New records and genetic diversity of Mycoplasma ovis in free-ranging deer in Brazil

Cervids represent a mammal group which plays an important role in the maintenance of ecological balance. Recent studies have highlighted the role of these species as reservoirs for several arthropods-borne pathogens. Globally, hemotropic mycoplasmas (haemoplasmas) are emerging or remerging bacteria that attach to red blood cells of several mammals species causing hemolytic anaemia. Therefore, the aim of this study was to investigate the occurrence and assess the phylogenetic positioning of Mycoplasma ovis in free-ranging deer from Brazil. Using a polymerase chain reaction targeting the 16S rRNA region, 18 (40%) out of 45 sampled deer were positive to M. ovis. Among the nine sequences analysed, four distinct genotypes were identified. The sequences detected in the present study were closely related to sequences previously identified in deer from Brazil and the USA. On the other hand, the Neighbour-Net network analysis showed that the human-associated M. ovis genotypes were related to genotypes detected in sheep and goats. The present study shows, for the first time, the occurrence of M. ovis in Mazama gouazoubira and Mazama bororo deer species, expanding the knowledge on the hosts harbouring this haemoplasma species. Once several deer species have your population in decline, additional studies are needed to evaluate the pathogenicity of M. ovis among deer populations around the world and assess its potential as reservoir hosts to human infections.

Mycoplasma ovis, a zoonotic pathogen frequently detected in sheep and goats [18 19], have been detected in O. virginianus [20,21] and Rangifer tarandus species [22] kept in captivity in the USA and in free-ranging Cervus nippon species in Japan [23]. In Brazil, M. ovis has already been detected in free-ranging B. dichotomus and O. bezoarticus from three distinct Brazilian areas, namely Pantanal (state of Mato Grosso do Sul, Midwestern Brazil), Emas National Park (state of Goiás state, Midwestern Brazil) and Paraná river basin (São Paulo state, southeastern Brazil) [24]. Additionally, M. ovis DNA was detected in blood samples from M. nana, M. americana and B. dichotomus species maintained in captivity at Bela Vista Biological Sanctuary (Paraná state, Southern Brazil) [25].
The present study aimed to investigate the occurrence and assess the genetic diversity of M. ovis in free-ranging deer sampled in four Brazilian states.

Ethical statement
Deer blood sampling was conducted by Professor José Maurício Barbanti Duarte (IBAMA Registration number 263703), from the Department of Animal Sciences, FCAV -UNESP, Jaboticabal, with license number 10636-1 provided by IBAMA, between 1996 and 2011.

Number and origin of sampled deer
In total 34 and 11 DNA samples were extracted from Mazama spp. and Ozotoceros bezoarticus buffy coats samples, respectively.   16S rRNA gene was used for detection of M. ovis DNA [24]. Briefly, 5 µl of DNA was used as a template in 25 µl reaction mixtures containing 10X PCR buffer, 1.0 mM MgCl 2 , 0.6 mM deoxynucleotide triphosphate (dNTPs) mixture, 1.5U of TaqDNA polymerase (Life Technologies) and 0.5 µM 16S-Fw 5 ′ -ATGC AAGTCGAACGAGTAGA-3 ′ , and 16S-Rv 5 ′ -TGATACTTTC TTTCATAGTTTG-3 ′ primers. PCR amplifications were performed at 94°C for 5 min followed by 39 repetitive cycles of 94°C for 1 min, 51.6°C for 30 s and 72°C for 1 min, followed by a final extension at 72°C for 5 min. Mycoplasma ovis DNA obtained from a naturally infected Brazilian marsh deer [25] and ultra-pure sterile water were used as positive and negative controls, respectively.

Sequencing and analyses of sequences
Randomly selected PCR products were purified using Silica Bead DNA Gel Extraction Kit (Fermentas, São Paulo, SP, Brazil). Purified amplified DNA fragments from positive samples were submitted to sequence confirmation in an automatic sequencer (ABI Prism 310 Genetic Analyser -Applied Byosystem/Perkin Elmer) in both directions. Lastly, in order to correctly determine the nucleotide composition, the electropherograms were submitted to PhredPhrap program [27]. The Phred quality score (peaks around each base call) was established higher than 20 (99% in accuracy of the base call). Subsequently, the sequences were submitted to phylogenetic analyses. The sequences amplified in the present study were deposited in GenBank data base under accession numbers: (MK919446-MK919454).

Phylogenetic analyses
Haemoplasmas-16S rRNA sequences were identified by BLASTn using the Megablast (following default parameters), aligned with sequences available in GenBank using Clustal/W [28] and adjusted in Bioedit v. 7.0.5.3 [29]. The phylogenetic analysis was performed using Maximum Likelihood (ML) method. The ML phylogenetic analysis was inferred with RAxML-HPC BlackBox 7.6.3 [30]. The analysis (ML) was performed in CIPRES Science Gateway [31]. The Akaike Information Criterion (AIC) available on MEGA software was applied to identify the most appropriate model of nucleotide substitution. GTR + G + I model was chosen as the most appropriate for the phylogenetic analysis of the 16S rDNA alignment.

Identification and genetic relationship of M. ovis genotypes
The 16S rRNA aligned sequences amplified in the present study were utilised to identify the number of genotypes, calculate the nucleotide diversity (π), the polymorphic level (genotype diversity -[Gd]) and the average number of nucleotide differences (K ) using the DnaSP v5.10 [32]. To investigate the genetic relationship among M. ovis genotypes detected in deer in the present study and those previously detected in sheep, goats and humans and retrieved from GenBank, a Neighbour-Net network was constructed using the pairwise genetic distances with SplitsTree v4.10 [33]. Additionally, the different genotypes identified were submitted to TCS network inferred using the Population Analysis with Reticulate Trees (popART) (v. 1.7) [34].

Phylogenetic and genotype analyses
The amplified sequences were positioned within the M. suis group and clustered with others M. ovis sequences detected in animals and humans. The sequences detected in the present study were closely related to sequences previously identified in deer from Brazil and the USA, albeit lightly apart from M. ovis genotypes detected in sheep, goats and humans. The ML analysis was supported by high bootstrap values (Fig. 1). In agreement with ML analysis, but with a marked separation among M. ovis sequences identified in sheep, goats, humans and those amplified in deer, the network analysis showed that the M. ovis sequences were divided into four groups. All analysed M. ovis 16S rRNA sequences detected in sheep, goats and humans were clustered into group I. In addition, the groups II, III and IV comprise the M. ovis sequences identified in deer (Fig. 2). Finally, the TCS network showed similar results, since all deer genotypes clustered together and separated from those genotypes detected in sheep, goats and human and identified in different countries (Fig. 3). Besides, the findings are supported by the divergence scores among the different genotypes (Table 1).
Among the nine sequences detected in the present study, four distinct genotypes were identified ( Table 2). The genotype diversity (Gd), nucleotide diversity (per site = π) and the average number of nucleotide differences (K ), were 0.694, 0.002 and 2.22, respectively.

Discussion
Recent studies have suggested a possible co-evolution among haemoplasmas and their respective hosts [11,21,35]. However, little is known about the origin, dispersion and evolutionary aspects of hemotropic mycoplasmas [12].
Globally, haemoplasmas comprise emerging or re-emerging zoonotic pathogens that affect domestic and wild animals. Previously, based upon molecular diagnosis, M. ovis was detected in a veterinarian in Texas, USA, which was coinfected with Bartonella henselae [36]. More recently, M. ovis was detected in patients without and with extensive arthropods or animal contact [10]. Although the pathogenicity and reservoirs of M. ovis infecting humans are still unknown, the present study showed, for the first time, that the human-associated M. ovis genotypes were closely related to those detected in goat and sheep. However, further studies are needed in order to assess this issue as well as the transmission routes of this haemoplasma species.
The occurrence of M. ovis detected in the present study (40%) was lower than that previously detected among free-ranging (58%) and captive cervids (87%) from Brazil [ 24 25]. A high  Marcos Rogério André et al.  [24]. Interesting, the phylogenetic relationship of the 16S rRNA sequences amplified in the present study showed that the M. ovis genotypes detected in Brazilian deer clustered together and were lightly distant from those detected in goats, sheep and humans, suggests a possible specificity among the different genotypes of M. ovis and their respective vertebrate hosts. In agreement to ML analysis, the networks also confirmed this result and showed clearly that M. ovis detected in goats, sheep and humans are genetic related each other, whereas M. ovis from deer could be classified as a distinct genogroup. However, more studies targeting different genes, analysing additional sequences and verifying other biological aspects are needed.
Although analysing few sequences, a low genetic diversity was observed in M. ovis sequences amplified from Brazilian deer. These results were expected since the 16S rRNA region show low genetic variation. However, it seems like different genotypes circulate in deer populations in the Pantanal region, state of Mato Grosso do Sul state. Two different genotypes were identified circulating on O. bezoarticus from the same region. On the other hand, only one genotype was detected in M. gouazobira individuals caught between 1996 and 2010, suggesting possible maintenance of this genotype over time among deer population in this region.
Among the eight Brazilian deer species, M. ovis has already been detected in four of them, namely B. dichotomus, O. bezoarticus, M. nana and M. americana [24 25]. Additionally, the present study shows, for the first time, the occurrence of M. ovis in M. gouazoubira and M. bororo species. The wide distribution and the number of deer species infected support the role of cervids in the maintenance of M. ovis transmission cycle in the environment. Once several deer species have your population in decline or are classified as vulnerable, additional studies are needed to evaluate the pathogenicity of M. ovis among deer populations from Brazil and around the world, as well as the environmental and biological factors which contribute to deer infection.

Conclusion
The present study shows, for the first time, the occurrence of M. ovis in M. gouazoubira and M. bororo deer species, expanding the knowledge on the hosts harbouring this haemoplasma species. The M. ovis genotypes found in deer in Brazil clustered with other sequences previously detected in cervids, albeit slightly apart from humans, sheep and goats-associated genotypes, suggesting a probable specificity of M. ovis genotypes to groups of vertebrate hosts.