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Abstract

To a better insight into the epidemiology and genetic diversity of protozoan hemoparasites infections in wild mammals, this study aimed to the post mortem detection of DNA from species of the order Piroplasmida (Babesia sp., Cytauxzoon sp., and Theileria sp.) and suborder Adelorina (Hepatozoon sp.) using polymerase chain reaction based on the 18S rRNA gene followed by genetic sequencing of blood and spleen samples collected from carcasses of 164 free-ranging and captive wild mammals from Mato Grosso state. Among them, one Leopardus pardalis, three Panthera onca, two Puma concolor were positive for Cytauxzoon sp., and six Tapirus terrestris tested positive for Piroplasmida, while one L. pardalis was positive for Hepatozoon sp. Furthermore, an uncharacterized piroplasmid genetically related to Theileria sp. previously detected in cats from Brazil was described in lowland tapirs. Despite the controversy regarding the epidemiological threat of these protozoa, the detection of these tick-borne agents in wild free-living and captive mammals, even when asymptomatic, demonstrates the importance of monitoring, particularly in hotspots such as the state of Mato Grosso, to verify the circulation and genetic diversity, to anticipate the possible emergence of diseases, and even their consequences to other animals as well as humans.

Keywords:
Cytauxzoon sp.; Hepatozoon sp.; Theileria sp.; PCR

Resumo

Para uma melhor compreensão da epidemiologia e diversidade genética das infecções por hemoprotozoários em mamíferos selvagens, este estudo teve como objetivo a detecção post mortem de DNA de espécies da ordem Piroplasmida (Babesia sp., Cytauxzoon sp. e Theileria sp.) e subordem Adelorina (Hepatozoon sp.), utilizando-se a reação em cadeia pela polimerase, baseada no gene 18S rRNA, seguido de sequenciamento genético de amostras de sangue e baço, coletadas de 164 carcaças de mamíferos selvagens de vida livre e cativos do estado de Mato Grosso. Entre eles, um Leopardus pardalis, três Panthera onca, dois Puma concolor foram positivos para Cytauxzoon sp., e seis Tapirus terrestris testaram positivos para Piroplasmida, enquanto um L. pardalis foi positivo para Hepatozoon sp. Além disso, foi descrito em antas, um piroplasmídeo não caracterizado geneticamente, relacionado à Theileria sp., previamente detectado em gatos do Brasil. Apesar da controvérsia quanto à ameaça epidemiológica desses protozoários, a detecção desses agentes em mamíferos silvestres e cativos, mesmo quando assintomáticos, demonstra a importância do monitoramento, principalmente em hotspots, como no estado de Mato Grosso, para verificar a circulação e a diversidade genética, a fim de antecipar o possível surgimento de doenças e, até mesmo, suas consequências para outros animais, bem como os humanos.

Palavras-chave:
Cytauxzoon sp.; Hepatozoon sp.; Theileria sp.; PCR

Living beings interact, and these interactions are the result of natural selection and the activities of evolution. Among these relationships, parasitism is one of the most successful, and one of the main factors responsible for modulating communities (Timi & Poulin, 2020) and reducing host fitness in various ways. Significant environmental changes and consequent increases in the interaction between domestic and wild animals (André et al., 2015) can increase the emergence of new diseases in new hosts. Molecular studies of hemoparasite genera belonging to the Apicomplexa have demonstrated the wide distribution and variety of hosts and vectors (Wang et al., 2017; van As et al., 2020), however, much remains to be elucidated.

Considering the expanse of Brazil and the great biodiversity of mammals in the state of Mato Grosso, there are currently few studies on apicomplexan protozoans and their relationship with large wild mammals in this region (André et al., 2010; Furtado et al., 2017b). Therefore, polymerase chain reaction (PCR)-based methods were used to investigate the diversity and occurrence of the apicomplexan parasites Babesia, Cytauxzoon, Hepatozoon, and Theileria in blood and spleen samples collected from carcasses of wild mammals in the state of Mato Grosso, Brazil.

From December 2019 to July 2021, tissue samples (blood and spleen) were collected from carcasses of road-killed free-roaming and captive wild animals from six municipalities in the State of Mato Grosso, Brazil, attended at Veterinary Hospital and sent for routine necropsy at the Veterinary Pathology sector of the Federal University of Mato Grosso, located in the Cuiabá municipality, as depicted in Table 1 and shown in Figure 1.

Thumbnail Table 1
Molecular detection by polymerase chain reaction (PCR) of Piroplasmida (genera Babesia, Cytauxzoon, and Theileria) and Hepatozoon spp. in blood (B) and spleen (S) samples of free living (FL) and captive (C) wild mammals from Mato Grosso state, Brazil, during 2019-2021.
Figure 1
Map of the municipalities within the State of Mato Grosso, Brazil, where blood and tissue samples (spleen) of wild mammals were collected for molecular detection by polymerase chain reaction (PCR) of Piroplasmida (genera Babesia, Cytauxzoon, and Theileria) and Hepatozoon spp., during 2019–2021. 1. Barão de Melgaço; 2. Cáceres; 3. Chapada dos Guimarães; 4. Cuiabá; 5. Nova Xavantina; 6. Poconé.

Procedures in this study were previously approved by the Ethics Committee on Animal Research of the Federal University of Mato Grosso (CEUA protocol no. 23108.015878/2019-65) and “Instituto Chico Mendes de Conservação da Biodiversidade” (ICMBio permit no. 55104-1).

DNA extraction from blood and tissue samples (spleen) was performed using the DNA extraction PureLink™ Genomic DNA Mini Kit (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. To verify the success of extraction, an initial PCR targeting a fragment of the mammalian glyceraldehyde-3-phosphate dehydrogenase (gapdh) gene was performed as previously described (Birkenheuer et al., 2003). Extracted DNA samples were then subjected to further PCR assays to amplify fragments of the small ribosomal DNA subunit 18S of Piroplasmida members (Babesia sp., Cytauxzoon sp., and Theileria sp.) and Hepatozoon spp. Negative controls (nuclease-free water) and appropriate positive controls for each PCR assay were included, as follows: Babesia caballi (GenBank accession number MG906574) and Hepatozoon canis (GenBank accession number MG496257) from blood of naturally infected horse and dog, respectively. DNA samples were subjected to nested PCR (nPCR) to amplify a fragment (~800 base pairs - bp) gene for Piroplasmida (Babesia sp., Cytauxzoon sp., and Theileria sp.), as previously described (Jefferies et al., 2007). Furthermore, biological samples were screened using a previously described conventional PCR (cPCR) protocol (Ujvari et al., 2004) by targeting a fragment (~600 bp) of the 18S rDNA region of the Hepatozoon spp. The PCR products were resolved on 1.5% agarose gels stained with GelRed™ Nucleic Acid Gel Stain (Biotium Inc, Fremont, CA, USA) and visualized using a ChemiDoc XRS system (Bio-Rad, Hercules, CA, USA). Amplicons of the expected sizes were purified using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Life Sciences, Pittsburgh, PA, USA) and prepared for sequencing according to the instructions provided in the BigDye™ kit (Applied Biosystems, Foster, CA, USA). An ABI PRISM 3500 Genetic Analyzer (ABI DNA Model 3500 Series Genetic Analyzer, Applied Biosystems, Inc., Foster City, CA, USA) was employed to conduct the sequencing procedures using the same primers used for the PCR. The obtained sequences were then subjected to BLAST analyses to determine the closest identities by comparison to organisms available in GenBank.

Sequences of the 18S rRNA gene generated in this study and homologue sequences retrieved from GenBank were used to construct alignments for Theileria spp. representatives. The selected sequences were aligned using Clustal X (Thompson et al., 1997), and manually adjusted with GeneDoc (Nicholas et al., 1997). Two phylogenetic inferences were performed for alignment. Inferences by maximum parsimony were constructed according to their implementation in PAUP version 4.0b10 (Swofford, 2002), using a heuristic search with 1000 replicates, 500 bootstrap replicates, random stepwise addition starting trees (with random addition sequences), and tree bisection and reconnection (TBR) branch swapping. MrBayes v3.1.2 was used to perform Bayesian analyses (Huelsenbeck & Ronquist, 2001) with four independent Markov chain runs for 1,000,000 metropolis-coupled MCMC generations, sampling a tree every 100th generations. The first 25% of trees represented burn-in, and the remaining trees were used to calculate the Bayesian posterior probability. GTR+I+G was the standard model used in MrBayes software. The tree was rooted in Toxoplasma gondii as an out-group. GenBank accession numbers for all sequences used for the phylogenetic analyses were embedded in each tree.

A total of 159 blood and 160 spleen samples from 164 specimens of wild mammals belonging to at least 31 different species were subjected to DNA extraction. DNA from all samples tested for gapdh internal control amplified the predicted product. Table 1 provides a list of all tested animals and the results of their molecular analyses grouped according to species, origin, and locality.

Amplicons for Piroplasmida were detected in four species of wild mammals (Leopardus pardalis, Panthera onca, Puma concolor, and Tapirus terrestris). Among the animals that exhibited positive results for this protozoa, one L. pardalis showed a positive spleen sample, and three P. onca and two P. concolor individuals had positive results in the spleen and blood, respectively. Among the six specimens of T. terrestris, Piroplasmida DNA was present in the blood and spleen samples from one animal, two specimens were positive for Piroplasmida in the blood, and three specimens had positive results in the spleen, as determined by the nPCR assay.

Partial sequences of the 18S rRNA gene from L. pardalis, P. onca, and P. concolor were identical to each other and 100% (721/721 bp) identical with sequences of Cytauxzoon felis (MT904037, AF399930, and AY679105). Since the present study relied on a small fragment of the 18S rRNA gene, herein we referred to as Cytauxzoon sp. isolate MT (MZ489665). Furthermore, partial sequences of the 18S rRNA gene obtained from six specimens of T. terrestris yielded two different haplotypes with 99% (743/747 bp) similarity with sequences of the Theileria genera (KP410271, KP410272, and KP410273) detected in free-roaming domestic cats in Midwestern Brazil. The GenBank accession numbers for the partial sequences generated for the Theileria isolates in the present study, herein designated Theileria sp. isolate tapir MT1 and Theileria sp. isolate tapir MT2 are MZ490586 and MZ491096, respectively. The phylogenetic analyses of a partial 18S rDNA obtained from wild felids (L. pardalis, P. onca, and P. concolor) show Cytauxzoon sp. isolate MT clustered in a clade with other C. felis sequences. Furthermore, the phylogenetic analyses of partial 18S rDNA sequences obtained from T. terrestris indicate that Theileria sp. isolate tapir MT1 and Theileria sp. isolate tapir MT2 formed a clade with other Theileria spp. recently detected in domesticated and stray cats in the states of Mato Grosso Sul and São Paulo, Brazil (Figure 2), and genetically related to Theileria equi (6.38% of divergence).

Figure 2
Maximum parsimony and Bayesian tree constructed for an alignment of sequences of Cytauxzoon spp. and Theileria spp. species using 18S rRNA gene sequences. Numbers at nodes are the support values for the major branches (bootstrap over 500 replicates). The sequence obtained in this study is highlighted in bold. Numbers in brackets correspond to GenBank accession numbers.

Among the mammal samples molecularly tested for Hepatozoon spp., only one L. pardalis yielded amplicons in blood samples after the 18S rRNA-based cPCR that revealed 100% (554/554 pb) identity with Hepatozoon felis (AB771570, AB771562). However, considering the small size of the fragment sequenced from 18S rRNA gene, as described above, herein we referred to as Hepatozoon sp. isolate ocelot MT (MZ490540). Co-infection with Hepatozoon and piroplasmid agents was not detected among the samples tested.

The present study demonstrated the presence of apicomplexan parasites in blood and/or spleen samples from wild mammals from the state of Mato Grosso, Midwestern Brazil. Infections found in L. pardalis, P. onca, and P. concolor by Cytauxzoon sp. corroborate previous studies that demonstrated a high occurrence of this agent in wild felids in Brazil (de Sousa et al., 2018; Furtado et al., 2017b; Santos et al., 2021).

In North America, bobcat (Lynx rufus) is the most common natural host for C. felis, with both Amblyomma americanum and Dermacentor variabilis ticks as suitable vectors (Wang et al., 2017). Despite the pathogenicity of the genotypes that circulate in domestic cats and wild felids in the country is still unknown (André et al., 2015), one case of death of cytauxzoonosis has been described in Brazil in lions from Rio de Janeiro State (Peixoto et al., 2007). In Brazil, high infection rates in P. onca incriminate this felid as a possible reservoir host for Cytauxzoon (Furtado et al., 2017b), although this assumption should be verified, along with the role of other neotropical felid species serving as natural reservoirs (André et al., 2009), as well as possible clinical signs of infection in wild Brazilian felines. Finally, little is known about the natural vectors of Brazilian isolates of Cytauxzoon sp.. It is possible that ticks of the genus Amblyomma are responsible for maintaining and transmitting this pathogen (Furtado et al., 2017b).

Tapirus terrestris (lowland tapir) is a wide-ranging herbivore ungulate of the order Perissodactyla, that is found in all biomes in Brazil and is highly susceptible to anthropogenic threats, including cohabitation and increased exposure to domestic and wild animal pathogens (Medici, 2011). In this study, a natural infection is described with Theileria genus positioned by phylogenetic analysis with Theileria sp. detected in cats (André et al., 2014, 2015). Piroplasms have been described infecting domestic cats worldwide, being a major veterinary concern mainly in South Africa, with animals presenting severe clinical abnormalities (Penzhorn & Oosthuizen, 2020). In Brazil, molecular occurrences (11.9-19%) of Babesia/Theileria spp., genetically related to Theileria molecularly detected in the present study, were described in clinically health domesticated and stray cats (André et al., 2014, 2015), suggesting that cats are able to maintain the infection with no discernible untoward effects (Penzhorn & Oosthuizen, 2020).

Regardless the natural infections with Theileria (molecularly identified as Theileria equi) have previously been reported in lowland tapirs (Gonçalves et al., 2020), neither clinical manifestation nor the impact were described of these infections in tapirids with this protozoan. Furthermore, there was no evidence of lowland tapirs as possible natural reservoirs or even potential vectors for Theileria sp., since these mammals have the greatest richness of tick species in South America (Labruna et al., 2021).

The genus Hepatozoon can infect a wide range of domestic and wild animals, including avians, mammals, and reptiles worldwide, and despite its frequent presence in wild and domestic felids (van As et al., 2020), few studies on Hepatozoon infection have been conducted with neotropical felines (Metzger et al., 2008; André et al., 2010; Furtado et al., 2017a; de Sousa et al., 2018). At least four species of Hepatozoon are capable of infecting domestic and wild felines worldwide, as follows: H. felis was described in Spain (Felis catus), India (Panthera tigris tigris, Panthera leo persica, and Panthera pardus fusca), Brazil (Felis catus), and Thailand (Panthera leo persica); Hepatozoon silvestris was detected in Felis silvestris silvestris from Bosnia and Herzegovin; Hepatozoon ingwe and Hepatozoon luiperdjie were detected in Panthera pardus pardus in South Africa (van As et al., 2020). A putative new species of Hepatozoon sp. have been detected in wild felids from Brazil (André et al., 2010). Despite several tick genera, including Amblyomma, Dermacentor, Haemaphysalis, Ixodes, and Rhipicephalus have been reported to harbor H. felis, the vectors of this protozoan species remain unknown (Bhusri et al., 2017). The infection with H. felis has previously been described in L. pardalis from Brazil (Metzger et al., 2008; Santos et al., 2021). Although Hepatozoon infections are often subclinical in wild felids (Metzger et al., 2008; van As et al., 2020), studies have demonstrated that it can be fatal in domestic cats, particularly immunocompromised animals or those with concomitant infections (Wang et al., 2017). Considering that some Brazilian felids are threatened, especially keystone species such as P. onca, studies are recommended, especially based on postmortem investigations, due to the inherent difficulties to obtain samples from wild animals, to clarify whether Hepatozoon or other pathogens infections represent a risk to these animals.

The present study showed that Cytauxzoon sp. and Hepatozoon sp. circulate among wild felids, while an uncharacterized piroplasmid genetically related to T. equi was detected in lowland tapirs in the state of Mato Grosso. Despite controversy regarding the epidemiological threat of these protozoan infections, the detection in free-living and captive wild mammals demonstrates the importance of monitoring, especially in regions of conservation interest, such as the state of Mato Grosso, to verify the circulation and genetic diversity of these agents in order to anticipate the possible emergence of diseases, and even their consequences. Furthermore, although clinical disease does not always develop in wildlife, mammals can be important reservoirs by contributing to the spread of the disease to other wild and domestic animals, as well as humans.

Acknowledgements

A. Marcili, L. Nakazato, R. C. Pacheco, and V. Dutra are in receipt of productivity scholarships from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). This research was financially supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) – financial code 001.

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