Mycoplasma detection in cell culture by concomitant use of bisbenzamide and fluoresceinated antibody

Summary Conditions are presented for application of both bisbenzamide (Hoechst 33258) stain and a specific fluoresceinated anti-Mycoplasma hyorhinis IgG to a single cell culture preparation. This allows the same field on a slide to be viewed for presumptive diagnosis of any cell culture contaminant mycoplasma by bisbenzamide staining and for definitive diagnosis ofM. hyorhinis strains using fluoresceinated antibody. The use of this method plus a cultural procedure will permit identification of the “noncultivable”M. hyorhinis strain DBS 1050.     

Application of PCR for detection and identification of mycoplasma contamination in virus stocks

Summary A nested polymerase chain reaction (PCR) was used to detect and identify mycoplasma contaminants in viral stocks. The results of the PCR assay proved to be a sensitive and accurate indicator of the true status of the stock tested. Those samples positive by agar culture or Hoechst stain were also positive by PCR. Those samples that were inconclusive by Hoechst stain (10.05%) could be clearly determined to be mycoplasma positive or negative by PCR. The PCR assay also detected those fastidious species of mycoplasma that gave false negative results by the direct culture method. In many respects the PCR-based mycoplasma detection method described is superior to the agar culture and Hoechst staining detection methods. In this study, the PCR assay detected substantially more mycoplasma-positive viral stocks than did the agar culture assay. Due to its speed, sensitivity, and reliability, the PCR assay is of particular value in monitoring the process of removing mycoplasma from contaminated stocks. Furthermore, the PCR amplification products can be analyzed by restriction analysis to rapidly identify the species of the mycoplasma contaminating the stock tested.     

Sodium ion-dependent hydrogen production in Acidaminococcus fermentans

Acidaminococcus fermentans is able to ferment glutamate to ammonia, CO₂, acetate, butyrate, and H₂. The molecular hydrogen (approximately 10 kPa; E′ = –385 mV) stems from NADH generated in the 3-hydroxybutyryl-CoA dehydrogenase reaction (E°′ = –240 mV) of the hydroxyglutarate pathway. In contrast to growing cells, which require at least 5 mM Na⁺, a Na⁺-dependence of the H₂-formation was observed with washed cells. Whereas the optimal glutamate fermentation rate was achieved already at 1 mM Na⁺, H₂ formation commenced only at > 10 mM Na⁺ and reached maximum rates at 100 mM Na⁺. The acetate/butyrate ratio thereby increased from 2.0 at 1 mM Na⁺ to 3.0 at 100 mM Na⁺. A hydrogenase and an NADH dehydrogenase, both of which were detected in membrane fractions, are components of a model in which electrons, generated by NADH oxidation inside of the cytoplasmic membrane, reduce protons outside of the cytoplasmic membrane. The entire process can be driven by decarboxylation of glutaconyl-CoA, which consumes the protons released by NADH oxidation inside the cell. Hydrogen production commences exactly at those Na⁺ concentrations at which the electrogenic H⁺/Na⁺-antiporter glutaconyl-CoA decarboxylase is converted into a Na⁺/Na⁺ exchanger. Received: 3 May 1996 / Accepted: 12 August 1996     

Molecular survey of haemoplasmas in shelter dogs and associations with Rhipicephalus sanguineus sensu lato

This study aimed to assess the occurrence of canine haemoplasma infection in domestic dogs and its possible trans‐stadial transmission by Rhipicephalus sanguineus sensu lato (Ixodida: Ixodidae) in shelter dogs in Diyarbak?r Province in southeast Turkey. Blood samples (n = 282) collected from domestic dogs were analysed by polymerase chain reaction (PCR) for the presence of canine haemoplasma. Fully engorged nymphs (n = 204) were removed from dogs that were positive for canine haemoplasma by PCR and maintained in an incubator at 28 °C for moulting. Unfed ticks (n = 2185) comprising 2100 nymphs and 85 adults collected from the grounds of the same shelter were also screened. Of 282 dogs, 108 [38.3%, 95% confidence interval (CI) 32.6–44.2] were PCR‐positive for canine haemoplasmas. Mycoplasma haemocanis (Mhc) infection (26.2%, 95% CI 21.2–31.8) was observed in a significantly higher number of dogs than was Candidatus Mycoplasma haematoparvum (CMhp) infection (6.7%, 95% CI 4.1–10.3). Co‐infections were seen in 15 (5.3%, 95% CI 3.0–8.6) dogs. None of the tick specimens examined were found to be positive for haemoplasma. Partial sequences of the 16S ribosomal RNA (rRNA) gene shared 99–100% identity with the corresponding published sequences for Mhc and CMhp. The present results revealed no trans‐stadial transmission of canine haemoplasma species by R. sanguineus s.l. in field conditions.     

Comparative studies to determine the efficiency of 6 methylpurine deoxyriboside to detect cell culture mycoplasmas

Summary Studies were performed to compare three methods to detect mycoplasmal infection of cell cultures. The methods included microbiological assay by inoculation into broth and onto agar with anaerobic incubation, fluorescent DNA staining by Hoechst 33258, and mycoplasmal mediated cytotoxicity by 6 methylpurine deoxyriboside (6MPDR). Fluorescent DNA staining and 6MPDR assays were performed in an indicator cell culture system. A total of 2589 cell cultures were assayed. Mycoplasmas were detected in 174, an incidence of 6.7%. Species isolated were:Acholeplasma laidlawii, Mycoplasma orale, M. arginini, M. hyorhinis, M. fermentans, M. pirum, and M. pneumoniae. In separate studies, 6MPDR also detected infection withSpiroplasma mirum when this organisms was deliberately inoculated into cell cultures. The efficiencies of microbiological testing, fluorescent DNA assays, and 6MPDR were 43.1, 98. 8, and 97.1%, respectively. The work was supported by grant AI-15748 from the National Institutes of Health, Bethesda, MD     

An ultramicrochemical test for mycoplasmal contamination of cultured cells

Summary A sensitive ultramicrochemical enzyme test for mycoplasmal contamination of cultured cells, based on the determination of the activity of adenosine phosphorylase, is described. The test was performed by assaying the enzymatic conversion of [8-¹⁴C]adenine and ribose-1-phosphate to [8-¹⁴C]adenosine by incubating a plastic leaflet carrying a counted number of cells (1 to 10). These leaflets were isolated from the bottom of the same plastic film dish in which the cells were cultured for experimental or diagnostic purposes, e.g. prenatal diagnosis or inborn errors of metabolism. The present test should be several 1000-fold more sensitive than the originally reported enzymatic method because (a) the adenosinephosphorylase reaction is measured in the nucleoside forming direction which is by far the most active; and (b) the assay is performed with the cells and not with the culture medium. The latter is of special importance for the detection of those low-grade contaminations in which most of the mycoplasma particles are attached to cell membranes. This investigation was partly supported by FUNGO (The Netherlands), INSERM (France) (A. T. P. 36.76.68), and DGRST (France) (No. 75.50.004).