Elimination of mycoplasmas from cell cultures utilizing hyperimmune sera

Eighteen cell lines contaminated with various mycoplasmas have been treated with hyperimmune sera and mycoplasmas have been eradicated from all. After treatment the cell lines have been observed for a least one year and they are still free from mycoplasma contamination as ascertained by four independent mycoplasma detection assays. The hyperimmune sera used were of high titer, type-specific and growth-inhibiting. These sera were produced by immunization of rabbits with purified membranes from Mycoplasma orale, M. arginini, M. hominis, M. fermentans, M. hyorhinis and Acholeplasma laidlawii. In addition to elimination of mycoplasmas from cell cultures we have successfully used these sera for detection and typing of mycoplasma contamination in cell cultures.     

Isolation of acholeplasmas and a mycoplasma from vegetables.

The isolation of Mollicutes from food has not been reported. To isolate Mollicutes in the presence of high levels of unwanted bacteria, we first incubated fresh vegetables in liquid culture media containing lysozyme, ampicillin, and thallous acetate. Culture fluids were than separated from the vegetable samples, subjected to one freeze-thaw cycle, and passed through a filter of 0.4-micron porosity. Filtered samples were cultured in SP4 medium and in a conventional medium containing horse serum. With this procedure 21 acholeplasma isolations representing three species were obtained from endive, broccoli, and kale. Of 35 food samples tested, 11 were positive for acholeplasmas; acholeplasmas isolated from 6 of these samples were recovered only in SP4 medium. In seven single vegetable samples, two or more Acholeplasma spp. were isolated. A. laidlawii was isolated from all three vegetables and A. axanthum was found in broccoli and kale. Four isolates were serologically identified as A. oculi. Mycoplasma verecundum was the only Mycoplasma species recovered. Several isolates could not be typed serologically, as they reacted with antisera to both A. morum and A. hippikon. these isolates may include new Acholeplasma spp.     

Tumor-associated blocking factors: isolation from sera of tumor-bearing mice.

An isolation and partial purifications of tumor-associated blocking factors from the sera of tumor-bearing mice is described. Columns for affinity chromatography were prepared by coupling syngeneic tumor-immune antibodies to Sepharose 4B. Passage of serum through such immunoadsorbent columns removed all blocking activity from tumor-bearers' sera; subsequent elution of the absorbent with 3 M NaSCN recovered the activity. The blocking material was further purified on Sephadex G-200. The data provide evidence for the presence of antigen in tumor-associated blocking factors and are compatible with the hypothesis that blocking factors often consist of antigen and antibodies in the form of immune complexes.     

Direct isolation of Francisella spp. from environmental samples

Aims:  To develop a selective medium for isolation of F. tularensis, F. novicida and F. philomiragia from environmental samples.
Methods and Results:  A selective media, cysteine heart agar with 9% chocolatized sheep blood, containing polymyxin B, amphotericin B, cyclohexamide, cefepime and vancomycin (CHAB-PACCV) was developed and evaluated for growth of Francisella spp. No differences were observed in recovered colony forming units (CFUs) for F. tularensis , F. novicida and F. philomiragia on CHAB-PACCV vs nonselective CHAB. Growth of non- Francisella species was inhibited on CHAB-PACCV. When environmental samples were cultured on CHAB and CHAB-PACCV, only CHAB-PACCV allowed isolation of Francisella spp. Three new Francisella strains were isolated directly from seawater and seaweed samples by culture on CHAB-PACCV.
Conclusions:  CHAB-PACCV can be used for direct isolation of Francisella spp from environmental samples.
Significance and Impact of the Study:  Francisella spp. show a close association with environmental sources. Future utilization of CHAB-PACCV for isolation of Francisella spp. directly from environmental samples should prove valuable for investigating outbreaks and human infections attributed to environmental exposure.     

A novel method for the determination of electrical potentials across cellular membranes. II. Membrane potentials of acholeplasmas, mycoplasmas, streptococci and erythrocytes

The membrane potentials of Acholeplasma laidlawii, Mycoplasma mycoides subsp. capri, Mycoplasma gallisepticum, Streptococcus faecalis and human erythrocytes have been determined by applying a novel technique. The membrane potentials were calculated simply from potassium concentrations determined by atomic absorption spectroscopy, and gravimetry. The versatility of the new technique is demonstrated by comparing our results with data obtained by different techniques.     

Origins of the mycoplasmas: sterol-nonrequiring mycoplasmas evolved from streptococci.

We report the establishment of a phylogenetic relationship between the sterol-nonrequiring mycoplasmas (Acholeplasma species) and streptococci. Three specific antisera prepared against purified Streptococcus faecalis fructose diphosphate aldolase and glyceraldehyde-3-phosphate dehydrogenase and Pediococcus cerevisiae glyceraldehyde-3-phosphate dehydrogenase were used for comparative enzyme immunological studies; the Ouchterlony double-diffusion technique and the quantitative microcomplement fixation procedure were employed. The reactions obtained provide evidence showing that all seven ACholeplasma species studied (A. laidlawii, A. granularum, A. modicum, A. oculi, A. axanthum. A. hippikon, and A. equifetale) are phylogenetically related to streptococci and that they evolved from streptococci. The data strongly suggest that the acholeplasmas comprise a distinct evolutionary group that has diverged from streptococci belonging to Lancefield group D or N. No reactions were observed between these enzyme antisera and cell extracts from six fermentative Mycoplasma species. These results support the view that mycoplasmas are derived from various bacteria.     

Direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis from meat.

Studies were done to determine the usefulness of dilute alkali (KOH) treatment of meat samples for direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis, without enrichment. Virulent Y. enterocolitica and Y. pseudotuberculosis in pork contaminated with 10(2), 10(3), and 10(4) cells per g survived the direct KOH treatment and were never recovered by using KOH postenrichment treatment. From 6 (4.8%) of 125 samples of retail ground pork, four biotype 4 serotype O3 and one biotype 3B serotype O3 strains of Y. enterocolitica and one Y. pseudotuberculosis serotype 4b strain were recovered by using direct KOH treatment without enrichment. As these isolations were attained without using enrichment cultural procedures, they represent an important time-saving alternative to simplify and speed isolation of Yersinia spp. from meat.     

Distribution and composition of lipopolysaccharides from mycoplasmas.

Polymeric carbohydrates containing glycerol and fatty acids were isolated from whole cells and membranes of mycoplasmas by hot aqueous phenol extraction and gel filtration. Lipopolysaccharides were found to occur in four species of Acholeplasma, two of Anaeroplasma, and in Mycoplasma neurolyticum. None were detected in Spiroplasma citri or in five species of Mycoplasma. All lipopolysaccharides contained both neutral and N-acylated amino sugars in ratios varying from 1:1 to 3:1. The neutral sugars found in varying distribution were glucose, galactose, and mannose. The amino sugars included fucosamine, an unidentified deoxyhexosamine, galactosamine, and glucosamine. Fucosamine and glucose were the only sugars common to all lipopolysaccharides. The fatty acids were similar to those found in the lipids of each organism.     

Direct isolation of Yersinia pseudotuberculosis from fresh water in Japan.

Yersinia pseudotuberculosis was recovered from KOH-treated precipitates of 20.6% of 500 freshwater samples. KOH treatment of precipitates is a simple and expedient means of recovering Y. pseudotuberculosis from such samples.     

Direct isolation of Yersinia pseudotuberculosis from fresh water in Japan.

Yersinia pseudotuberculosis was recovered from KOH-treated precipitates of 20.6% of 500 freshwater samples. KOH treatment of precipitates is a simple and expedient means of recovering Y. pseudotuberculosis from such samples.     

Phase separation, ion permeability, and the isolation of membranes from osmotically stable mycoplasmas

The osmotic stability of M. gallisepticum was found to be a consequence of the synthesis of disaturated phosphatidylcholine incorporated into the cell membrane. The disaturated lipid induces the formation of segregated lipid domains, thus providing the sites for increased permeation of ions. Such permeation reduces the internal pressure so as to minimize cell swelling and subsequent lysis in a hypotonic medium. Purified membranes of M. gallisepticum can be prepared from cells suspended in an iso-osmotic NaCl solution containing either dicyclohexylcarbodiimide (DCCD), which blocks ATPase activity, or a mild alkaline buffer. Both conditions seem to interfere with cell volume regulation. These procedures can be used also to isolate membranes of other osmotically stable mycoplasmas.     

Direct isolation of neuronal microtubule skeletons.

The microtubule skeletons of cultured neuronal cells have been isolated in their original form utilizing taxol and rigorous extraction conditions. Continuous microtubules are visible throughout the cell body and into the processes. These preparations include the complex set of microtubule-associated proteins of 69,000 and 80,000 daltons previously identified in these cells.     

Demonstration of cross-reactive antibodies to mycoplasmas in human sera by ELISA and immunoblotting

Varying levels of cross-reactivity to some mycoplasma species were observed in the sera of patients infected with Mycoplasma pneumoniae and even in normal human sera by enzyme-linked immunosorbent assay (ELISA). The absorption of the patients' sera with M. pneumoniae lysate showed the decrease in ELISA titers not only to M. pneumoniae, but also to other mycoplasma species. These results suggested the existence of cross-reactive antibodies to mycoplasmas in human sera. Cross-reactive antibodies to M. pneumoniae and other mycoplasmas in the patients' sera were also demonstrated by Western blotting technique.     

Direct isolation of tobacco necrosis virus from soils

Summary In TNV-bearing soils, the virus occurred adsorbed to soil colloids in low levels. By direct assay, the TNV could be more readily isolated from the rhizosphere of naturally infected cluster bean plants. The level of reaction of the TNV isolated from the rhizosphere soil was the same as TNV-D (cb isolate) in precipitin ring tests with antisera against TNV-A and TNV-D. The phenomenon of release of TNV from the infected roots into the soil and adsorption of TNV particles to colloidal particles in the soil are discussed from the point of ecology and stability of TNV in soils.     

Direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis from meat

Studies were done to determine the usefulness of dilute alkali (KOH) treatment of meat samples for direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis, without enrichment. Virulent Y. enterocolitica and Y. pseudotuberculosis in pork contaminated with 10(2), 10(3), and 10(4) cells per g survived the direct KOH treatment and were never recovered by using KOH postenrichment treatment. From 6 (4.8%) of 125 samples of retail ground pork, four biotype 4 serotype O3 and one biotype 3B serotype O3 strains of Y. enterocolitica and one Y. pseudotuberculosis serotype 4b strain were recovered by using direct KOH treatment without enrichment. As these isolations were attained without using enrichment cultural procedures, they represent an important time-saving alternative to simplify and speed isolation of Yersinia spp. from meat.     

Selective isolation and analysis of glycoprotein fractions and their glycomes from hepatocellular carcinoma sera

As one of the most important post‐translational modifications, the discovery, isolation, and identification of glycoproteins are becoming increasingly important. In this study, a Con A‐magnetic particle conjugate‐based method was utilized to selectively isolate the glycoproteins and their glycomes from the healthy donor and hepatocellular carcinoma (HCC) case sera. The isolated glycoproteins and their N‐linked glycans were identified by LC‐ESI‐MS/MS and MALDI‐TOF/TOF‐MS, respectively. A total of 93 glycoproteins from the healthy donors and 85 glycoproteins from the HCC cases were identified. There were 34 different glycoproteins shown between the healthy donors (21/34) and the HCC cases (13/34). Twenty‐eight glycans from the healthy donors and 30 glycans from the HCC cases were detected and there were 22 different glycans shown between the healthy donors (10/22) and HCC cases (12/22). Among these glycoproteins, 50 were known to be N‐linked glycoproteins and three novel glycopeptides from two predicted potential glycoproteins were discovered. Moreover, lectin blotting, Western blotting and lectin/glyco‐antibody microarrays were applied to definitely elucidate the change of selective protein expressions and their glycosylation levels, the results indicated that the differences of the identified glycoproteins between the healthy donors and HCC cases were caused by the change of both protein expression and their glycosylation levels.     

Direct detection and isolation of plasmid-bearing virulent serotypes of Yersinia enterocolitica from various foods.

A procedure was developed for direct detection, isolation, and maintenance of plasmid-bearing virulent serotypes of Yersinia enterocolitica from different food sources. Plasmid-bearing virulent strains of Y. enterocolitica representing five serotypes were simultaneously detected and isolated from enriched swab samples of artificially contaminated pork chops, ground pork, cheese, and zucchini, using Congo red binding and low-calcium-response tests. The method was also effective in isolating plasmid-bearing virulent strains of Y. enterocolitica from naturally contaminated porcine tongues. Virulence of the strains isolated from these foods was confirmed by PCR, the expression of plasmid-associated phenotypes, and mouse pathogenicity.