---
title: "Cave-dwelling bats in southeastern Nigeria harbor Trypanosoma livingstonei: molecular evidence fro"
id: "plos-one-10-cave-dwelling-bats-as-hosts-of-trypanosoma-livingstonei-evidence-from-peri"
canonical_url: "https://medichelpline.com/clinical-feed/plos-one-10-cave-dwelling-bats-as-hosts-of-trypanosoma-livingstonei-evidence-from-peri"
content_type: "clinical_feed_article"
specialty: "Infectious Disease"
source_name: "PLOS ONE (Medicine)"
source_url: "https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0355840"
published_at: "2026-08-12T14:00:00.000Z"
evidence_level: "Journal Feed"
license: "CC-BY-NC-4.0 / Informational Use"
---
# Cave-dwelling bats in southeastern Nigeria harbor Trypanosoma livingstonei: molecular evidence fro
## Provenance & Clinical Metadata
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- **Specialty:** [Infectious Disease](https://medichelpline.com/clinical-feed/infectious-disease.md)
- **Primary Source:** PLOS ONE (Medicine)
- **Source URL:** [Original Journal Publication](https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0355840)
- **Published At:** 2026-08-12T14:00:00.000Z
- **Evidence Rating:** Journal Feed
## Executive GIST (TL;DR)
- Researchers screened blood and ectoparasite samples from 57 bats representing 10 species captured in nine caves and one residential roost 0.4–2 km from human dwellings in Enugu State, southeastern Nigeria. - **Trypanosoma** DNA was detected in 22 of 57 bats (38.6%), spanning six bat species across four families: Rousettus aegyptiacus, Hipposideros abae, H. jonesi, H. ruber, Rhinolophus landeri, and Mops condylurus. - Phylogenetic analyses placed all Nigerian trypanosome sequences within the **Trypanosoma livingstonei** species group, which is part of the early-diverging lineages of the **Trypanosoma cruzi clade**. - The Nigerian sequences did not form a single homogeneous lineage; species delimitation recovered them together with other T. livingstonei / T. cf. livingstonei references across several putative molecular lineages, indicating phylogenetic structuring within the group. - The exclusive detection of members of the T. livingstonei species group across multiple bat hosts suggests relatively low trypanosome diversity in these cave systems but highlights lineage-level variation. - Study methods included PCR screening and phylogenetic and species delimitation analyses; sequence data were deposited in GenBank (accessions PV790209–PV790224) and supporting data are available on OSF (DOI: 10.17605/OSF.IO/3794C). - Authors conclude broader geographic sampling, multilocus data, and ecological measurements are needed to clarify drivers of trypanosome diversity in cave-dwelling bats and to better resolve host–parasite associations.
## Clinical Analysis & Structured Key Points
Cave-dwelling bats as hosts of Trypanosoma livingstonei: Evidence from peri-domestic roosts in southeastern Nigeria | PLOS One Browse Subject Areas ? Click through the PLOS taxonomy to find articles in your field. For more information about PLOS Subject Areas, click here . Article Authors Metrics Comments Media Coverage Reader Comments Figures Figures Abstract Bats play important roles in ecosystem functioning and host a remarkable diversity of blood parasites, including a limited number of bat-associated Trypanosoma species that have been described from Africa. Cave roosts provide critical habitat for bats and often support large, multispecies aggregations that may influence parasite transmission dynamics. Nevertheless, the diversity and host associations of trypanosomes within these environments remain poorly characterized, particularly in West Africa. To address this gap, we screened blood and ectoparasite samples from 57 bats representing ten species captured in nine caves located 0.4–2 km from human residences, as well as from one residential building in southeastern Nigeria, using PCR. Trypanosome DNA was detected in 22/57 bats (38.6%) and in six bat species across four families: Rousettus aegyptiacus (1/2), Hipposideros abae (11/23), H. jonesi (1/1), H. ruber (5/11), Rhinolophus landeri (2/10), and Mops condylurus (2/6). Phylogenetic analyses placed the trypanosome sequences within the Trypanosoma livingstonei species group, a clade of African bat-associated trypanosomes representing early-diverging lineages within the Trypanosoma cruzi clade, which includes the causative agent of Chagas disease. The Nigerian trypanosome sequences were not recovered as a single homogeneous lineage, and species delimitation analysis recovered them together with other T. livingstonei/ T. cf. livingstonei references across several putative molecular lineages. The exclusive detection of parasites of the T. livingstonei species group across multiple bat species indicates low trypanosome diversity within this cave system but reveals phylogenetic structuring within this parasite group. The findings highlight the need for broader geographic sampling, multilocus data, and ecological measurements to clarify the drivers of trypanosome diversity in cave-dwelling bats. Citation: Okwuonu ES, Nnabuife HE, Obitte BC, Werb O, Tanshi I, Kingston T, et al. (2026) Cave-dwelling bats as hosts of Trypanosoma livingstonei : Evidence from peri-domestic roosts in southeastern Nigeria. PLoS One 21(8): e0355840. https://doi.org/10.1371/journal.pone.0355840 Editor: Daniel Oladimeji Oluwayelu, University of Ibadan Faculty of Veterinary Medicine, NIGERIA Received: February 28, 2026; Accepted: July 27, 2026; Published: August 12, 2026 Copyright: © 2026 Okwuonu 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 data underlying the findings are fully available without restriction. The R script used to generate Fig 2 and associated files is available from the Open Science Framework (OSF) repository (DOI: 10.17605/OSF.IO/3794C ). The nucleotide sequence alignment used for phylogenetic analyses, as well as all files associated with the PTP analysis, are also available from the same repository. Sequence data generated in this study have been deposited in GenBank under accession numbers PV790209–PV790224. All other relevant data are included within the manuscript and its Supporting information files. Funding: This research project was funded by the Rufford Foundation [38035-1] and Bat Conservation International [SS2210]. JS was supported by the German Research Foundation [437846632]. The project equally received funding from the West African Mammal Fellowship (WAMF) of the Small Mammal Conservation Organisation (SMACON) and the African Research Fellowship (ARF) of the American Society of Mammalogists’ (ASM). Idea Wild provided an equipment grant to support this project. The Berlin Centre for Global Engagement (BCGE) awarded Flexible Travel Funds to ESO to cover travel and accommodation expenses at Humboldt University of Berlin, Germany, where the molecular analyses were conducted. 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 Trypanosomes are protozoan parasites of medical and veterinary importance [ 1 ]. Members of the genus Trypanosoma exhibit remarkable genetic diversity in bats, with several lineages closely related to species infecting other mammals, including livestock and humans [ 2 – 6 ]. Phylogenetic evidence further supports bats as ancestral hosts in the evolutionary history of the Trypanosoma cruzi clade , the group that includes the agent of Chagas disease [ 7 ]. Although Chagas disease occurs primarily in rural areas of Mexico, Central and South America, multiple lineages within the T. cruzi clade, including Trypanosoma livingstonei , are thought to have originated in African bats [ 5 , 7 , 8 ]. This evolutionary context highlights the importance of systematic sampling and molecular characterization of trypanosomes in African bat communities. Since its first description in 2013 from two bat species in Mozambique , T. livingstonei has been reported from several bat species across Africa [ 6 , 8 – 10 ]. However, its host associations, patterns of occurrence within multispecies assemblages, and potential ecological drivers remain insufficiently understood. The potential for interspecific transmission is likely enhanced in bat species that roost together in stable environments such as caves. Cave systems provide constant microclimatic conditions, high host densities, and repeated interspecific contact, all of which may influence parasite dynamics. Expanding research on bat trypanosomes in understudied regions, including southeastern Nigeria, is therefore important for clarifying host–parasite associations and understanding how ecological context shapes parasite distribution [ 6 , 7 , 11 ]. Bats also host a remarkable diversity of ectoparasites, reflecting their wide geographic distribution, social behavior, and ecological versatility [ 12 , 13 ]. Bat flies (Nycteribiidae and Streblidae), mites (Spinturnicidae), fleas (Ischnopsyllidae), and ticks (Argasidae) are common and often form intimate, co-evolved associations with their hosts [ 14 , 15 ]. These ectoparasites form close associations with their hosts and represent potential components of bat-associated parasite systems. In sub-Saharan Africa, cave-dwelling bats represent a particularly suitable system for studying host-parasite interactions. Enugu State in southeastern Nigeria, characterized by extensive sandstone cave systems, supports diverse bat assemblages. Despite Nigeria supporting a high bat species richness of approximately 100 bat species [ 16 , 17 ], protozoan blood parasites of cave-roosting bats remain poorly studied. While investigations on bat trypanosomes have been conducted in parts of West Africa, including the arid lands of northern Nigeria, comparable studies are lacking for the wet tropical ecosystems of southern Nigeria, where bat species richness is among the highest on the continent [ 16 , 18 , 19 ]. Here, we investigate the presence and diversity of trypanosomes and associated ectoparasites in cave-dwelling bats from southeastern Nigeria. Using molecular approaches, we aim to characterize parasite diversity, assess host associations across multispecies cave assemblages, and examine their phylogenetic relationships within the broader context of African bat-associated trypanosomes. Materials and methods Study area The study was conducted in nine caves and one residential building used as bat roosts across eight communities in Enugu State, southeastern Nigeria ( Fig 1 ). Enugu State is situated on the Udi-Nsukka Plateau, whose geology comprises crystalline basement rocks with localized sedimentary cover and extensive sandstone formations. These geological features create a fractured, relief-rich landscape supporting numerous cave systems. Download: PNG larger image TIFF original image Fig 1. Map of Enugu State, southeastern Nigeria. Locations of nine cave roosts and one residential building where bats were sampled are indicated. Sampling sites are marked with symbols, as shown in the legend. LGA = Local Government Area. Map created by Chukwu, Matthew Tochukwu, Cpraise Evaluations and Geospatial Aid (CEVGEO™), reproduced under the Creative Commons Attribution 4.0 International License (CC BY 4.0). https://doi.org/10.1371/journal.pone.0355840.g001 Caves occur along escarpments, river valleys, and fault corridors and include both natural formations and anthropogenic voids (e.g., abandoned buildings, quarry pits, and building crevices). Peri-domestic roosts, caves and cave-like structures near settlements, were prioritized to characterize cave-dwelling bat communities and assess the presence of Trypanosoma parasites. Caves in the region are used for a variety of purposes, including recreation and exploration, educational and cultural activities led by schools and guides, spiritual or seasonal practices, and as places for shelter or temporary storage. They also support local economies and protein needs through tourism and hunting. Visit frequency includes regular local use, occasional visits, seasonal trends, and events, which are shaped by factors such as trail ease, safety considerations, and the availability of managed paths. Ethics statement Ethical approval for this study was obtained from the Ethics and Biosafety Committee, Faculty of Biological Sciences, University of Nigeria, Nsukka (approval number: UNN/FBS/EC/1014). Fieldwork and sample collection were conducted with permission from the relevant institutional and regulatory authorities in Nigeria, including the State Ministries of Health and Environment. Where required, access to sampling sites was granted with the approval and support of local community leadership. All bat capture, handling, and sampling procedures followed the guidelines of the American Society of Mammalogists for the use of wild mammals in research [ 20 ], and all efforts were made to minimize stress and harm to the animals. Extracted DNA samples were transferred to Germany in 2024 for molecular analysis under a formal Material Transfer Agreement (MTA) between the University of Nigeria, Nsukka, and Humboldt University of Berlin (signed 16 July 2024). The transferred material consisted solely of extracted DNA and not whole biological specimens. The samples were derived from bat species that are not listed as threatened, and to our knowledge, no specific export or import permits were required for this type of material. The extracted DNA was fully utilized during the analyses, and no material remains for return or further transfer. Sampling (field methods) The study is part of a broader investigation of cave-dwelling bat ecology in Enugu State ( S1 Table ). Sampling was conducted between 2020 and 2022 (January-February 2020, July-August 2021, and February-September 2022). Caves were selected based on proximity to human settlements, evidence of bat activity, and permission granted by community leaders. The nine selected caves were located approximately 0.4–2 km (5–30 minutes walking distance, depending on terrain) from human residences. Bats were captured using harp traps and ground-level mist nets of varying lengths (6 m, 9 m, and 12 m) placed at cave entrances [ 21 ]. Triple-high mist nets were deployed at the residential building. Harp traps were primarily used for insectivorous bat species, whereas mist nets were used for the fruit bat species Rousettus aegyptiacus . Vegetation was strategically arranged to reduce exit routes and improve capture efficiency [ 22 ]. Nets were opened at sunset (6:00–8:00 PM) and monitored continuously until approximately 20 individuals were captured, after which nets were closed. Sampling was conducted over two consecutive nights per site. Captured bats were placed individually in breathable cloth bags and identified to species level using morphological identification keys [ 23 , 24 ]. A 3 mm biopsy punch was used to collect wing tissue for identification of recaptured individuals. Ectoparasites were collected with fine forceps and brushes and preserved in absolute ethanol. Blood samples were obtained via venipuncture of the cephalic vein [ 25 ]. Blood volumes did not exceed 0.6–1% of body mass, remaining within established safety margins relative to estimated total blood volume [ 20 ]. To minimize discomfort and prevent infection, a lignocaine–amoxicillin paste was applied to the puncture site. Whole blood was stored in EDTA tubes. All procedures were performed by trained personnel. Most bats were released at the site of capture immediately after sampling. A subset of individuals (14/57) was retained as voucher specimens; these individuals were euthanized using isoflurane and preserved in absolute ethanol and are deposited in the SMACON (the Afrotropical Biodiversity Center) museum collection, where they retain their original specimen identification numbers ( S1 Table ). The use of isoflurane followed established guidelines for humane euthanasia of small mammals [ 20 ]. All remaining individuals were released at the sampling site following recovery. Fruit bats (Pteropodidae) were provided with a sugar solution prior to release, whereas no supplemental feeding was provided to insectivorous species. Microscopy and ectoparasite identification Thin blood smears were prepared, air-dried, and fixed in absolute methanol for 60 seconds. Slides were then stained with Giemsa for 45 minutes, gently rinsed with phosphate-buffered saline (PBS), and allowed to air-dry. The stained smears were examined microscopically using a 100 × oil immersion objective (1,000 × total magnification). Each slide was systematically screened across the entire smear for the presence of trypanosomes. Bat fly specimens were subsequently examined under a stereomicroscope and identified to family level (e.g., Nycteribiidae, Streblidae) based on morphological characteristics using published taxonomic keys [ 26 ]. DNA extracted from selected ectoparasite specimens was subjected to PCR amplification of the mitochondrial cox1 gene to confirm and, where possible, refine these identifications. Molecular methods Genomic DNA was extracted from EDTA-preserved blood using the QIAamp Mini Kit (Qiagen) following the manufacturer’s protocol. DNA was eluted in 100 µL of elution buffer and stored at −20 °C. Genomic DNA from ethanol-preserved ectoparasites was extracted using the Quick-DNA Miniprep Plus Kit (Zymo Research). Ectoparasites were rinsed three times in sterile phosphate-buffered saline (PBS), air-dried under sterile conditions, and individually homogenized in 1.5 mL tubes using sterile pestles. Homogenates were incubated with 95 µL nuclease-free water, 95 µL solid tissue buffer and 10 µL Proteinase K at 55 °C for 5 h before further processing according to the manufacturer’s protocol. DNA was eluted in 100 µL elution buffer and stored at −20 °C. PCR amplification was performed using the AllTaq Master Mix Kit (Qiagen) with 4–5 µL of genomic DNA and 1 µL of each primer (10 µM). Trypanosome screening employed a nested PCR targeting approximately 600 bp of the small subunit 18S ribosomal RNA gene (18S rRNA) following [ 27 ]. The primary reaction used primers TRY927F/R, and the nested reaction used SSU561F/R. Selected ectoparasites were genotyped using the standard DNA barcoding primers LCO1490/HCO2198 [ 28 ], amplifying approximately 720 bp of the mitochondrial cox1 gene. Primer sequences are provided in S2 Table . PCR products of the expected size were visualized by agarose gel electrophoresis and purified using the QIAquick PCR Purification Kit (QIAGEN) according to the manufacturer’s instructions. Purified amplicons were subsequently subjected to Sanger sequencing in both directions using the same primers as for amplification. Sequencing was performed at LGC Genomics (Berlin, Germany). Nucleotide sequence analysis Nucleotide sequences were manually curated in Geneious Prime 2024.04. Ambiguous base calls were coded using standard ambiguity codes. Samples with low-quality reads were re-amplified and sequenced where possible. Sequences exhibiting ambiguous signals, including double peaks, elevated background noise, or a high proportion of unresolved nucleotides, were considered low quality. Although some of these sequences showed highest similarity to trypanosome sequences in BLAST searches, they were omitted from the phylogenetic inference due to insufficient quality for reliable analyses. These samples were, however, retained as positive detections for the purpose of prevalence estimation. Sequence identity was assessed using BLASTn (NCBI). Mitochondrial cox1 gene sequences obtained from ectoparasites were used for molecular identification. Edited sequences were compared against the NCBI database using BLASTn to determine their closest matches and confirm taxonomic assignment. Sequence identity values and top hits were used to support species-level or genus-level identification. Trypanosome sequences were aligned using MAFFT implemented in Geneious Prime [ 29 , 30 ]. Reference sequences were retrieved from GenBank and included in the 18S rRNA alignment (accession numbers are provided in the respective phylogenetic tree figure and S3 Table ). The final 18S rRNA dataset comprised 98 sequences (including 16 representative sequences from this study) with a total length of 821 nucleotides (nt) including 139 gap positions. The final alignment used for phylogenetic analysis is available in FASTA format from the OSF repository. Model selection was performed using ModelTest-NG 0.1.7 [ 31 ] implemented in raxmlGUI v2.0.14 [ 32 ]. Maximum Likelihood (ML) analysis was conducted under the model TIM3 + I (proportion of invariant) + Gamma (rate heterogeneity). Trypanosoma microti and Trypanosoma lewisi were used as outgroups. Node support was assessed with 1,000 bootstrap replicates (thorough bootstrap). The phylogenetic tree was visualized in FigTree v.1.4.4 ( http://tree.bio.ed.ac.uk/software/figtree/ ). Putative molecular lineages were inferred using the Bayesian implementation of the Poisson Tree Processes model on the bPTP web server ( https://species.h-its.org/ptp/ ). Because PTP-based approaches may overestimate the number of delimited lineages when terminal sampling is uneven [ 33 ], a reduced 18S rRNA dataset was generated for the bPTP analysis. The dataset was screened for identical and highly similar sequences, and phylogenetically redundant reference terminals from overrepresented species or lineages were pruned. Representative sequences from the major named species/lineages, the T. livingstonei / T. cf . livingstonei -related clade, and all newly generated Nigerian sequences included in the analysis were retained. The final reduced alignment contained n = 65 terminals and had a length of 821 nt. A maximum-likelihood tree was re-estimated from this reduced alignment using the same settings as described above. The tree included selected outgroup taxa and was rooted using Trypanosoma microti and Trypanosoma lewisi as outgroups. The rooted tree was submitted to the bPTP server in Newick format, and the outgroup taxa were excluded from the delimitation analysis using the outgroup-removal option of the server. The bPTP analysis was run for 200,000 MCMC generations, with a thinning value of 100 and a burn-in of 25%. Convergence of the MCMC chain was assessed visually using the diagnostic plots provided by the server. The resulting groups were interpreted as putative molecular lineages/spe
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