Expanding the Universe of Hemoplasmas: Multi-Locus Sequencing Reveals Putative Novel Hemoplasmas in Lowland Tapirs (Tapirus terrestris), the Largest Land Mammals in Brazil

Abstract

The lowland tapir (Tapirus terrestris) is the largest land mammal in Brazil and classified as a vulnerable species, according to the assessment of the risk of extinction. The present study aimed at investigating the occurrence and genetic diversity of hemoplasmas in free-ranging T. terrestris from the Brazilian Pantanal and Cerrado biomes. Blood samples were collected from 94 living and eight road-killed tapirs, totalizing 125 samples Conventional PCR targeting four different genes (16S rRNA, 23S rRNA, RNAse P, and dnaK) were performed, and the obtained sequences were submitted for phylogenetic, genotype diversity, and distance analyses. The association between hemoplasma positivity and possible risk variables (age, gender, and origin) was assessed. Out of 122 analyzed samples, 41 (41/122; 33.61% CI: 25.84-42.38%) were positive in the 16S rRNA-based PCR assay for hemoplasmas. Positivity for hemoplasmas did not differ between tapirs' gender and age. Tapirs from Pantanal were 5.64 times more likely to present positive results for hemoplasmas when compared to tapirs sampled in Cerrado. BLASTn, phylogenetic, genotype diversity, and distance analyses performed herein showed that the sampled lowland tapirs might be infected by two genetically distinct hemoplasmas, namely 'Candidatus Mycoplasma haematoterrestris' and 'Candidatus Mycoplasma haematotapirus'. While the former was positioned into "Mycoplasma haemofelis group" and closely related to 'Candidatus Mycoplasma haematoparvum, the latter was positioned into "Mycoplasma suis group" and closely related to 'Candidatus Mycoplasma haematobos'. The impact of both putative novel species on tapir health status should be investigated.

Keywords: Cerrado; Pantanal; hemotropic Mycoplasma; lowland tapirs.

Figure 1

Phylogenetic tree based on partial 16S rRNA gene fragments of Mycoplasma sp. Tree was constructed by Bayesian Inference and a sequence from Bacillus subtillis (AB042061) was used as outgroup. Sequences obtained in the present study (Ca1 and Ca2) are highlighted in colors: Ca1 are highlighted in purple, pink and red colors, differentiating each subclade formed. Ca2 is highlighted in blue.

Figure 2

Phylogenetic tree based on partial 23S rRNA gene fragments of Mycoplasma sp. Tree was constructed by Bayesian Inference and a sequence from Bacillus subtillis (NR103037) was used as outgroup. Sequences obtained in the present study are highlighted in colors: sequences that fit in Ca1 by the 16S rRNa phylogeny are highlighted in purple, pink and red colors. Sequences that fit in Ca2 by the 16S rRNA phylogeny are highlighted in blue.

Figure 3

Phylogenetic tree based on partial RNAse P gene fragments of Mycoplasma sp. Tree was constructed by Bayesian Inference and a sequence from Clostridium innocuum (U64878) was used as outgroup. Sequences obtained in the present study are highlighted in colors: the sequence that fit in Ca1 by the 16S rRNA phylogeny is highlighted in pink. The sequence that fit in Ca2 by the 16S rRNA phylogeny is highlighted in blue.

Figure 4

Phylogenetic tree based on partial dnaK gene fragments of Mycoplasma sp. Tree was constructed by Bayesian Inference and a sequence from Klebisiella pneumonia (KJ690086) was used as outgroup. Sequences obtained in the present study is highlighted in pink, once it fits in Ca1 by the 16S rRNA phylogeny.

Figure 5

Genotype diversity among 16S rRNA gene sequences detected herein. Analysis was made using DnaSP6. Inference and graphic representation were made by TCS network method on PopART software. Genotypes in blue were obtained from samples from tapirs in Pantanal regions meanwhile genotypes in green were obtained from samples from tapirs in Cerrado regions.

Figure 6

Representation of the genotype distribution along all sampling areas. Map was constructed using PopART software based on GPS coordinates data of each sampling.

Figure 7

Representation of the genotype distribution of Pantanal biome areas. Map was constructed using PopART software based on GPS coordinates data of each sampling.

Figure 8

Representation of the genotype distribution of Cerrado biome areas. Map was constructed using PopART software based on GPS coordinates data of each sampling.

Figure 9

Distance analysis of 16S rRNA fragments from different Mycoplasma species was made using SplitsTree v4.14.6 software. Sequences from Ca1 and Ca2 are indicated in the tree. Regarding Ca1 sequences, colors from the Splitstree graph match with the subclades highlighted in 16S rRNA phylogeny (purple, pink and red). The Ca2 sequences are highlighted in blue. The species M. suis and M. haemofelis are highlighted in green. The species ‘Ca. M. haematoparvum’ and ‘Ca. M. haematobos’ were also highlighted in green to demonstrate their position compared to Ca1 and Ca2.

Figure 10

A heatmap constructed using the distance matrix based on p-value between all sequences obtained herein. The scale on the right demonstrates the color shade transition between obtained values.