title: Rapid detection of haemotropic mycoplasma infection of feline erythrocytes using a novel flow cytometric approach.
abstract: BACKGROUND
  The 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.
  
  
  METHODS
  We 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.
  
  
  RESULTS
  Here, 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.
  
  
  CONCLUSIONS
  The 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.
authors.affiliation: Discipline of Physiology and Bosch Institute, School of Medical Sciences, University of Sydney, Camperdown, NSW, Australia.
authors.forename: Angeles
authors.initials: A
authors.lastname: Sánchez-Pérez
authors.forename: Graeme
authors.initials: G
authors.lastname: Brown
authors.forename: Richard
authors.initials: R
authors.lastname: Malik
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Assinder
authors.forename: Katherine
authors.initials: K
authors.lastname: Cantlon
authors.forename: Christine
authors.initials: C
authors.lastname: Gotsis
authors.forename: Samantha
authors.initials: S
authors.lastname: Dunbar
authors.forename: Stuart T
authors.initials: ST
authors.lastname: Fraser
mesh_terms: D000818:Animals; D000880:Anthraquinones / Q000378:metabolism; D001431:Bacteriological Techniques / Q000379:methods; D002371:Cat Diseases / Q000175:diagnosis* / Q000382:microbiology; D002415:Cats; D004912:Erythrocytes / Q000382:microbiology*; D005434:Flow Cytometry / Q000379:methods*; D009174:Mycoplasma / Q000302:isolation & purification*; D009175:Mycoplasma Infections / Q000175:diagnosis / Q000662:veterinary*; D013194:Staining and Labeling / Q000379:methods; D014730:Veterinary Medicine / Q000379:methods*
chemical_list: C520537:1,5-bis((2-(methylamino)ethyl)amino)-4,8-dihydroxyanthracene-9,10-dione; D000880:Anthraquinones