Haemoplasma infection detected in an owl monkey from over 40 year old blood smears

Haemotropic mycoplasmas (’haemoplasmas’) infect a variety of mammals including cats, dogs, rodents, cattle, sheep and the opossum. ‘Candidatus Mycoplasma kahaneii’, a haemoplasma species from the haemominutum clade of haemoplasmas, has previously been shown to infect squirrel monkeys (Saimiri sciurcus). In the late 1960s an owl monkey (Aotus trivirgatus), imported from Colombia, was splenectomised and experimentally infected with Plasmodium falciparum. Whilst observing the course of malaria infection, erythrocyte-associated ’eperythrozoon’-like bodies were described following examination of Giemsa-stained blood smears by light and electron microscopy [Peters et al.(1974) Ann Trop Med Parasitol 64, 47-50]. Smears of fixed, stained whole blood from this owl monkey made in 1967 were available for analysis in this study. An optimized method using a commercially available kit (Macherey-Nagel Nucleospin Blood kit, ABgene, Epsom, UK) was used to extract DNA from the blood smears. The extracted DNA was subjected to a newly developed generic haemoplasma real-time quantitative PCR (qPCR) incorporating an internal amplification control for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The qPCR confirmed the extraction of adequate amounts of host DNA from the blood smear, and the presence of a haemoplasma species from the haemominutum clade. Positive and negative controls were appropriately positive and negative. Conventional PCR and sequencing were performed to obtain partial ribonuclease P gene sequence data. The gene sequence demonstrated that the haemoplasma was most closely related to ‘Candidatus M. kahaneii’ and ‘Candidatus Mycoplasma haemominutum’ (82.4% & 77.6% identities respectively). This study has provided molecular confirmation of the existence of a novel haemoplasma species in the owl monkey previously suspected by cytology. Additionally, this report confirms the use of the generic haemoplasma qPCR to successfully amplify haemoplasma and host DNA from blood smears archived for over 40 years.