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Article

Molecular Survey of Hemopathogens in Bats from the Western Brazilian Amazon

by
Abraão Isaque da Silva
1,
Eliz Oliveira Franco
1,
Ana Cláudia Calchi
1,
Francisco Chagas Bezerra dos Santos
2,
Rair de Sousa Verde
3,
Victória Valente Califre de Mello
1,
Daniel Antônio Braga Lee
1,
Caroline Tostes Secato
1,
Clara Morato Dias
1,
Rosangela Zacarias Machado
1,
André Luiz Rodrigues Roque
2 and
Marcos Rogério André
1,*
1
Vector-Borne Bioagents Laboratory (VBBL), Department of Pathology, Reproduction and One Health, School of Agrarian and Veterinary Sciences, São Paulo State University, Jaboticabal CEP 14884-900, Brazil
2
Laboratory of Trypanosomatid Biology, Instituto Oswaldo Cruz (FIOCRUZ), Rio de Janeiro CEP 21040-900, Brazil
3
Graduate Program in Health and Sustainable Animal Production in the Amazon, Federal University of Acre, Rio Branco CEP 69920-900, Brazil
*
Author to whom correspondence should be addressed.
Pathogens 2025, 14(6), 527; https://doi.org/10.3390/pathogens14060527
Submission received: 18 April 2025 / Revised: 14 May 2025 / Accepted: 22 May 2025 / Published: 25 May 2025
(This article belongs to the Special Issue Vector Control and Parasitic Infection in Animals)
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Versions Notes

Abstract

:
Bats are considered reservoirs of several emerging zoonotic pathogens. Previous studies on blood parasites such as Anaplasmataceae, hemoplasmas, piroplasmids, and Hepatozoon spp. in South American bats have revealed high genetic diversity. This study investigated the molecular occurrence of these agents in 278 bats of 32 species captured in the state of Acre in the Western Brazilian Amazon. Spleen DNA samples were screened by PCR for different pathogens and tested negative for Anaplasma spp., Ehrlichia spp., Neorickettsia spp., piroplasmids, and Hepatozoon spp. However, 84 of the 208 samples (40.4%) were positive for hemoplasmas based on the 16S rRNA gene, and 15 out of the 84 (17.85%) were positive for the 23S rRNA gene. Seventeen 16S rRNA sequences, corresponding to 12 genotypes, were grouped with hemotropic Mycoplasma sp. previously detected in bats from Brazil, Peru, and Belize. Three 23S rRNA sequences represent three distinct genotypes clustered with hemotropic Mycoplasma sp., previously detected in Desmodus rotundus. This is the first molecular report of hemoplasmas in six bat species, namely Dermanura cinereus, Lophostoma silviculum, Phyllostomus elongatus, Phyllostomus hastatus, Rhinophylla fischerae, and Sturnira tildae.

1. Introduction

The order Chiroptera represents the second most diverse group of mammals described in Brazil, composed of 9 families, 68 genera, and 186 species identified in different regions of the country [1,2]. These mammals play an important role in the recovery and maintenance of forest environments, pollination, control of insect populations, and maintenance of the balance and functionality of ecosystem dynamics [3]. Due to the unique characteristics of the life history, such as long life expectancy (average of 10 to 20 years), large population densities, roosting behavior, and ability to fly, bats are considered ideal reservoirs for several emerging zoonotic pathogens [4].
The genera Anaplasma, Ehrlichia, and Neorickettsia (Rickettsiales: Anaplasmataceae) encompass obligate intracellular alphaproteobacteria capable of infecting a wide variety of animal and human blood cells [5,6]. While Ehrlichia and Anaplasma species are transmitted by Ixodida ticks [7], Neorickettsia spp. are transmitted to vertebrates through the ingestion of larval forms of digenetic trematodes infected by the agent [8,9]. Although such agents have been detected in bats and associated ectoparasites or trematodes in South America [10,11,12,13] and Europe [14,15,16,17], few studies have been conducted in Brazil [18,19]. In this sense, Ikeda et al. [20] detected that Ehrlichia spp. were phylogenetically associated with Ehrlichia minasensis, Ehrlichia ruminantium, and Anaplasma spp., related to A. phagocytophilum and Neorickettsia spp. in non-hematophagous bats and associated ectoparasites in Central–Western Brazil. Recently, Ehrlichia spp. phylogenetically related to E. minasensis, Anaplasma marginale, and Neorickettsia spp. phylogenetically associated with N. risticii that have been detected in hematophagous bats in the northern region of Brazil [19].
Hemoplasmas (Mycoplasmatales: Mycoplasmataceae) are epi-erythrocytic Gram-negative bacteria that can cause hemolytic anemia in animals and humans [21]. Although these bacteria have been reported in bats in several Brazilian states [22,23,24,25], the genetic diversity of such detected agents seems higher than previously noticed. The incrimination of bats of the Pteromalidae family in New Caledonia as reservoirs of ‘Candidatus Mycoplasma haematohominis’, a species of hemoplasma that causes clinical disease in humans [26], reinforced the need to investigate the diversity of hemoplasmas in bats.
Although piroplasmids (Piroplasmorida: Babesiidae/Theileriidae) have been extensively investigated in mammals worldwide [27], few studies have been conducted to detect these tick-borne apicomplex protozoa in Neotropical bats. Babesia vesperuginis has been pointed out as the main piroplasmid species detected in bats from Europe and Asia [28,29,30,31,32,33,34,35]. However, little is known regarding the molecular identity of piroplasmids in bats from other continents. A supposed novel Babesia sp. related to the Babesia microti group has been described in bats from Madagascar [36]. In South America, Babesia sp. was found in blood smears from bats sampled in Colombia [37]. At the same time, a putative novel Piroplasmorida species was molecularly detected in Phyllostomus discolor in Central–Western Brazil [38]. In contrast, Babesia spp. and Theileria spp., phylogenetically grouped in the clades “South American Marsupialia”, “Theileria sensu stricto”, and “Tapirus terrestris”, have been detected in hematophagous bats sampled in the northern region of Brazil [24].
Hepatozoon spp. (Adeleorina: Hepatozoidae) are apicomplexan protozoa transmitted by ingesting definitive invertebrate hosts (ticks, mosquitoes, flies, fleas, lice) and predation [39,40,41,42,43]. Few studies have reported Hepatozoon spp. in bats worldwide. So far, Hepatozoon spp. has only been detected in bats from Borneo [44] and Brazil [45,46]. The 18S rRNA sequences detected by Hepatozoon spp. were positioned in the same clade as Hepatozoon spp., detected in reptiles, rodents, amphibians, and marsupials [44,45,46].
Considering that Brazil has a huge bat fauna and plays a potential role in the maintenance and transmission of distinct hemopathogens worldwide, the present study aimed to investigate the occurrence and molecular identity of Anaplasmataceae, hemoplasmas, piroplasmids, and Hepatozoon spp. agents in non-hematophagous bats sampled in the state of Acre, in the Western Brazilian Amazon Forest.

2. Materials and Methods

2.1. Ethical Statement

The bat captures were approved by the Chico Mendes Institute for the Conservation of Brazilian Biodiversity (ICMBio—SISBIO), licenses 44089-1 and 47377-1. All procedures carried out followed protocols approved by the Ethics Committee on the Use of Animals of FIOCRUZ (LW81-12) and by the Ethics Committee on the Use of Animals of the Faculty of Agrarian and Veterinary Sciences of UNESP (CEUA FCAV/UNESP 3911/23).

2.2. Sampling and Study Area

Between 2014 and 2015, 367 non-hematophagous bats were captured in two municipalities in the state of Acre: Rio Branco (09°58′29″ S/67°48′36″ W) and Xapuri (10°10′95″ S/68°30′16″ W) [47] (Figure 1). Morphological characters were used to identify the bats [48], which were classified into 23 genera and 30 species. Of the 367 bats captured [47], 278 of those for which spleen samples were available were included in the present study. Of this total, 196 samples were collected in Rio Branco and 82 in Xapuri (Table 1).

2.3. DNA Extraction and PCR for the Endogenous Mammalian Gene

DNA extraction from bat spleen samples was performed using the BioPur Mini Spin Plus Extraction Kit (Mobius Life Science, Pinhais, PR, Brazil), following the manufacturer’s instructions. After DNA extraction, the concentration and quality of the DNA (260/280 ratio) were evaluated using a spectrophotometer (Nanodrop, Thermo Fisher Scientific, San Jose, CA, USA). To verify the presence of amplifiable DNA and the absence of PCR inhibitors in the extracted samples, a conventional PCR assay targeting the endogenous mammalian glyceraldehyde-3-phosphate dehydrogenase (gapdh) gene was performed [50], as previously described [51]. Only positive samples for these assays were used in other PCR reactions to detect the infections.

2.4. PCR Tests for Screening and Characterisation of Haemopathogens

PCR assays targeting the 16S rRNA gene of Anaplasma spp. (548 bp) [52], Neorickettsia spp. (529 bp) [53] and hemoplasmas (620 bp) [54], the dsb gene of Ehrlichia spp. (409 bp) [55], the 18S rRNA gene of piroplasmids (800 bp) [56], Hepatozoon spp. (800 bp) [57], and (600 bp) [58] were performed. In addition, PCR assays based on the 16S rRNA (1380 bp) [54] and 23S rRNA (800 bp) [59] genes were conducted for further molecular characterization of the hemoplasma-positive samples.
Each PCR reaction had a total volume of 25 µL, containing 10× PCR buffer (Promega®, Madison, WI, USA), sterilized ultrapure water (Invitrogen®, Carlsbad, CA, USA), 0.2 mM of each deoxynucleotide, 0.4 µM of each oligonucleotide, 3.0 mM of MgCl2, 1.25 U Go Taq Hot Start Polymerase (Promega®, Madison, WI, USA), and 3 µL of DNA template. In nPCR assays for piroplasmids and Neorickettsia spp., 1 µL of the amplified product of the first PCR reaction was used as the target DNA in the second reaction. DNA samples of Ehrlichia spp., obtained from bats [19], hemotropic Mycoplasma spp., obtained from Tapirus terrestris [60], Babesia vogeli (Jaboticabal strain) [61], and Hepatozoon sp. detected in Cerdocyon thous [62] were used as positive controls. gBlocks® (Integrated DNA Technologies, Coralville, IA, USA) containing an insert of the target fragments of the 16S rRNA gene from Anaplasma phagocytophilum and Neorickettsia risticii were also used as positive controls. Sterile ultrapure water (Invitrogen®, Carlsbad, CA, USA) was used as a negative control in all PCR assays.

2.5. Sequencing and Phylogenetic Analysis

The PCR products were purified using the ExoSAP-IT™ PCR Product Cleaning Reagent (Thermo Scientific, San Jose, CA, USA). Sequencing of the amplified products was performed using an automated technique based on the Sanger dideoxynucleotide chain termination method [63] in an ABI PRISM 3700 (Applied Biosystems, Waltham, MA, USA) DNA analyzer in the Human Genome and Stem Cell Research Center, Institute of Biosciences, University of São Paulo (USP), São Paulo, SP, Brazil.
An analysis of the electropherograms generated in the sequencing was performed, observing the quality of the peaks corresponding to each sequenced base using Bioedit v. 7.0.5.3 software [64]. To construct the consensus sequences, the Phred-Phrap version 23 [65,66] program was used, analyzing the “forward” and “reverse” strands sequenced from the same sample, respecting the minimum quality value of 20 for each nucleotide to determine the nucleotide sequence. The consensus sequences were subjected to analysis by BLASTn [67] for comparison with those deposited in GenBank “http://www.ncbi.nlm.nih.gov/genbank (Accessed on 25 February 2025)”.
Sequences saved in “FASTA” mode were aligned with other homologous sequences of the same sequenced gene retrieved from the GenBank database using MAFFT-Multiple Sequence Alignment Software version 7.0 available online “https://mafft.cbrc.jp/alignment/server/index.html” (Accessed on 25 February 2025) [68]. The phylogenetic analysis of maximum likelihood was performed using the W-IQ-Tree software available online “http://iqtree.cibiv.univie.ac.at/” (Accessed on 25 February 2025) [69,70]. Clade support for maximum likelihood analysis was evaluated using bootstrap analysis [71] with 1000 replicates. Phylogenetic tree editing, as well as rooting (via external group), was performed using the Treegraph 2.13.0 beta software [72].

2.6. Analysis of Genetic Diversity

The analysis of genetic diversity was performed using the sequences obtained in the analysis of the hemoplasm 16S rRNA and 23S rRNA. The calculation of nucleotide diversity (π), polymorphism level (haplotype diversity—[dh]), number of haplotypes (h), and the average number of nucleotide differences (K) between the sequences obtained was performed using the DnaSP v5 software [73].

3. Results

In total, 208 (74.8%) spleen samples were positive for the endogenous gapdh gene and submitted to diagnostic PCR reactions. All bats were negative in molecular screening assays targeting Anaplasma spp. 16S rRNA, Ehrlichia spp. dsb, Neorickettsia spp. 16S rRNA, piroplasmid 18S rRNA, and Hepatozoon spp. 18S rRNA. On the other hand, 84/208 (40.4%) of the bats were positive in PCR directed to the 16S rRNA gene of hemoplasmas (~620 bp), comprising different species of bats. Of these, 15/84 (17.85%) were positive in the molecular characterization based on the 23S rRNA gene (800 bp) (Table 2).
We were able to retrieve 20 high-quality consensus sequences: 17 for the 16S rRNA genes and 3 for the 23S rRNA genes. The number of sequences obtained, as well as the results of the BLASTn analysis, including the target gene, the coverage of the consultation, the E-value, and the percentage of identity, are described in Table 3.
The phylogenetic analysis based on the 16S rRNA gene (620 bp alignment) inferred by the Maximum Likelihood method and using GTR + F + I + G4 as an evolutionary model, grouped the sequences obtained PV388069 (C. perspicillata), PV388082 (P. infuscus), and PV388084 (C. beikeith c.f.) in the same clade with a sequence of Mycoplasma spp., detected in D. rotundus in Brazil, with bootstrap clade support of 88. In addition, the sequences PV388075 (R. fischerae) and PV388076 (R. fischerae) were positioned in the same clade, sister to the clade containing the PV388069 sequence and the G. soricina sequence from Brazil, supported by a bootstrap value of 100. The PV388083 sequence (D. cinereus) was positioned in a sister group with Mycoplasma sp. sequences previously detected in Artibeus lituratus from Belize and Platyrrhinus lineatus from Brazil, supported by a bootstrap value of 100. The sequences PV388070 (P. elongatus), PV388071 (P. discolor), PV388072 (P. hastatus), PV388073 (P. hastatus), PV388074 (L. silviculum), PV388078 (P. elongatus), PV388079 (P. elongatus), PV388080 (P. hastatus), and PV388085 (A. caudifer) were positioned in the same subclade as the sequences of Mycoplasma sp. detected in P. discolor from Brazil, with a bootstrap support value of 95. The PV388077 sample (S. tildae) was positioned in a sister clade with Mycoplasma sp. sequences detected in A. lituratus in Brazil and D. rotundus in Peru. Finally, the PV388081 sequence (A. lituratus) was positioned in the sister clade of a clade containing sequences of Mycoplasma spp., detected in A. planirostris in Brazil and Platyrrhunus helleri in Belize (Figure 2).
The phylogenetic analysis based on the 23S rRNA gene (800 bp alignment) inferred by the Maximum Likelihood method and the GTR + F + G4 evolutionary model positioned the sequences PV364143 (C. perspicillata), PV364145 (C. perspicillata), and PV364144 (C. perspicillata), in the same subclade, sister of a subclade containing sequences of Mycoplasma spp., previously detected in D. rotundus and G. soricina, supported by a bootstrap value of 99 (Figure 3).
The genetic diversity analysis revealed the presence of 12 genotypes among the sixteen 16S rRNA sequences, with a genotypic diversity ranging from 0.9667 ± 0.03, while 3 genotypes were found among the three sequences of the 23S rRNA gene with high genotypic diversity ranging from 1 ± 0.740 (Table 4).

4. Discussion

The present work contributes to the understanding of the genetic diversity of hemoplasmas in non-hematophagous bats in Western Amazonia, a geographic location still little explored in the Brazilian territory with regard to the theme addressed in this study. We found an occurrence of 84/208 (40.4%) for hemoplasmas in bats in the region studied, bringing the first report of these agents in bats of the species Dermanura cinereus, Lophostoma silviculum, Phyllostomus elongatus, Phyllostomus hastatus, Rhinophylla fischerae, and Sturnira tildae. Previous studies from different regions of Brazil have reported molecular occurrences of hemoplasmas in bats ranging from 6.1% to 80% [21,22,23,24]. Although these data cannot be compared, they indicate a relatively high occurrence of hemoplasmas in the bat populations studied.
Phylogenetic analyses showed no distinction between the phylogenetic positioning of the hemoplasma sequences detected in bats of different feeding habits since the hemoplasma sequences obtained from non-hematophagous bats in this study were positioned close to those obtained from vampire and non-hematophagous bats from Belize, Peru, and Brazil [24,74,75,76]. In fact, although genotype analysis showed that hemoplasmas detected in bats of the same genus tended to cluster together in a previous study, the results of unipartite and bipartite analyses did not robustly support this hypothesis [76]. Although the route of transmission of hemoplasmas between bats is unknown, the behavior of co-perching between different bat species, direct contact (e.g., grooming between females), and environmental exposure may favor the transmission of hemoplasmas between bats [76].
The high genotypic diversity of the 16S and 23S rRNA sequences observed in this study corroborates the findings of Ikeda et al. [76], who identified 12 genotypes among 24 16S rRNA sequences. Similarly, high genotypic diversity was found among the 16S rRNA sequences detected in hematophagous bats in the Brazilian Amazon, where 19 genotypes were found among 23 sequences of the 16S rRNA gene [25].
This study reported the absence of DNA from agents of Anaplasmataceae, piroplasmids, and Hepatozoon spp. in non-hematophagous bats from the state of Acre, Northern Brazil. This lack of detection of these hemopathogens may be related to the absence of competent vectors in the region studied or the presence of a parasitemia below the detection threshold of the PCR assays used here. Future studies with the aim of using higher sensitivity techniques (e.g., digital PCR) should be carried out to confirm the results obtained. Similarly, these agents were not detected in vampire bat liver samples collected in several Brazilian regions [18]. On the other hand, Ikeda et al. [20] detected DNA from Anaplasma spp. and a genotype of Ehrlichia sp. phylogenetically associated with E. ruminantium in bats from the Central-Western region of Brazil. In the northern region of Brazil, Anaplasma spp. and different genotypes of Ehrlichia spp. were detected molecularly in spleen samples of D. rotundus. Interestingly, sequences of Ehrlichia minasensis and Anaplasma marginale were detected in the sampled hematophagous bats, leading to suspicions of transmission of these agents from cattle to D. rotundus via hematophagy [19]. In addition, distinct genotypes of Ehrlichia spp. have also been detected, suggesting that they may circulate specifically among bats [19].
Although bats are incriminated as important links in the biological cycle of N. risticii, acting not only as definitive hosts of trematodes infected with this parasite but also as probable natural reservoirs for N. risticii [10,12,13,77], all bat spleen samples were negative for Neorickettsia spp. Previously, Neorickettsia spp., phylogenetically associated with N. risticii, was detected in spleen samples from non-hematophagous bats in the Cerrado biome [20], Central-Western Brazil, and from hematophagous bats in the Amazon, Northern Brazil [19].
In this work, we did not detect the presence of piroplasmids and DNA of Hepatozoon spp. in Amazonian bats. Studies carried out with blood samples from bats from China, and liver samples from hematophagous bats captured in several Brazilian geographic areas were also negative for the investigated protozoa [24,78]. In fact, the low occurrence of these agents has been reported in bats, ranging from 3% to 20.6% for Hepatozoon spp. [24,30,31] and 2.7% to 18.8% for piroplasmids [32,33,34]. Additional studies have also observed similar low prevalence rates [36,38,45,46]. On the other hand, only one study conducted in Lithuania reported high positivity rates for piroplasmids in bats, with 35.2% (44/125) and 52.3% (45/86) positivity in blood and tissues, respectively [35]. In this study, qPCR based on the 18S rRNA gene was used to detect B. vesperuginis, a method that showed higher sensitivity compared to nPCR. However, previous studies conducted by our research group have shown the occurrence of piroplasmids of different phylogenetic lineages in non-hematophagous bats sampled in the Brazilian Cerrado biome [38] and in specimens of Desmodus rotundus sampled in the Brazilian Amazon [25].

5. Conclusions

The present study provides the first molecular evidence of hemoplasma infection in the bat species Dermanura cinereus, Lophostoma silviculum, Phyllostomus elongatus, Phyllostomus hastatus, Rhinophylla fischerae, and Sturnira tildae. Bats sampled in two localities in the state of Acre, in the Western Amazon Forest of Brazil, do not seem to be exposed to infection by Anaplasmataceae agents, piroplasmids, and Hepatozoon spp., which may be related to the absence of competent vectors for such agents in the localities studied.

Author Contributions

A.I.d.S., conceptualization, methodology, visualization, formal analysis, writing—original version, writing—review and editing; E.O.F., conceptualization, methodology, visualization, formal analysis, writing—review and editing; A.C.C., conceptualization, methodology, visualization, formal analysis, writing—original version; F.C.B.d.S., methodology, writing—review and editing; R.d.S.V., methodology, writing—review and editing; V.V.C.d.M., methodology; D.A.B.L., methodology; C.T.S., methodology; C.M.D., methodology; R.Z.M., conceptualization, writing—review and editing; A.L.R.R., conceptualization, writing—review and editing; M.R.A., conceptualization, fundraising, supervision, writing—original version, writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by the São Paulo Research Foundation (FAPESP), Brazil (Process No. #2020/12037-0; #2022/08543–2; #2020/07826-5; #2023/01545-2; #2024/04537-3). We also thank the Coordination for the Improvement of Higher Education Personnel—Brazil (CAPES)—Finance Code 001; and CNPq (National Council for Scientific and Technological Development; Productivity Scholarship awarded to Dr. Marcos Rogério André [CNPq Process No. 303701/2021–8]).

Institutional Review Board Statement

The bats captured were approved by the Chico Mendes Institute for the Conservation of Brazilian Biodiversity (ICMBio—SISBIO), licenses 44089-1 and 47377-1. All procedures performed with bats followed protocols approved by the Ethics Committee on the Use of Animals of Fiocruz (LW81-12) and by the Ethics Committee on the Use of Animals of the Faculty of Agrarian and Veterinary Sciences of UNESP (CEUA FCAV/UNESP 3911/23).

Informed Consent Statement

Not applicable.

Data Availability Statement

The sequences generated during the present study were deposited in the NCBI Genbank “https://www.ncbi.nlm.nih.gov/genbank/ (Accessed on 21 March 2025). The sequences can be accessed through the following access numbers: PV388069; PV388070; PV388071; PV388072; PV388073; PV388074; PV388075; PV388076; PV388077; PV388078; PV388079; PV388080; PV388081; PV388082; PV388083; PV388084, PV388085 (16S rRNA sequences), and PV364143; PV364144 and PV364145 (23S rRNA sequences).

Acknowledgments

The authors would like to thank Armando Muniz Calouro and Ana Maria Jansen for providing the bat samples for this study and the São Paulo Research Foundation (FAPESP) for funding this research.

Conflicts of Interest

The authors declare that there are no conflicts of interest.

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Figure 1. Representative image of Brazil (highlighted in green in South America) and location of the municipalities of Rio Branco (in orange) and Xapuri (in blue), state of Acre (in yellow), where the bats were sampled. QGIS Geographic Information System. Open Source Geospatial Foundation Project. http://qgis.osgeo.org (Accessed on 29 March 2025).
Figure 1. Representative image of Brazil (highlighted in green in South America) and location of the municipalities of Rio Branco (in orange) and Xapuri (in blue), state of Acre (in yellow), where the bats were sampled. QGIS Geographic Information System. Open Source Geospatial Foundation Project. http://qgis.osgeo.org (Accessed on 29 March 2025).
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Figure 2. Phylogenetic tree based on a sequence alignment of the 16S rRNA gene (620 bp) of Mycoplasma spp., using the Maximum Likelihood (ML) method and GTR + F + I + G4 as an evolutionary model. The sequences detected in the present study are highlighted in green. Mycoplasma pneumoniae (AB680604) and Bacillus subtilis (AB042061) were used as outgroups.
Figure 2. Phylogenetic tree based on a sequence alignment of the 16S rRNA gene (620 bp) of Mycoplasma spp., using the Maximum Likelihood (ML) method and GTR + F + I + G4 as an evolutionary model. The sequences detected in the present study are highlighted in green. Mycoplasma pneumoniae (AB680604) and Bacillus subtilis (AB042061) were used as outgroups.
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Figure 3. Phylogenetic tree based on a sequence alignment of the 23S rRNA gene (800 bp) of Mycoplasma spp. using the Maximum Likelihood (ML) method and GTR + F + G4 as an evolutionary model. The sequences detected in the present study are highlighted in red. Mycoplasma pneumoniae (CP008895) and Bacillus subtilis (NR103037) were used as outgroups.
Figure 3. Phylogenetic tree based on a sequence alignment of the 23S rRNA gene (800 bp) of Mycoplasma spp. using the Maximum Likelihood (ML) method and GTR + F + G4 as an evolutionary model. The sequences detected in the present study are highlighted in red. Mycoplasma pneumoniae (CP008895) and Bacillus subtilis (NR103037) were used as outgroups.
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Table 1. Number of bat species sampled in Rio Branco and Xapuri, in the state of Acre, Brazil. The table presents the number of individuals captured for each species in each locality, the total by region, and the total number of individuals sampled, including the geographic coordinates and the Trophic specialization of each species.
Table 1. Number of bat species sampled in Rio Branco and Xapuri, in the state of Acre, Brazil. The table presents the number of individuals captured for each species in each locality, the total by region, and the total number of individuals sampled, including the geographic coordinates and the Trophic specialization of each species.
Location
SpeciesRio BrancoXapuriTrophic Specialization
[49]
Anoura caudifer (Saint-Hilaire, 1818)01Nectarivore
Artibeus lituratus (Olfers, 1818)443Frugivore
Artibeus obscurus (Schinz, 1821)22Frugivore
Artibeus planirostris (Spix, 1823)4410Frugivore
Carollia brevicauda (Schinz, 1821)72Opportunistic frugivore
Carollia cf. benkeithi (Solari and Baker, 2006)01Opportunistic frugivore
Carollia perspicillata (Linnaeus, 1758)5912Frugivore
Chiroderma villosum (Peters, 1860)21Frugivore
Dermanura cinerea (Gervais, 1856)102Frugivore
Gardnerycteris crenulatum (1803)01Frugivore
Glossophaga soricina (Pallas, 1766)43Nectarivore
Hysunycteris thomasi (J.A. Allen, 1904)02Nectarivore
Lophostoma silvicolum (d’Orbigny, 1836)22Frugivore
Mesophylla macconnelli (Thomas, 1901)20Frugivore
Phyllostomus discolor (Wagner, 1843)14Frugivore
Phyllostomus elongatus (É. Geoffroy, 1810)59Frugivore
Phyllostomus hastatus (Pallas, 1767)06Frugivore
Plathyrrhinus incarum (Thomas, 1912)12Frugivore
Plathyrrhinus infuscus (Peters, 1880) 20Frugivore
Rhinophylla fischerae (Carter, 1966)04Insectivorous and Frugivore
Rhinophylla pumilio (Peters, 1865)40Insectivorous and Frugivore
Saccopteryx leptura (Schreber, 1774)10Insectivore
Sturnira giannae (Velazco and Patterson, 2019)02Frugivore
Sturnira tildae (de la Torre, 1959)02Frugivore
Tonatia maresi (Williams, Willig and Reid, 1995)01Frugivore
Trachops cirrhosus (Spix, 1823)10Carnivorous
Uroderma bilobatum (Peters, 1866)58Frugivore
Vampyressa thyoune (Thomas, 1909)01Frugivore
Spectre of Vampires (Linnaeus, 1758)01Carnivorous
Total/Region19682
Total number of individuals278
Table 2. Positive samples in molecular assays for hemoplasmas according to bat species and location in the state of Acre, Northern Brazil.
Table 2. Positive samples in molecular assays for hemoplasmas according to bat species and location in the state of Acre, Northern Brazil.
LocalityBat SpeciesNumber of cPCR Positive Samples for Hemoplasma Screening (16S rRNA)/TotalNumber of cPCR-Positive Samples for Hemoplasma Characterization (23S rRNA)/Total
Xapuri
10°10′95″ S/68°30′16″ W
(n = 40)		Anoura caudifer1/10/1
Artibeus lituratus1/30/1
Artbeus planirostis2/100/2
Carollia beikeith c.f.1/10/1
Carollia perspicillata12/122/12
Dermanura cinereus2/20/2
Glossophaga soricina3/31/3
Lophostoma silviculum2/21/2
Phyllostomus discolor4/40/4
Phyllostomus elongatus2/91/2
Phyllostomus hastatus6/60/6
Rhinophylla fischerae3/40/3
Stumira tildae1/20/1
Rio Branco
09°58′29″ S/
67°48′36″ W
(n = 44)		Artibeus lituratus8/442/8
Artibeus planirostis8/442/8
Carollia brevicauda3/70/3
Carollia perspicillata19/594/19
Phyllostomus elongatus5/51/5
Platyrhinus infuscus1/21/1
Total 8415
Table 3. Sequence identity of the positive samples of 16S rRNA and 23S rRNA most closely related to hemotropic Mycoplasma spp.
Table 3. Sequence identity of the positive samples of 16S rRNA and 23S rRNA most closely related to hemotropic Mycoplasma spp.
GenBank Access NumberHost/LocationTarget Gene
(Sequence Size)		Consultation CoverageValue andIdentityHost/Country
PV388069Carollia perspicillata/
Xapuri		16S rRNA
(563 bp)		100%0.0100% Mycoplasma sp. strain A100 (MH245134)Carollia sowelli/
Belize
PV388070Phyllostomus elongatus/
Xapuri		16S rRNA
(566 bp)		100%0.099.12% Mycoplasma sp. strain S61 (MZ048305)Phyllostomus discolor/
Campo Grande, Brazil
PV388071Phyllostomus discolor/
Xapuri		16S rRNA
(558 bp)		100%0.099% Mycoplasma sp. strain B72 (MZ048296)Phyllostomus discolor/
Campo Grande, Brazil
PV388072Phyllostomus hastatus/
Xapuri		16S rRNA
(561 bp)		100%0.098.22% Mycoplasma sp. strain S61 (MZ048305)Phyllostomus discolor/
Campo Grande, Brazil
PV388073Phyllostomus hastatus/
Xapuri		16S rRNA
(561 bp)		100%0.098.22% Mycoplasma sp. strain S71 (MZ048307)Phyllostomus discolor/
Campo Grande, Brazil
PV388074Lophostoma silviculum/
Xapuri		16S rRNA
(561 bp)		100%0.098% Mycoplasma sp. strain A35 (MH245145)Trachops cirrhosus/
Belize
PV388075Rhinophylla fischerae/
Xapuri		16S rRNA
(563 bp)		100%0.098.40% Mycoplasma sp. strain D159 (KY932722)Desmodus rotundus/
Belize
PV388076Rhinophylla fischerae/
Xapuri		16S rRNA
(564 bp)		100%0.098.40% Mycoplasma sp. estirpe D159 (KY932722)Desmodus rotundus/
Belize
PV388077Stumira tildae/
Xapuri		16S rRNA
(561 bp)		100%0.099.29% Mycoplasma sp. strain KAS 745 (MH24513)Sturnira parvidens/
Belize
PV388078Phyllostomus elongatus/
Xapuri		16S rRNA
(563 bp)		100%0.099.11% Mycoplasma sp. Strain S61(MZ048305)Phyllostomus discolor/
Campo Grande, Brazil
PV388079Phyllostomus elongatus/
Xapuri		16S rRNA
(567 bp)		100%0.098.59% Mycoplasma sp. strain S61 (MZ048305)Phyllostomus discolor/
Campo Grande, Brazil
PV388080Phyllostomus hastatus/
Xapuri		16S rRNA
(461 bp)		100%0.098.70% Mycoplasma sp. strain S61 (MZ048305)Phyllostomus discolor/
Campo Grande, Brazil
PV388081Artibeus lituratus/
Rio Branco		16S rRNA
(558 bp)		100%0.0100% Mycoplasma sp. strain A12_b (MH245185)Artibeus phaeotis/
Belize
PV388082Platyrhinus infuscus/
Rio Branco		16S rRNA
(561 bp)		100%0.099.47% Mycoplasma sp. strain A100 (MH245134)Carollia sowelli/
Belize
PV388083Dermanura cinereus/
Xapuri		16S rRNA
(562 bp)		100%0.097.86% Mycoplasma sp. strain A100 (MH245134)Carollia sowelli/
Belize
PV388084Carollia beikeith c.f./
Xapuri		16S rRNA
(561 bp)		100%0.099.82% Mycoplasma sp. strain A100 (MH245134)Carollia sowelli/
Belize
PV388085Anoura caudifer/
Xapuri		16S rRNA
(562 bp)		100%0.097.70% Mycoplasma sp. strain S61 (MZ048305)Phyllostomus discolor/
Brazil
PV364143Carollia perspicillata/
Xapuri		23S rRNA
(835 bp)		100%0.088.32% ‘Candidatus Mycoplasma haematomolossi’ (OQ518944)Molossus rufus/
Belize
PV364144Carollia perspicillata/
Xapuri		23S rRNA
(801 bp)		97%0.094.03% Mycoplasma sp. strain 144 23S (OR753267,1)Desmodus rotundus/
Brazil
PV364145Carollia perspicillata/
Xapuri		23S rRNA
(666 bp)		99%0.091.82% Mycoplasma sp. strain 144 23S (OR753267,1)Desmodus rotundus/
Brazil
Table 4. Results of genetic diversity analysis. N = Number of sequences analyzed; VS = number of variable sites; GC% = G + C content; g = number of genotypes; gd = genotypic diversity; SD = standard deviation; π = nucleotide diversity (by sites); K = nucleotide difference number.
Table 4. Results of genetic diversity analysis. N = Number of sequences analyzed; VS = number of variable sites; GC% = G + C content; g = number of genotypes; gd = genotypic diversity; SD = standard deviation; π = nucleotide diversity (by sites); K = nucleotide difference number.
GenebpNVSGC%ggd (Mean ± SD)π (Mean ± SD)K
16S rRNA541165146.5120.9667 ± 0.0310.05057 ± 0.0053022.80
23S rRNA66631047.431 ± 0.7400.01003 ± 0.003136.666
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MDPI and ACS Style

Silva, A.I.d.; Franco, E.O.; Calchi, A.C.; Santos, F.C.B.d.; Verde, R.d.S.; de Mello, V.V.C.; Lee, D.A.B.; Secato, C.T.; Dias, C.M.; Machado, R.Z.; et al. Molecular Survey of Hemopathogens in Bats from the Western Brazilian Amazon. Pathogens 2025, 14, 527. https://doi.org/10.3390/pathogens14060527

AMA Style

Silva AId, Franco EO, Calchi AC, Santos FCBd, Verde RdS, de Mello VVC, Lee DAB, Secato CT, Dias CM, Machado RZ, et al. Molecular Survey of Hemopathogens in Bats from the Western Brazilian Amazon. Pathogens. 2025; 14(6):527. https://doi.org/10.3390/pathogens14060527

Chicago/Turabian Style

Silva, Abraão Isaque da, Eliz Oliveira Franco, Ana Cláudia Calchi, Francisco Chagas Bezerra dos Santos, Rair de Sousa Verde, Victória Valente Califre de Mello, Daniel Antônio Braga Lee, Caroline Tostes Secato, Clara Morato Dias, Rosangela Zacarias Machado, and et al. 2025. "Molecular Survey of Hemopathogens in Bats from the Western Brazilian Amazon" Pathogens 14, no. 6: 527. https://doi.org/10.3390/pathogens14060527

APA Style

Silva, A. I. d., Franco, E. O., Calchi, A. C., Santos, F. C. B. d., Verde, R. d. S., de Mello, V. V. C., Lee, D. A. B., Secato, C. T., Dias, C. M., Machado, R. Z., Roque, A. L. R., & André, M. R. (2025). Molecular Survey of Hemopathogens in Bats from the Western Brazilian Amazon. Pathogens, 14(6), 527. https://doi.org/10.3390/pathogens14060527

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