Tick-borne protozoans of the genus Hepatozoon are obligate hemoparasites that can infect domestic and wild terrestrial vertebrates. Main hepatozoonosis affects canids and involves mainly Hepatozoon canis and Hepatozoon americanum. However, molecular studies revealed the capacity of H. canis to infect a wide range of wild mammals. In July 2018, we conducted an epidemiological survey for tick-borne pathogens in wild hosts, assaying Hepatozoon sp. occurrence in 34 bats captured in different habitats within a conservation unit in the state of Espírito Santo, southeastern Brazil. Blood and spleen tissue DNA samples were submitted to PCR amplifications of Babesia/Theileria and Hepatozoon 18S rRNA gene and 21% (7/34) were positive for Hepatozoon sp. Phylogenetic inferences grouped the obtained sequences from Seba's short-tailed bat (Carollia perspicillata) with the H. canis cluster, and from the great fruit-eating bat (Artibeus lituratus) with rodent-associated Hepatozoon cluster. Further studies are needed to characterize the epidemiological role of Seba's short-tailed bat and the great fruit-eating bat in the wild transmission cycle of these hemoparasites in Brazil.

The genus Hepatozoon comprises obligate hemoparasites that can infect domestic and wild terrestrial vertebrates. The Hepatozoon life cycle has a definitive hematophagous invertebrate host, and an intermediate vertebrate host (Serra-Freire 1979; Alencar et al. 1997; Dantas-Torres 2008), which becomes infected through a trophic cycle involving the ingestion of infected invertebrates or vertebrate prey, which can lead to hepatozoonosis (Smith 1996; Almeida et al. 2013; Maia et al. 2014).

Hepatozoonosis affects canids and typically involves Hepatozoon canis and Hepatozoon americanum, whose transmission occurs by ingestion of infected ticks (Rubini et al. 2005; Demoner et al. 2016). However, molecular studies revealed the potential of H. canis to infect a wide range of wild mammals such as the white-eared opossum (Didelphis albiventris), hoary fox (Pseudalopex vetutus), and bush dog (Speothos venaticus; Silva et al. 2017). The first evidence of Hepatozoon infection in Chiroptera was during a molecular survey of Trypanosoma sp. in mammals' tissues, which resulted in detection of Hepatozoon sp. in fawn leaf-nosed bat (Hipposideros cervinus) from Malaysia, without sequencing (Pinto et al. 2013). In Brazil, although molecular studies have shown the occurrence of Hepatozoon species in wild canines and felids, rodents, coatis, marsupials, crocodiles and amphibians (André et al. 2010; Azevedo et al. 2018; Perles et al. 2019), Hepatozoon-like organisms were detected in Chiroptera (by morphological analysis) in little yellow-shouldered bat (Sturnira lilium) from São Paulo state (Torres et al. 1983).

Between 28 July and 10 August 2018, an epidemiological survey was conducted for tick-borne pathogens inhabiting wild hosts and, in this study, we evaluate the occurrence of Hepatozoon in bats captured in different habitats types from a biological reserve of Espirito Santo state, southeastern Brazil. During an epidemiological survey of Babesia occurrence, field expeditions were conducted to collect ectoparasites, blood, and spleen tissues from bats, in the Biological Reserve of Duas Bocas, municipality of Cariacica, Espírito Santo State, Southeast Brazil (Fig. 1). The area is comprised of protected Atlantic Rainforest biome, with lightly anthropized fragments.

Figure 1

Map of Brazil highlighting Espírito Santo State at Brazil's Southeast Region, with the municipality of Cariacica outlined, emphasizing the investigated sites of bat sampling in Duas Bocas Biological Reserve during the babesiosis epidemiologic research in July 2018. The numbers indicate the collection sites within the Reserve: accommodation for researchers (1 and 2), anthropized area trail (3), preserved forest trail (4), and cave with pipes (5). The bat species captured are indicated as: square=Artibeus lituratus; pentagon=Carollia perspicillata; triangle=Desmodus rotundus; star=Molossus rufus; circle=Myotis lucifugus.

Figure 1

Map of Brazil highlighting Espírito Santo State at Brazil's Southeast Region, with the municipality of Cariacica outlined, emphasizing the investigated sites of bat sampling in Duas Bocas Biological Reserve during the babesiosis epidemiologic research in July 2018. The numbers indicate the collection sites within the Reserve: accommodation for researchers (1 and 2), anthropized area trail (3), preserved forest trail (4), and cave with pipes (5). The bat species captured are indicated as: square=Artibeus lituratus; pentagon=Carollia perspicillata; triangle=Desmodus rotundus; star=Molossus rufus; circle=Myotis lucifugus.

Close modal

Bats were captured and euthanized for this study by the authorization of State Institute for the Environment and Water Resources of Espírito Santo (research license GRN no. 026a-2017, process no. 755612/1/16—Atuali-zação) and by Chico Mendes Institute for Biodiversity Conservation of Brazilian Ministry of Environment (authorization for scientific purpose activity no. 63023-1). Bats were captured for 7 consecutive d, between 0800 hours and 1600 hours with nets at sleeping sites, in artificial tunnels and natural caves, and at night, with mist nets. The nets were set up before dusk, around 1700 hours, and were checked every 20 min until 2300 hours, when they were closed. Captured bats were sedated with an intraperitoneal injection of 10% ketamine (200 mg/kg) and 2% xylazine (20 mg/kg) solution at a ratio of 2:1, and blood was collected by cardiac puncture and stored at –20 C until DNA purification. Taxonomic identifications were based on analyses of skull and dental characters and on morphometric measurements (Moratelli et al. 2011; Reis et al. 2017; Loureiro et al. 2018). Subsequently, specimens were euthanized with 2.5% sodium thiopental (120 mg/kg) and spleen samples were collected and fixed in 99% pure isopropyl alcohol and submitted to DNA purification.

We isolated DNA from chiropteran blood and spleen samples using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) and used as template for semi-nested PCR amplifications of Babesia/Theileria and Hepatozoon partial nuclear 18S rRNA gene (Criado-Fornelio et al. 2003b). The PCR products of the expected size (approximately 410 base pairs; Criado-Fornelio et al. 2003a) were purified using the Wizard® SV Gel and PCR Clean-Up System (Promega, Madison, Wisconsin, USA) and sequenced with the BigDye Terminator–Cycle Sequencing Ready Reaction kit (Applied Biosystems, Carlsbad, California, USA) in an automatic sequencer (Applied Biosystems 3730xl DNA Analyzer). Sequences were edited using Seq-Man software (DNASTAR Lasergene, Madison, Wisconsin, USA), and identity values were obtained using BLAST (National Center for Biotechnology Information 2018). Sequence alignment was performed using Clustal Omega (Madeira et al. 2019) and phylogenies were assessed applying the maximum-likelihood method, with T92+G correction model selected by MEGA version 7 software (Kumar et al. 2016), which was also used to produced phylogenetic trees from 1,000 bootstrap replicates for node support estimation.

We collected 34 Chiroptera specimens identified as the great fruit-eating bat (Artibeus lituratus), Seba's short-tailed bat (Carollia perspicillata), vampire bat (Desmodus rotundus), black mastiff bat (Molossus rufus), and black myotis (Myotis nigricans; Table 1). Of all examined bats, only one black mastiff bat was found parasitized by a female of Mesostigmata mite; however, the specimen was damaged and further identification could not be performed.

Table 1

Detection of partial nuclear 18S rRNA gene of Hepatozoon sp. in bats from Biological Reserve of Duas Bocas, Espírito Santo State, Southeast Brazil, between 28 July and 10 August 2018.

Detection of partial nuclear 18S rRNA gene of Hepatozoon sp. in bats from Biological Reserve of Duas Bocas, Espírito Santo State, Southeast Brazil, between 28 July and 10 August 2018.
Detection of partial nuclear 18S rRNA gene of Hepatozoon sp. in bats from Biological Reserve of Duas Bocas, Espírito Santo State, Southeast Brazil, between 28 July and 10 August 2018.

Hepatozoon DNA was detected in one Seba's short-tailed bat blood sample and four spleen tissue samples; it was also detected in two great fruit-eating bat spleen tissue samples, totaling seven Hepatozoon-positive samples (Table 1). The BLAST analysis of Seba's short-tailed bat sequences that we obtained (MN369545, MN369546, and MN369547) showed 99% identity (410/416, 402/408, and 403/409 base pairs, respectively) with H. canis (KU893123). In addition, the great fruit-eating bat-derived sequences (MN369548 and MN369549) showed 99% identity (407/411 and 406/411, respectively) with Hepatozoon sp. strain BV2 (AY600625) and BV1 (AY600626), originally isolated from a cricetid rodent in Spain. Indeed, our phylogenetic reconstructions supported grouping our Hepatozoon 18S rRNA sequences from Seba's short-tailed bat with the H. canis cluster from canids, and our Hepatozoon sequences from great fruit-eating bat with the Hepatozoon sp. BV2/BV1 cluster, from rodents (Fig. 2).

Figure 2

Phylogenetic inferences by maximum-likelihood method from 1,000 replicated trees based on nucleotide sequences of Hepatozoon 18S rRNA gene isolated of bats from Biological Reserve of Duas Bocas, Espírito Santo State, Southeast Brazil, between 28 July and 10 August 2018. Evolutionary distances were estimated by T92+G model. Bootstrap values greater than 70% are shown. Sequences obtained are highlighted with black triangle and GenBank accession numbers precede the sequence names. Scale bar indicates nucleotide substitutions per site.

Figure 2

Phylogenetic inferences by maximum-likelihood method from 1,000 replicated trees based on nucleotide sequences of Hepatozoon 18S rRNA gene isolated of bats from Biological Reserve of Duas Bocas, Espírito Santo State, Southeast Brazil, between 28 July and 10 August 2018. Evolutionary distances were estimated by T92+G model. Bootstrap values greater than 70% are shown. Sequences obtained are highlighted with black triangle and GenBank accession numbers precede the sequence names. Scale bar indicates nucleotide substitutions per site.

Close modal

Immunologically, spleen is an important organ for vector-borne pathogen control, because it is the organ where several stages of H. canis are found and, together with the bone marrow, it is one of the most frequently parasitized organs, harboring chronic infections (Levine 1973; Levi et al. 2018). Here, we recorded a higher number of positive samples (six of seven positive samples) of Hepatozoon strains from spleen (Table 1) than blood, which was a better tissue for detection than blood in this study, as has been observed for other mammalian species (Hodžić et al. 2018; Levi et al. 2018). It was not possible to include the Hepatozoon sequences of Chiroptera from Malaysia (Pinto et al. 2013) in our alignments (Fig. 2) because these sequences represent a different portion of 18S rRNA gene fragment than the sequences obtained in this study, impeding identification and comparison of the Chiroptera isolates.

In our study, nearly half of the great fruit-eating bat and Seba's short-tailed bat captured were infected individuals (Table 1). This intrinsic relationship of Hepatozoon strains detected in the two infected Chiroptera species might be related to the enzootic cycle of each group, because spleen can harbor chronic infections (O'Dwyer 2011; Movilla et al. 2017; Levi et al. 2018), and due to the feeding habits and behavior of bats in the investigated area. The great fruit-eating bat is distributed throughout the Neotropical region, from Mexico to northern Argentina, including all regions of Brazil. The species is abundant in conserved areas as well as in altered and urban environments and, although primarily frugivores, can also feed on insects. Similarly, Seba's short-tailed bat is known to occur widely in the Neotropics, including all regions of Brazil, predominantly in altered and urban environments, feeding mainly on pepper plants (Piperaceae), but also able to feed on nectar and insects (Reis et al. 2007).

Although few studies investigating the tick-borne protozoans infection have reported the presence of Hepatozoon in bats (Torres et al. 1983; Pinto et al. 2013), our results indicated that great fruit-eating bat and Seba's short-tailed bat are susceptible to infection by organisms genetically related to Hepatozoon sp. strain BV2 and H. canis, respectively, and probably, the hygienic practices of these mammals could increase Hepatozoon infection mechanisms via ingestion of infected hematophagus ectoparasites (Pinto et al. 2013).

It is not possible to establish a relation with vectors, paratenic, and reservoir hosts for Hepatozoon in this region, because these remain unknown. Further studies are needed to clarify whether Seba's short-tailed bat and great fruit-eating bat have any eco-epidemiologic importance in the cycle of these hemoparasites in Brazil.

E.C.F.S. is a graduate fellow supported by supported by the Brazilian Federal Agency for Support and Evaluation of Graduate Education within the Ministry of Education of Brazil, and this work is part of her PhD thesis at Oswaldo Cruz Foundation, Brazil. The authors thank the Secretary of State for Health of Espírito Santo (Brazilian Ministry of Health) and the Laboratory of Biology and Parasitology of Reservoir Wild Mammals (Oswaldo Cruz Foundation, Brazil) for assistance on bat captures and sampling. We thank Daniela Dias (Wild Reservoir Taxonomy and Diagnosis Reference Service of Oswaldo Cruz Foundation, Brazil) for bat taxonomic identifications; to Genomic Platform DNA Sequencing (PDTIS/Oswaldo Cruz Foundation, Brazil) for sequencing support; and to Adrian Paul Ashton Barnett for the English review and comments on the manuscript. Financial support for the epidemiological survey for tick-borne pathogens investigation of wild hosts came from a project funded the Ministry of Health of Brazil.

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Author notes

5These authors contributed equally to this work