Flow cytometric method for quantifying viable Mycoplasma agassizii, an agent of upper respiratory tract disease in the desert tortoise (Gopherus agassizii).

AIMS: Mycoplasma agassizii can cause upper respiratory tract disease in the threatened desert tortoise of the Southwestern United States. Two technical challenges have impeded critical microbiological studies of this microorganism: (i) its small size limits the use of light microscopy for cell counting and (ii) its extremely 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. METHODS AND RESULTS: Here, we demonstrate that the nonfluorescent molecule 5-carboxyfluorescein (5-CF) diacetate acetoxymethyl ester penetrates M. agassizii cell membranes and it is converted in the cytoplasm to the fluorescent molecule 5-CF by the action of intracellular esterases. Labelled mycoplasma cells can be easily detected by flow cytometry, and cultures with as few as 100 viable mycoplasma cells ml(-1) can be labelled and counted in less than 1 h. Experiments using temperature-induced cell death demonstrated that only viable M. agassizii cells are labelled with this procedure. CONCLUSIONS: A rapid and sensitive flow cytometric technique has been developed for enumerating viable M. agassizii cells. SIGNIFICANCE AND IMPACT OF THE STUDY: This technique should facilitate basic immunological, biochemical and pharmacological studies of this important pathogen which may lead to new diagnostic and therapeutic methods.

Effect of thyroidectomy on 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced lipid peroxidation.

Thyroid hormones have been implicated in the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Therefore, the effects of methimazole (MMI) and propylthiouracil (PTU) induced hypothyroidism and surgical thyroidectomy on several toxic manifestations of TCDD were investigated. Female rats were treated with MMI (0.50 mg/kg) for 10 or 28 days, or PTU (5.0 mg/kg) for 10 days. Other animals were surgically thyroidectomized. The animals received TCDD (100 micrograms/kg) orally or the corn oil vehicle 6 days prior to sacrifice. MMI and PTU decreased serum thyroxine (T4) levels by 27-33% while surgical thyroidectomy decreased T4 levels by 66%. TCDD alone decreased T4 levels by 67%, and similar effects occurred in MMI and PTU treated animals. TCDD produced a 9% increase in serum triiodothyronine (T3) concentrations, and neither MMI or PTU treatment for 10 days modified this effect. Neither antithyroid drug prevented TCDD induced weight loss. TCDD administration resulted in over a 300% increase in hepatic malondialdehyde (MDA) content and a 60% decrease in glutathione peroxidase activity. Neither antithyroid compound affected TCDD-induced alterations in these two parameters. TCDD enhanced MDA content by 220% and inhibited glutathione peroxidase activity by 39% in surgically thyroidectomized rats. Thus, only severe hypothyroidism produced by surgical thyroidectomy was able to partially prevent the effects of TCDD on hepatic MDA content and glutathione peroxidase activity.

Glutathione peroxidase and reactive oxygen species in TCDD-induced lipid peroxidation.

Previous studies have shown that high doses of TCDD induce hepatic lipid peroxidation and inhibit selenium dependent glutathione peroxidase (GSH-Px) activity. The dose dependent effects of TCDD on hepatic lipid peroxidation (malondialdehyde content) and GSH-Px activity were determined. A dose as low as 1 microgram/kg induced hepatic lipid peroxidation and inhibited GSH-Px. Based on the use of scavengers of reactive oxygen species, lipid peroxidation (malondialdehyde formation) by hepatic microsomes from both control and TCDD-treated rats appears to be due primarily to H2O2. The results indicate that superoxide, hydroxyl radical and singlet oxygen are also involved. The differences in the reactive oxygen species involved in microsomal lipid peroxidation between control and TCDD treated animals appear to be quantitative rather than qualitative. A 5.9-fold greater rate of malondialdehyde (MDA) formation by microsomes from TCDD treated animals occurred as compared to controls, while livers of TCDD rats had an MDA content that was 5.0-fold greater than the controls. These differences may be due in part to an enhanced production of H2O2 as well as a decrease in the activity of selenium dependent glutathione peroxidase which metabolizes H2O2.