Rapid detection of haemotropic mycoplasma infection of feline erythrocytes using a novel flow cytometric approach

Abstract

BackgroundThe haemotropic mycoplasmas Mycoplasma haemofelis and Candidatus Mycoplasma haemominutum cause feline infectious anaemia with infection rates in feline populations reflecting widespread subclinical infection. Clinically significant infections are much rarer but can be life-threatening. Current diagnosis is dependent upon visualising organisms in stained blood smears, PCR or quantitative PCR (qPCR). These procedures are labour-intensive and time-consuming. Furthermore, PCR-based approaches offer limited insight into the disease burden of the infected animal.MethodsWe have developed a novel and rapid flow cytometric system that permits diagnosis of haemotropic mycoplasma infections and quantitation of the percentage of erythrocytes that are parasitized. The method exploits the fact that mature mammalian erythrocytes, the host cell for haemoplasmas, are enucleated and thus lack nucleic acid. DRAQ5 is a synthetic anthrocycline dye which rapidly crosses cell membranes and binds to nucleic acids. The presence of exogenous bacterial DNA in mammalian erythrocytes can, therefore, be detected by DRAQ5 uptake and flow cytometric detection of DRAQ5 fluorescence.ResultsHere, we show that this system can detect epi-erythrocytic infection of companion felines by haemotropic mycoplasma. Due to their differences in size, and hence the quantity of DNA, the two major feline hemoplasmas M. haemofelis and Candidatus M. haemominutum can be distinguished according to DRAQ5 fluorescence. We have also shown the usefulness of DRAQ5 uptake in monitoring a cat infected with M. haemofelis sequentially during treatment with doxycycline.ConclusionsThe technique described is the first report of a flow cytometric method for detecting haemotropic mycoplasmas in any species and could be applied to widespread screening of animal populations to assess infection by these epi-erythrocytic parasites.

Quantitative Assessment of Mycoplasma Hemadsorption Activity by Flow Cytometry

“…Despite this, there is an increasing number of works showing that FC is an accurate method to detect and quantify mycoplasma cells in broth medium [23] , [24] or in natural samples [25] . FC has been used also to quantify the effect of several antibacterial agents [26] , [27] and to detect hemotropic mycoplasmas on red blood cells (RBCs) samples [28] . Here we show that FC could be used to measure rapidly and accurately the HA activity of mycoplasmas in the presence of SYBR Green I, a vital fluorochrome that stains nucleic acids, allowing to resolve RBCs and mycoplasma cells by their different size and fluorescence.…”

Avaliação Eritrocitária Através Da Biotecnologia De Citometria De Fluxo

“…Outro emprego da CF na parasitologia é a pesquisa dedicada à biologia e mecanismo de infecção, Moretta et al (2014) verificaram a evolução da parasitemia relacionando à redução nas contagens de eritrócitos e ao aumento de células infectadas por Anaplasma marginale, marcadas com SYTO 16, com tempo de evolução da doença e aparecimento de anemia, bem como a debelação parasitária após administração de antibióticos. -Pérez et al (2013) desenvolveram uma metodologia de marcação de micoplasmas hemotrópicos em eritrócitos de felinos através da marcação de DNA parasitário pelo corante DRAQ5, evidenciando células positivas para a infecção quando analisadas por CF. Ao contrário das metodologias usuais para o diagnóstico, que permitem apenas a identificação da espécie, a CF permitiu averiguar a proporção de eritrócitos infectados; além disso, esta metodologia mostrou-se mais acessível e rápida em relação aos testes de PCR, que são comumente utilizados no reconhecimento de micoplasmas.…”