Tsetse flies are the vectors of African trypanosomiasis affecting 36 sub-Saharan countries. Both wild and domestic animals play a crucial role in maintaining the disease-causing parasites (trypanosomes). Thus, the identification of animal reservoirs of trypanosomes is vital for the effective control of African trypanosomiasis. Additionally, the biotic and abiotic factors that drive gut microbiome diversity in tsetse flies are primarily unresolved, especially under natural, field conditions. In this study, we present a comprehensive DNA metabarcoding approach for individual tsetse fly analysis in the identification of mammalian blood meal sources and fly bacterial microbiome composition. We analyzed samples from two endemic foci, Kafue, Zambia collected in June 2017, and Hurungwe, Zimbabwe sampled in April 2014 (pilot study) and detected DNA of various mammals including humans, wild animals, domestic animals and small mammals (rat and bat). The bacterial diversity was relatively similar in flies with different mammalian species DNA, trypanosome infected and uninfected flies, and female and male flies. This study is the first report on bat DNA detection in wild tsetse flies. This study reveals that small mammals such as bats and rats are among the opportunistic blood meal sources for tsetse flies in the wild, and the implication on tsetse biology and ecology needs to be studied.

To improve our knowledge on the epidemiological status of African trypanosomiasis, better tools are required to monitor Trypanosome genotypes circulating in both mammalian hosts and tsetse fly vectors. This is important in determining the diversity of Trypanosomes and understanding how environmental factors and control efforts affect Trypanosome evolution. We present a single test approach for molecular detection of different Trypanosome species and subspecies using newly designed primers to amplify the Internal Transcribed Spacer 1 region of ribosomal RNA genes, coupled to Illumina sequencing of the amplicons. The protocol is based on Illumina’s widely used 16s bacterial metagenomic analysis procedure that makes use of multiplex PCR and dual indexing. Results from analysis of wild tsetse flies collected from Zambia and Zimbabwe show that conventional methods for Trypanosome species detection based on band size comparisons on gels is not always able to accurately distinguish between T. vivax and T. godfreyi. Additionally, this approach shows increased sensitivity in the detection of Trypanosomes at species level with the exception of the Trypanozoon subgenus. We identified subspecies of T. congolense, T. simiae, T. vivax, and T. godfreyi without the need for additional tests. Results show T. congolense Kilifi subspecies is more closely related to T. simiae than to other T. congolense subspecies. This agrees with previous studies using satellite DNA and 18s RNA analysis. While current classification does not list any subspecies for T. godfreyi, we observed two distinct clusters for these species. Interestingly, sequences matching T. congolense Tsavo (now classified as T. simiae Tsavo) clusters distinctly from other T. simiae Tsavo sequences suggesting the Nannomonas group is more divergent than currently thought thus the need for better classification criteria. This method presents a simple but comprehensive way of identification of Trypanosome species and subspecies-specific using one PCR assay for molecular epidemiology of trypanosomes. Tsetse flies are central actors in the transmission of Trypanosomes to vertebrate hosts. Therefore, detection of Trypanosomes in the tsetse flies is important for understanding the epidemiology of African trypanosomiasis as a component of new control or surveillance strategies. We have developed a method that combines multiplex PCR and next-generation sequencing for the detection of different Trypanosome species and subspecies. Similar to the widely used bacterial metagenomic analysis protocol, this method uses a modular, two-step PCR process followed by sequencing of all amplicons in a single run, making sequencing of amplicons more efficient and cost-effective when dealing with large sample sizes. As part of this approach, we designed novel Internal Transcribed Spacer 1 primers optimized for short read sequencing and have slightly better sensitivity than conventional primers. Taxonomic identification of amplicons is based on BLAST searches against the constantly updated NCBI’s nt database. Our approach is more accurate than traditional gel-based analyses which are prone to misidentification of species. It is also able to discriminate between subspecies of T. congolense, T. simiae, T. vivax, and T. godfreyi species. This method has the potential to provide new insights into the epidemiology of different Trypanosome genotypes and the discovery of new ones.

Ticks transmit many pathogens with public health and veterinary importance. Despite the wide distribution of tick-borne pathogens in Sudan, the information on the tick–pathogen relationship needs to be updated, particularly using modern molecular techniques. This cross-sectional study, conducted between September and November 2019, used morphology, PCR, and sequencing to confirm the identity of adult cattle ticks (male and female; n = 536) from Khartoum State (n = 417) and East Darfur State (n = 119). Moreover, the presence of Theileria annulata, Babesia bigemina, B. bovis, Anaplasma marginale, and Ehrlichia ruminantium was detected and confirmed in each tick using species-specific PCR or nested PCR and sequencing. The most economically important tick genera, Rhipicephalus, Hyalomma, and Amblyomma, were prevalent in the study area, and 13 different tick species were identified. The most prevalent tick species were Rhipicephalusevertsi evertsi (34.3%) and Hyalomma anatolicum (57.3%) in Khartoum State, and Rhipicephalus annulatus (27%), Rhipicephalus decoloratus (25%), and Hyalomma rufipes (29%) in East Darfur State. We detected all five pathogens in both states. To the best of our knowledge, this is the first study to report the presence of E. ruminantium, its vector Amblyomma variegatum, and B. bovis in Khartoum State. Further, this is the first report on most tick and pathogen species identified in East Darfur State. Our findings indicate the migration of some tick and pathogen species beyond their distribution areas in the country, and this consideration is necessary to develop future control strategies.

The World Health Organization has targeted stopping the transmission of Human African Trypanosomiasis by 2030. To achieve this, better tools are urgently required to identify and monitor Trypanosome infections in human, animals, and tsetse fly vectors. This study presents a single test approach for detection and identification of Trypanosomes and their comprehensive characterization at species and sub-group level. Our method uses newly designed ITS1 PCR primers (a widely used method for detection of African Trypanosomes, amplifying the ITS1 region of ribosomal RNA genes) coupled to Illumina sequencing of the amplicon. The protocol is based on the widely used Illumina’s 16s bacterial metagenomic analysis procedure that makes use of multiplex PCR and dual indexing. We analyzed wild tsetse flies collected from Zambia and Zimbabwe. Our results show that the traditional method for Trypanosome species detection based on band size comparisons on a gel is unable to distinguish between T. vivax and T. godfreyi accurately. Additionally, this approach shows increased sensitivity of detection at species level. Through phylogenetic analysis, we identified Trypanosomes at species and sub-group level without the need for any additional tests. Our results show T. congolense Kilifi sub-group is more closely related to T. simiae than to other T. congolense sub-groups. This agrees with previous studies using satellite DNA and 18s RNA analysis. While current classification does not list any sub-groups for T. vivax and T. godfreyi , we observed distinct subgroups for these species. Interestingly, sequences matching T. congolense Tsavo (now classified as T. simiae Tsavo) clusters distinctly from the rest of the T. simiae Tsavo sequences suggesting that the Nannomonas group is more divergent than currently thought thus the need for a better classification criteria. This approach has the potential for refining classification of Trypanosomes and provide detailed molecular epidemiology information useful for surveillance and transmission control efforts. Author summary: Detection of Trypanosomes in the tsetse flies plays an important role in the control of African trypanosomiasis by providing information on circulating Trypanosome species in a given area. We have developed a method that combines multiplex PCR and next-generation sequencing for Trypanosome species detection. The method is based on the widely used bacterial metagenomic analysis protocol and uses a modular, two-step PCR process followed by sequencing of all amplicons in a single run, making sequencing of amplicons more efficient and cost-effective when dealing with large sample sizes. As part of this approach, we designed novel primers for amplifying the ITS1 region of the Trypanosome rRNA gene that is more sensitive than conventional primers. Identification of Trypanosome species is based on BLAST searches against the constantly updated NCBI’s nt database, which facilitates the identification of Trypanosome subgroups. Our approach is more accurate than traditional gel-based analysis and shows how the latter is prone to misidentification. It is also sensitive and is able to discriminate between subgroups within Trypanosome species. Applied as an epidemiological tool, it has the potential to provide new, comprehensive and more accurate information on vector-pathogen-host interconnections which are key in the control and management of African trypanosomiasis.