MOLECULAR IDENTIFICATION OF HEMOTROPIC MYCOPLASMAS (HEMOPLASMAS) IN WILD MAMMALS

Abstract

Hemotropic mycoplasmas (hemoplasmas) are worldwide distributed bacteria affecting domestic and wildlife animals besides human beings. They still remain uncultivated in vitro. Hemoplasms have been described as potential causes of hemolytic anemia in domestic and wild mammals. The objective of this study was to detect by molecular methods the presence of hemotropic mycoplasmas in native and exotic wild mammals. This doctoral thesis presents three articles. The first article is of systematic review and meta-analysis on the molecular detection of hemoplasms in wild mammals, using articles indexed in MEDLINE and SCIELO, from December 1967 to October 2016. A total of 45/1235 articles (3.64%) related to molecular identification of hemoplasmas in wild mammals, and 78 wild mammal species were reported to be infected. The meta-analysis was performed using a random-effects model to compare the prevalence data available for wild mammals in capitivity and in free ranging between orders. A phylogenetic tree based on 16S rRNA gene sequences was constructed, compared and discussed. Hemoplasmas are distributed in wild mammals throughout the world, with prevalence of 29.92% (CI 24.53 – 33.74) for all reported animals: 31.00% (CI 24.97 – 37.76, I² p < 0.001) for wild animals and 22.33% (CI 17.20 – 28.47, I² p < 0.001) for captive animals. The second article refers to the research of hemotropic mycoplasmas in bats, being this the first study with hemotropic mycoplasmas in bats in Brazil. Blood samples (n=10) were taken from eight hematophagous bats: six males common vampire bat (Desmodus rotundus; Family Phyllostomidae), two males hairy-legged vampire bat (Diphylla ecaudata; Family Phyllostomidae); and two no-hematophagous females Pallas's mastiff bat (Molossus sp.; Family Molossidae), at Curitiba’s region, Parana State, southern Brazil. For anesthesia, bat cages were put inside a plastic container and isoflurane was infused with a machine with oxygen. Sedation maintenance was performed using inhalation mask. Intracardiac puncture was performed to obtain blood, sequentially, the bats were euthanized with lethal dose of intracardiac potassium chloride. Blood smears of two Desmodus rotundus, prepared immediately after blood collection, were stained with May-Grünwald-Giemsa and examined using light microscopy at 1,000x magnification for the presence of hemoplasma. DNA was extracted from 200 µL blood using a commercially available kit according to the manufacturer’s instructions. A PCR for the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase GAPDH), was performed to ensure successful DNA extraction. Thereafter, samples were screened by conventional pan-hemoplasma PCR targeting the 16S rDNA regions specific for hemoplasmas. ‘Candidatus M. haemovis’-positive goat blood sample and nuclease-free water were used as positive and negative control, respectively. Using universal primers for the 16S rRNA (Reference), samples from two bats of the species Desmodus rotundus that tested positive for Mycoplasma sp in the first PCR reaction, were amplified. The PCR products of 745 bp were purified from the 1.5% agarose gel and sequenced. The nucleotide sequences of the hemoplasmas isolates from bats were submitted to the GenBank database under the accession number KX722541. A phylogenetic tree based on 16S rRNA gene sequences was constructed. In overall, 8/10 (80.0%) bats tested positive to Mycoplasma sp. including 5/6 (83.3%) Desmodus rotundus, 2/2 (100%) Diphylla ecaudata and 1/2 (50.0%) Molossus sp. The analyses of the partial sequence of 16S rRNA gene have identified a potentially novel hemoplasma species infecting bats at Curitiba’s region, Parana State, Southern Brazil. In the third article, the objective of the study was to apply a PCR protocol of Mycoplasma ovis in 12 captive Barbary sheep (Ammotragus lervia) at Curitiba Zoo, in southern Brazil. A total of 12 blood samples with EDTA, previously searched for other pathogens were used. DNA extracted and a PCR protocol for the glyceraldehyde-3- phosphate dehydrogenase (GAPDH) gene was performed on all samples to ensure amplifiable DNA. Subsequently, all the samples were tested and found to be negative using a specific PCR protocol for M. ovis detection and amplification. In the supplement are two articles, the first supplement is a review of pathogens in aoudads, with articles published between September 1959 and October 2016, identified through a computerized search in the electronic databases PubMed and SciELO. Some pathogens detected in aoudads, such as Mycobacterium tuberculosis and Toxoplasma gondii, can also infect domestic animals and humans. The second supplement refers to the research of Plasmodium sp. in cervidae in Brazil. A captive herd of 22 deer-bororó (Mazama nana), four deer-mateiro (Mazama americana) and six marsh deer (Blastocerus dichotomus) from southern Brazil were evaluated using light microscopy and molecular approaches. Microscopic and molecular analyses were both negative for parasite presence. Keywords: Hemotropic Mycoplasma, Hemoplasma, Wild Mammals, PCR, Pathogens, Plasmodium sp