Detection of Hemotropic Mycoplasma sp. in white-eared opossums (Didelphis albiventris) from Southern Brazil

Opossums are marsupials from the New World of the genus Didelphis and known as synanthropic animals due to their proximity with human beings. To date, ‘Candidatus Mycoplasma haemodidelphis’ has been solely found infecting the North American opossum (Didelphis virginiana). Accordingly, the aim of this study was to screen eight white-eared opossums (Didelphis albiventris) from a public park in Maringa city, Paraná State, southern Brazil, for hemoplasma infection. Blood samples were taken from caudal venipuncture, and DNA was extracted and further screened by a pan-hemoplasma PCR assay. Seven out of eight (87.50%; CI 95%: 47.35-99.68%) white-eared opossums were positive for Mycoplasma spp. Sequencing of the 16S rRNA fragment showed 98,97% identity with ‘Ca. M. haemodidelphis’ detected in the USA. Three out of eight (37.50%; CI 95%: 8.52-75.51%) white-eared opossums were infested by Amblyomma dubitatum ticks. This is the first report on detection of a potentially novel hemotropic Mycoplasma sp. infecting opossums from South America.

This study was approved by the Ethics Committee for Animal Experimentation and Animal Welfare at the Universidade Federal do Paraná, Brazil (protocol number 053/2018). Animal and laboratory procedures were approved and performed under regulations of the Chico Mendes Institute for Biodiversity Conservation (ICMBio, protocol number 63433-2).
The study was carried out in an urban public park of Maringá city: Ingá park (51°55′59ʺ W, 23°25′28ʺ S). Maringá city is located in northwest region of Paraná State, which is characterized by semideciduous Atlantic Forest fragments and has a subtropical climate with an average temperature of 21.7 °C. The area has a diverse fauna, with populations of common marmoset (Callithrix jacchus), capybaras, and marsupials, as well as a wide variety of birds and fishes. D. albiventris is the only species of the genus Didelphis living in the park.
A total of eight adult (five females and three males) white-eared opossums (D. albiventris) were captured using wire mesh traps baited with fruit. Opossums were identified at species level based on morphological and phenotypical characteristics (LEMOS & CERQUEIRA, 2002). After chemical restraint (xylazine 4.0 mg/kg and ketamine 20 mg/kg), animals were individually microchipped with a subcutaneous implant (Animall TAG  , São Carlos, São Paulo, Brazil) and visually inspected for ectoparasites (ticks and fleas). Ticks were removed and stored in 70% ethanol-labeled tubes for further classification according to morphological taxonomic keys (ARAGÃO & FONSECA, 1961;GUIMARÃES et al., 2001;MARTINS et al., 2016). Thereafter, EDTA blood samples were collected by caudal venipuncture and stored at -20 °C until molecular analysis. After sampling, animals were monitored until total recovery from the chemical restraint and were then released at the park.
DNA was extracted from 200 μL blood using a commercially available kit (Illustra TM Blood Genomic Prep Mini Spin Kit, GE Healthcare Life Sciences, Little Chalfont, UK), according to the manufacturer's instructions. Ultrapure water was used as a negative control in parallel to monitor for cross-contamination.
To ensure successful DNA extraction, PCR for the opossum housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (gapdh) (BIRKENHEUER et al., 2003) was performed in all samples. Thereafter, samples were screened using conventional PCR with genus-specific primers targeting a portion of the 16S rRNA gene of Mycoplasma spp. (HOELZLE et al., 2011;MACHADO et al., 2017). The amplified PCR products were subjected to gel electrophoresis in 1.5% agarose gels for 1 h at 100 V, followed by SYBR safe staining (6 μg/ml; SYBR  Safe DNA Gel Stain, Invitrogen, CA, USA) and were viewed under a 312 nm UV light transilluminator.
Amplicons (871 bp) obtained from two Mycoplasma sp.-positive samples were purified from the agarose gel (Wizard  SV Gel and PCR Clean-Up System, Promega, Madison, EUA), evaluated by spectrophotometry for concentration and purity (NanodropTM 2000 Spectrophotometer, Thermo Fisher Scientific, Wilmington, MA, USA), and sequenced in both directions by the Sanger method. The assembled partial sequences of the 16S rRNA gene were subjected to BLASTn analysis (ALTSCHUL et al., 1990) to determine the identity with sequences deposited in the GenBank database. Nucleotide sequences of the Mycoplasma sp. amplified herein were submitted to the GenBank database (accession numbers: MH158514 and MH158515).
Herein, we report the molecular detection of hemotropic Mycoplasma sp. closely related to 'Ca. M. haemodidelphis' in free-ranging opossums from Brazil. To the authors' knowledge, molecular detection of hemoplasma in opossums (Didelphis sp.) has only been described in the United States (MESSICK et al., 2000;MESSICK, 2004). In the Brazilian Pantanal region, a previous study screened 30 marsupials for hemoplasma infection, including one white-eared opossum, and all tested negative for Mycoplasma sp. by conventional PCR targeting the 16S rRNA of these bacteria (SOUSA et al., 2017).
In the present study, 87.5% of opossums were positive for hemotropic Mycoplasma sp. Previous studies have suggested high hemoplasma prevalence rates in tropical regions, which may favor transmission by arthropod vectors. Although previous studies have implicated ticks in the transmission of hemoplasmas, such as Rhipicephalus sanguineus sensu lato as a vector of Mycoplasma haemocanis in dogs (SENEVIRATNA et al., 1973) and Haemaphysalis plumbeum and Rhipicephalus bursa as vectors of Mycoplasma ovis in small ruminants (NEIMARK et al., 2004), to date there has not been adequate evidence to support the contention that hemoplasmas are truly vector-borne pathogens. Herein, although 37% of opossums were infested by A. dubitatum and Amblyomma sp. ticks, further studies are needed to elucidate the role of A. dubitatum ticks in the transmission of hemoplasmas.
Raccoons are considered adapted to urbanized and suburbanized environments (GEHRT et al., 2010), similar to opossums. A previous study has shown that raccoons from undisturbed habitats were more likely to be infected by hemoplasmas than animals from urban areas (VOLOKHOV et al., 2017). Herein, a high prevalence of hemotropic Mycoplasma sp. was found in opossums from public parks located in an urban area of Paraná State, southern Brazil. Further studies are needed to elucidate differences in hemoplasma prevalence among environments.
Additionally, the analyses of the partial sequence of 16S rRNA gene have identified a potentially novel hemoplasma species infecting white-eared opossums from Southern Brazil. This data is supported since the inhabited areas for D. virginiana and D. albiventris do not overlap (COSTA et al., 2015;PÉREZ-HERNANDEZ et al., 2016), and thus, an infection of D. albiventris in Brazil with 'Ca. M. haemodidelphis' from D. virginiana by direct transmission is less likely. Further studies evaluating other housekeeping genes (e.g. rpoB, 23S rRNA) are needed for characterization of this potentially novel hemotropic Mycoplasma species.
A potentially novel hemotropic Mycoplasma sp. is very prevalent in white-eared opossums from southern Brazil. This is the first report on detection of a hemotropic Mycoplasma sp. infecting opossums from South America.