Kaoru Koshimizu

Detection of Mycoplasma hyopneumoniae DNA by the polymerase chain reaction.

DNA amplification by the polymerase chain reaction (PCR) was examined to detect DNA of Mycoplasma hyopneumoniae, an etiological agent of porcine pneumonia. A pair of synthetic primers was selected that specify the amplification of a 520-basepair DNA fragment in a reiterative sequence of M. hyopneumoniae genome. The PCR product was detected by direct gel electrophoresis or by blot hybridization to a synthetic oligonucleotide probe. The specificity of PCR for M. hyopneumoniae was confirmed by lack of cross-reactivity to DNA from other porcine mycoplasmas.

The Polymerase Chain Reaction for Mycoplasma pulmonis

in vitro DNA amplification by polymerase chain reaction was examined to detect Mycoplasma pulmonis. A pair of synthetic oligonucleotide primers was constructed, and used to amplify a unique sequence of M. pulmonis DNA. Amplified products were detected by agarose gel electrophoresis and verified by blot hybridization with a synthetic oligonucleotide probe. This system detected cellular DNA of M. pulmonis but not M. arthritidis or M. neurolyticum, and thus appears to be useful for M. pulmonis diagnosis.

Ureaplasma gallorale sp. nov. from the Oropharynx of Chickens

Seven ureaplasma strains isolated from the oropharynx of chickens (Gallus gallus var. domesticus) were characterized and compared with the two previously described species of this genus, Ureaplasma urealyticum from humans and Ureaplasma diversum from cattle. The avian isolates were found to have similar biological, biochemical, serological, and genomic properties and were readily distinguished from the two previously described species. These avian ureaplasma strains have been reported to hydrolyze urea, but not to catabolize arginine or glucose; they lack a cell wall, pass through a 450-nm membrane filter, require cholesterol for growth, and form minute colonies (15 to 60 μm) on agar medium. The seven avian strains constitute a homogeneous group based on their antigenic properties as determined by the metabolism and growth inhibition tests and by protein composition analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On the basis of these findings, we propose that ureaplasmas with these characteristics belong to a new species, Ureaplasma gallorale, with strain D6-1 (= ATCC 43346 = NCTC 11707) as the type strain.

Ureaplasma felinum sp. nov. and Ureaplasma cati sp. nov. isolated from the oral cavities of cats.

Seven ureaplasma strains isolated from the oral cavities of domestic cats (Felis domestica) were characterized and compared with the type strains of the three previously established species of this genus, Ureaplasma urealyticum (humans), Ureaplasma diversum (cattle), and Ureaplasma gallorale (chickens). The feline strains hydrolyzed urea but not arginine or glucose, were membrane bound, lacked cell walls, passed through 0.45-micron membrane filters, required cholesterol for growth, and formed minute (15- to 140-microns) colonies on agar medium. The seven feline strains fell into two distinct groups based on (i) their antigenic properties (determined by using the metabolism and growth inhibition and indirect immunoperoxidase procedures), (ii) their genomic properties (determined by using DNA-DNA hybridization and DNA cleavage pattern procedures), and (iii) their polypeptide profiles (determined by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses). Based on these properties, the two feline groups were unrelated to each other or to the three previously established species, and each group represents a distinct Ureaplasma species. Thus, we propose that ureaplasmas with these phylogenetic and genomic properties be given taxonomic status as Ureaplasma felinum and Ureaplasma cati, with strain FT2-B (= ATCC 49229 = NCTC 11709) and strain F2 (= ATCC 49228 = NCTC 11710) as the type strains, respectively.

A Reliable and Sensitive Method for Detecting Mycoplasmas in Cell Cultures

Mycoplasma contamination of cell cultures has been a serious problem in biomedical research. The incidence of mycoplasma contamination is higher than might be expected, and it is difficult to eliminate mycoplasmas from contaminated cell cultures (1, 12). Several techniques for the detection of mycoplasma contamination of cell cultures have been described (2). They are the direct culture procedure

DNA relatedness among established Ureaplasma species and unidentified feline and canine serogroups.

The levels of DNA relatedness among two unclassified feline ureaplasma serogroups, four unclassified canine ureaplasma serogroups, and the three previously established Ureaplasma species were examined and compared. The strains examined included five feline strains representing two feline serogroups, four canine strains representing four canine serogroups, and the type strains of the three established species. Each strain representing each species or serogroup exhibited 78% or more actual DNA homology with its homologous DNA, but less than 10% DNA homology with DNAs from the heterologous strains. These findings indicate that each of these human, bovine, avian, feline, and canine strains is genomically distinct. In addition, the three previously recognized species (Ureaplasma urealyticum [human], Ureaplasma diversum [bovine], and Ureaplasma gallorale [avian]), which were established on the basis of phenotypic properties, were also shown to be genomically distinct. The three feline serogroup SI strains were genomically related (from 89 to 100% DNA homology) to each other but were unrelated (less than 10% DNA homology) to the feline serogroup SII strains, indicating that these two feline serogroups are also genomically distinct. Conversely, the two feline serogroup SII strains were genomically very similar (from 83 to 100% DNA homology) to each other but were unrelated (less than 10% DNA homology) to the three feline serogroup SI strains. However, canine serogroup SI strain D1M-C exhibited 73% DNA homology with serologically distinct canine serogroup SII strain D29M, indicating that these strains representing two separate serogroups belong to the same genomic species.(ABSTRACT TRUNCATED AT 250 WORDS)

Ureaplasma canigenitalium sp. nov., Isolated from Dogs

Ureaplasma strains isolated from dogs (Canis familiaris) were characterized and compared with the type strains of five previously described species of the genus Ureaplasma, Ureaplasma urealyticum (isolated from humans), Ureaplasma diversum (isolated from cattle), Ureaplasma gallorale (isolated from chickens), Ureaplasma cati (isolated from cats), and Ureaplasma felinum (isolated from cats). The canine strains hydrolyzed urea but not arginine or glucose, were membrane bound, lacked a cell wall, passed through 450-nm-pore-size membrane filters, required cholesterol for growth, and formed minute colonies (diameter, 20 to 140 microns) on agar medium. These canine ureaplasma strains have been reported to be members of four serovars. The four serovars of canine strains fell into a single group on the basis of their genomic properties, as determined by DNA-DNA hybridization. On the basis of these findings, we propose that ureaplasmas with these characteristics belong to a new species, Ureaplasma canigenitalium, with strain D6P-C (= ATCC 51252) as the type strain.

Characterization of a Strain of Mycoplasma hominis Lacking 120 kDa Membrane Protein Isolated from Vero Cell Culture

A strain of Mycoplasma hominis lacking a major membrane protein of 120 kDa was isolated from a Vero cell culture. This strain showed very slow growth rate and formed nipple‐less colonies on agar medium.

A Non‐Radioactive DNA Probe for the Detection of Mycoplasma pulmonis in Murine Mycoplasmosis

A non‐radioactive DNA probe for the detection of Mycoplasma pulmonis was developed by using photobiotin. The probe detected 0.3 ng chromosomal DNA of M. pulmonis. No cross‐hybridization was observed between the probe and the other murine mycoplasma species, M. arthritidis and M. neurolyticum.

A proposed life cycle model of Spiroplasma mirum based on scanning electron microscopical observations of growth in liquid culture.

Cells of Spiroplasma mirum strain SMCA were grown in PPG broth and examined by scanning electron microscopy. Comparison of the results at different time intervals allowed for a model of the life cycle of S. mirum to be proposed. Under favorable growth conditions, helical filament formation was initially observed, followed by the formation of small spherical structures originating from each filament. In old culture, large spherical bodies appeared from entangled helical filaments. From the larger spherical bodies, granular bodies representing the smallest reproductive units were produced to continue the life cycle.