%) and G. pallidipes (9.22%) in east, central and southern Africa (Figure 3B, Table 6 and Supplementary Table 6).
Impact of co infection on Trypanosoma and Sodalis density
Attempts to assess the density of Trypanosoma and Sodalis under single (S-/T+) and (S+/T-) or double infection (S+/T+) was conducted using qPCR with primers mentioned in Supplementary Table 2. The results show that Sodalis infections do not have any significant impact on Trypanosoma density (X2= 0.648, df = 2, P = 0.723). However, Trypanosoma infections significantly reduced the density of Sodalis in flies with (S+/T+) comparing to the flies with (S+/T-) (P = 0.014) (Figure 4). No significant different was found in the Trypanosoma density determined by qPCR in the flies tested negative (S+/T-) or positive (S+/T+) and (S-/T+) with the standard PCR, however, Sodalis density showed significant difference between flies with different infection type (X2 = 14.54, df = 2, P < 0.001) (Figure 4). The results showed no correlation between Sodalis and Trypanosoma density (r = 0.007, t = 0.055, df = 69, P = 0.9561) Supplementary Figure 2, Supplementary File 1).