Theileria terrestris nov. sp.: A Novel Theileria in Lowland Tapirs (Tapirus terrestris) from Two Different Biomes in Brazil

Abstract

The low-land tapir (Tapirus terrestris) is the largest wild terrestrial mammal found in Brazil. Although T. terrestris has been already reported as a host of hemoparasites, the occurrence and genetic identity of Piroplasmida agents in this species is still cloudy. Although it is reported that Theileria equi, an endemic equid-infective agent in Brazil, is occurring in lowland tapirs, these reports are probably misconceived diagnoses since they are solely based on small fragments of 18S rRNA that may not achieve accurate topologies on phylogenetic analyses. The present study aimed to detect and investigate the identity of Theileria spp. in tapirs from Pantanal and Cerrado biomes. Blood-DNA samples from tapirs were screened for a partial (~800 bp) 18S rRNA gene fragment from Piroplasmida and 64 (64/122; 52.46% CI: 43.66-61.11%) presented bands of expected size. Samples were submitted to different protocols for molecular characterization, including near-full length 18S rRNA gene (~1500 bp), and the ema-1 gene from T. equi. Eight sequences were obtained for extended fragments (1182-1473 bp) from the 18S rRNA gene. Moreover, three sequences from partial cox-1 and five from partial hsp70 gene were obtained. None of the samples presented amplifications for the ema-1 gene. Phylogenetic and distance analyses from the 18S rRNA sequences obtained demonstrated a clear separation from tapirs' Theileria spp. and T. equi. Phylogenetic analyses of cox-1 and hsp70 sequences obtained herein also showed a unique clade formed by tapir's Theileria spp. Theileria terrestris sp. nov. is positioned apart from all other Theileria species in 18S rRNA, cox-1, and hps70 phylogenetic analyses. This novel proposed species represents a new Piroplasmida clade, yet to be characterized regarding biological features, vectors involved in the transmission cycles, additional vertebrate hosts, and pathogenicity.

Keywords: Cerrado; Pantanal; Piroplasmida; tapirs; theileriosis; wildlife.

Figure 1

Number of tapirs from each biome that presented blood DNA samples with bands of expected sizes on agarose gel electrophoresis when tested for partial Piroplasmida 18S rRNA gene by nested PCR analysis [13] in Mato Grosso do Sul State. This map was constructed using QGis v. 3.26 software (http://qgis.org, accessed on 26 October 2022).

Figure 2

Phylogenetic tree based on the 18S rRNA gene from Piroplasmida. Tree was constructed by Bayesian inference using an alignment size of 1460 bp. Clades (I–X) were identified according to the phylogenetic study of Piroplasmida conducted by Jalovecka et al. (2019). Sequences from the present study are highlighted in green. Post probability values >50 appear in tree. Identifications of the phylogenetic groups as proposed by Jalovecka et al. (2019) are indicated next to taxa or groups.

Figure 3

Phylogenetic tree based on partial cox-1 gene. Tree was constructed by Bayesian inference using an alignment size of 259 amino-acids. A sequence of Plasmodium falciparum (AAP57966) was used as outgroup. Sequences from the present study are highlighted in red. Post probability values >50 appear in tree. Clades (I, III, VII, VIII, IX, X) were identified according to the phylogenetic study of Piroplasmida conducted by Jalovecka et al. (2019) are indicated next to taxa or groups.

Figure 4

Phylogenetic tree based on partial hsp70 gene. Tree was constructed by Bayesian inference using an alignment size of 690 bp. A sequence of Cryptosporidium ratti (MT507483) was used as outgroup. Sequences from the present study are highlighted in bold. Post probability values >50 appear in tree. Clades (I, III, VIII, IX, X) were identified according to the phylogenetic study of Piroplasmida conducted by Jalovecka et al. (2019) are indicated next to taxa or groups.

Figure 5

Splitstree network based on ‘Neighbor-net’ and ‘Uncorrected-p’ parameters, using an alignment of 1460 bp of the 18S rRNA. Identifications of the phylogenetic groups as proposed by Jalovecka et al. (2019) are indicated next to taxons or groups. Sequences from the present study are circled in green.

Figure 6

Maltese-Cross inclusions suggestive of Theileria spp. found in erythrocytes of Tapirus terrestris (indicated by the red arrow). Visualization was made by using a light microscope (1000×), during blood smear examination. The animal (ID: PO-P) was positive in a PCR protocol that amplify a fragment of approximately 1500 bp of the 18S rRNA gene from piroplasmids [23,24] in the present study.