METAGENOMIC BLOOD SCREENING FOR MICROBIAL PATHOGENS IN BATS INHABITING THREE BULGARIAN CAVES

Authors

DOI:

https://doi.org/10.58395/9vk3tc02

Keywords:

bats, blood microbiome, metagenomics, zoonotic pathogens, Bulgarian caves

Abstract

Background: Bats are important reservoir hosts for numerous emerging pathogens with zoonotic potential. In Europe, 47 bat species have been recorded, 33 of which occur in Bulgaria. Previous research has shown that bats harbor several dangerous viruses, including SARS, Ebola, Nipah, Hendra, Marburg, and MERS, as well as various bacterial, fungal, and protozoan pathogens. Despite these findings, information about the composition of the bat blood microbiome remains limited. The present study aimed to characterize the microbial diversity present in the blood of bats inhabiting caves in Bulgaria.

Materials and Methods: Blood samples were collected from bat species belonging to the families Vespertilionidae and Rhinolophidae inhabiting three caves in Bulgaria. Between 10 and 30 µL of blood was obtained from each individual. Samples from 122 bats were combined into 14 pooled samples. Additionally, heart tissue and two spleens were collected. DNA extraction was performed, followed by metagenomic sequencing using Illumina shotgun technology with a sequencing depth of 6 Gb. Taxonomic classification of quality-filtered reads was conducted using the Kraken2 tool with the RefSeq NCBI bacterial database within the Galaxy platform (www.usegalaxy.eu). Operational taxonomic unit (OTU) tables were generated using Bracken and visualized with Pavian software.

Results: Metagenomic analysis revealed the presence of numerous microbial taxa in bat blood samples. A notable abundance of potentially pathogenic bacteria was detected, with sequencing reads exceeding 10000 for species belonging to the genera Mycobacterium, Mycoplasma, and Bartonella.

Conclusions: The results indicate that bats in Bulgaria may serve as hidden or potential reservoirs of bacterial pathogens with zoonotic relevance. These findings highlight the importance of continued surveillance of bat-associated microbiota to better understand potential public health risks.

Downloads

Download data is not yet available.

References

1. Panaiotov S, Hodzhev Y, Tsafarova B, Nenova R, Mikov O, Tolchkov V, Slavov G, Deleva S, Toshkova N, Nedyalkov N, Langurov M, Bekchiev R, Nikolova M, Yakici G, Stoev P, Simov N. Monitoring of extreme ecological niches for novel and reoccurring zoonoses. International Conference One Health and Zoology; 2023 Sep 27–29; Hissarya, Bulgaria

2. Brook CE, Dobson AP. Bats as ‘special' reservoirs for emerging zoonotic pathogens. Trends Microbiol. 2015; 23(3):172-180. https://doi.org/10.1016/j.tim.2014.12.004 DOI: https://doi.org/10.1016/j.tim.2014.12.004

3. Calisher CH, Childs JE, Field HE, Holmes KV, Schountz T. Bats: Important reservoir hosts of emerging viruses. Clin Microbiol Rev. 2006; 19(3):531-545. https://doi.org/10.1128/CMR.00017-06 DOI: https://doi.org/10.1128/CMR.00017-06

4. Popov VV. Bats in Bulgaria: Patterns of species distribution, richness, rarity, and vulnerability derived from distribution models. In: Voigt CC, Kingston T, editors. Bats. InTech; 2018. https://doi.org/10.5772/intechopen.73623 DOI: https://doi.org/10.5772/intechopen.73623

5. Deleva S, Toshkova N, Kolev M, Tanalgo KC. Important underground roosts for bats in Bulgaria: current state and priorities for conservation. Biodivers Data J. 2023; 11:e98734. https://doi.org/10.3897/BDJ.11.e98734 DOI: https://doi.org/10.3897/BDJ.11.e98734

6. Bazzoni E, Cacciotto C, Zobba R, Pittau M, Martella V, Alberti A. Bat ecology and microbiome of the gut: A narrative review of associated potentials in emerging and zoonotic diseases. Animals. 2024; 14(20):3043. https://doi.org/10.3390/ani14203043 DOI: https://doi.org/10.3390/ani14203043

7. Blehert DS, Hicks AC, Behr M, Meteyer CU, Berlowski-Zier BM, Buckles EL, Coleman JTH, Darling SR, Gargas A, Niver R, Okoniewski JC, Rudd RJ, Stone WB. Bat white-nose syndrome: An emerging fungal pathogen? Science. 2009;323(5911):227. https://doi.org/10.1126/science.1163874 DOI: https://doi.org/10.1126/science.1163874

8. Han HJ, Wen HL, Zhou CM, Chen FF, Luo LM, Liu JW, Yu XJ. Bats as reservoirs of severe emerging infectious diseases. Virus Res. 2015; 205:1-6. https://doi.org/10.1016/j.virusres.2015.05.006 DOI: https://doi.org/10.1016/j.virusres.2015.05.006

9. Plowright RK, Eby P, Hudson PJ, Smith IL, Westcott D, Bryden WL, Middleton D, Reid PA, McFarlane RA, Martin G, Tabor GM, Skerratt LF, Anderson DL, Crameri G, Quammen D, Jordan D, Freeman P, Wang LF, Epstein JH, McCallum H. Ecological dynamics of emerging bat virus spillover. Proc R Soc B Biol Sci. 2015; 282(1798):20142124. https://doi.org/10.1098/rspb.2014.2124

10. Olival KJ, Hosseini PR, Zambrana-Torrelio C, Ross N, Bogich TL, Daszak P. Host and viral traits predict zoonotic spillover from mammals. Nature. 2017; 546(7660):646-650. https://doi.org/10.1038/nature22975 DOI: https://doi.org/10.1038/nature22975

11. Stuckey MJ, Chomel BB, de Fleurieu EC, Aguilar-Setién A, Boulouis HJ, Chang CC. Bartonella, bats and bugs: A review. Comp Immunol Microbiol Infect Dis. 2017; 55:20-29. https://doi.org/10.1016/j.cimid.2017.09.001 DOI: https://doi.org/10.1016/j.cimid.2017.09.001

12. Sun H, Wang Y, Zhang Y, Ge W, Zhang F, He B, Li Z, Fan Q, Wang W, Tu C, Li J, Liu Q. Prevalence and genetic characterization of Toxoplasma gondii in bats in Myanmar. Appl Environ Microbiol. 2013; 79(11):3526-3528. https://doi.org/10.1128/AEM.00410-13 DOI: https://doi.org/10.1128/AEM.00410-13

13. Païssé S, Valle C, Servant F, Courtney M, Burcelin R, Amar J. Comprehensive description of blood microbiome from healthy donors assessed by 16S targeted metagenomic sequencing. Transfusion. 2016; 56(5):1138-1147. https://doi.org/10.1111/trf.13477 DOI: https://doi.org/10.1111/trf.13477

14. Schaer J, Perkins SL, Decher J, Leendertz FH, Fahr J, Weber N, Matuschewski K, Mätz-Rensing K. High diversity of West African bat malaria parasites and a tight link with rodent Plasmodium taxa. Proc Natl Acad Sci USA. 2013; 110(43):17415-17419. https://doi.org/10.1073/pnas.1311016110 DOI: https://doi.org/10.1073/pnas.1311016110

15. Yu Z, Morrison M. Improved extraction of PCR-quality community DNA from digesta and fecal samples. BioTechniques. 2004; 36:808-812. https://doi.org/10.2144/04365ST04 DOI: https://doi.org/10.2144/04365ST04

16. Wood DE, Lu J, Langmead B. Improved metagenomic analysis with Kraken 2. Genome Biol. 2019; 20:257. https://doi.org/10.1186/s13059-019-1891-0 DOI: https://doi.org/10.1186/s13059-019-1891-0

17. Afgan E, Baker D, Batut B, van den Beek M, Bouvier D, Čech M, Chilton J, Clements D, Coraor N, Grüning BA, Guerler A, Hillman-Jackson J, Hiltemann S, Jalili V, Rasche H, Soranzo N, Goecks J, Taylor J, Nekrutenko A, Blankenberg D. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Res. 2018; 46(W1):W537-W544. https://doi.org/10.1093/nar/gky379 DOI: https://doi.org/10.1093/nar/gky379

18. Breitwieser FP, Salzberg SL. Pavian: Interactive analysis of metagenomics data for microbiome studies and pathogen identification. Bioinformatics. 2020; 36(4):1303-1304. https://doi.org/10.1093/bioinformatics/btz715 DOI: https://doi.org/10.1093/bioinformatics/btz715

19. Lu J, Breitwieser FP, Thielen P, Salzberg SL. Bracken: Estimating species abundance in metagenomics data. PeerJ Comput Sci. 2017; 3:e104. https://doi.org/10.7717/peerj-cs.104 DOI: https://doi.org/10.7717/peerj-cs.104

20. Szentivanyi T, McKee C, Jones G, Foster JT. Trends in Bacterial Pathogens of Bats: Global Distribution and Knowledge Gaps. Transbound Emerg Dis. 2023, 2023:9285855. https://doi.org/10.1155/2023/9285855 DOI: https://doi.org/10.1155/2023/9285855

21. Corduneanu A, Zając Z, Kulisz J, Wozniak A, Foucault-Simonin A, Moutailler S, Wu-Chuang A, Peter Á, Sándor AD, Cabezas-Cruz A. Detection of bacterial and protozoan pathogens in individual bats and their ectoparasites using high-throughput microfluidic real-time PCR. Microbiol Spectr. 2023; 11(5):e0153123. https://doi.org/10.1128/spectrum.01531-23. DOI: https://doi.org/10.1128/spectrum.01531-23

22. O'Shea TJ, Cryan PM, Cunningham AA, Fooks AR, Hayman DTS, Luis AD, et al. Bat flight and zoonotic viruses. Emerg Infect Dis. 2014; 20(5):741-745. https://doi.org/10.3201/eid2005.130539 DOI: https://doi.org/10.3201/eid2005.130539

23. Luis AD, Hayman DTS, O'Shea TJ, Cryan PM, Gilbert AT, Pulliam JRC, Mills JN, Timonin ME, Willis CKR, Cunningham AA, Fooks AR, Rupprecht CE, Wood JLN, Webb CT. A comparison of bats and rodents as reservoirs of zoonotic viruses: Are bats special? Proc R Soc B Biol Sci. 2013; 280(1756):20122753. https://doi.org/10.1098/rspb.2012.2753

24. Moreno KR, Weinberg M, Harten L, Salinas Ramos VB, Herrera MLG, Czirják GÁ, Yovel Y. Sick bats stay home alone: fruit bats practice social distancing when faced with an immunological challenge. Ann N Y Acad Sci. 2021; 1505:178-190. https://doi.org/10.1111/nyas.14600 DOI: https://doi.org/10.1111/nyas.14600

25. Luo J, Greif S, Ye H, et al. Flight rapidly modulates body temperature in freely behaving bats. Anim Biotelemetry. 2021; 9:45. https://doi.org/10.1186/s40317-021-00268-6 DOI: https://doi.org/10.1186/s40317-021-00268-6

26. Péter Á, Mihalca AD, Sándor AD. First report of the bat fly species Basilia italica in Romania. Biodivers Data J. 2021; 9:e57680. https://doi.org/10.3897/BDJ.9.e57680 DOI: https://doi.org/10.3897/BDJ.9.e57680

27. Sándor AD, Péter Á, Corduneanu A, Barti L, Csősz I, Kalmár Z, Hornok S, Kontschán J, Mihalca AD. Wide distribution and diversity of malaria-related haemosporidian parasites (Polychromophilus spp.) in bats and their ectoparasites in Eastern Europe. Microorganisms. 2021; 9(2):230. https://doi.org/10.3390/microorganisms9020230 DOI: https://doi.org/10.3390/microorganisms9020230

Downloads

Published

2026-09-19

Issue

Section

Articles

How to Cite

(1)
Panaiotov, S.; Hodzhev, Y.; Tsafarova, B.; Mikov, O.; Nedyalkov, N.; Langurov, M.; Bekchiev, R.; Stoev, P.; Zhelyazkova, M.; Petrova, M.; Generalova, A.; Simov, N. METAGENOMIC BLOOD SCREENING FOR MICROBIAL PATHOGENS IN BATS INHABITING THREE BULGARIAN CAVES. Probl Infect Parasit Dis 2026, 54 (2), 21-29. https://doi.org/10.58395/9vk3tc02.

Most read articles by the same author(s)