ABSTRACT Mycoplasma agassizii is a cause of upper respiratory tract disease in the threatened desert tortoise (Gopherus agassizii) of the Southwestern United States. Two technical challenges have impeded critical microbiological studies of this microorganism. First, its small size (<300 nm) limits the use of light microscopy for cell counting. Second, its slow growth in broth and agar cultures impedes colony counting. Our aim was to develop a rapid and sensitive flow cytometric method using a vital fluorescent dye to enumerate viable M. agassizii cells. We discovered that the non-fluorescent molecule 5-carboxyfluorescein diacetate acetoxymethyl ester penetrates M. agassizii cell membranes and is converted in the cytoplasm to the fluorescent molecule 5-carboxyfluorescein by the action of intracellular esterases. Labeled mycoplasma cells can be easily detected by flow cytometry, and cultures with fewer than 100 viable mycoplasma cells µl-1 can be labeled and counted in less than 1 h. Experiments using temperature-induced cell death demonstrated that this procedure labels only viable M. agassizii cells. This technique should facilitate basic immunological, biochemical, and pharmacological studies of this desert tortoise pathogen which may lead to new diagnostic and therapeutic methods. To facilitate seroepidemiologic studies of infectious diseases caused by microorganisms like M. agassizii, isotype-specific antibody reagents are needed. Our goal was to prepare isotype-specific rabbit polyclonal antibodies to desert tortoise IgM and IgY. A 50% saturated ammonium sulfate precipitate of desert tortoise serum was fractionated by Sephacryl A-300 gel filtration chromatography and IgM-rich and IgY-rich fractions were collected. SDS-PAGE confirmed the molecular weights of the heavy chains of tortoise IgM and IgY to be 71 kD and 68 kD respectively. Rabbits immunized with these separate fractions produced high titer sera that cross-reacted slightly with the opposite heavy chain and strongly with the conserved 22 kD light chain. To make heavy chain isotype-specific reagents, the immunoglobulin fraction of each antiserum was cross-absorbed using polystyrene microspheres coated with the opposite isotype. SDS-PAGE and Western blots confirmed the heavy chain specificity of the cross-absorbed antibodies. These high avidity rabbit polyclonal antibody reagents were used in ELISA and Western blots to document the primary and secondary isotype-specific antibody responses to M. agassizii infection and ovalbumin immunizations in desert tortoises. The availability of rabbit polyclonal antibodies to desert tortoise IgM and IgY will allow more detailed studies of the humoral immune response of the desert tortoise to microbial pathogen
