Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Is a Superior Diagnostic Tool for the Identification and Differentiation of Mycoplasmas Isolated from Animals.

In veterinary diagnostic laboratories, identification of mycoplasmas is achieved by demanding, cost-intensive, and time-consuming methods that rely on antigenic or genetic identification. Since matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) seems to represent a promising alternative to the currently practiced cumbersome diagnostics, we assessed its applicability for the identification of almost all mycoplasma species isolated from vertebrate animals so far. For generating main spectrum profiles (MSPs), the type strains of 98 Mycoplasma, 11 Acholeplasma, and 5 Ureaplasma species and, in the case of 69 species, 1 to 7 clinical isolates were used. To complete the database, 3 to 7 representatives of 23 undescribed Mycoplasma species isolated from livestock, companion animals, and wildlife were also analyzed. A large in-house library containing 530 MSPs was generated, and the diversity of spectra within a species was assessed by constructing dendrograms based on a similarity matrix. All strains of a given species formed cohesive clusters clearly distinct from all other species. In addition, phylogenetically closely related species also clustered closely but were separated accurately, indicating that the established database was highly robust, reproducible, and reliable. Further validation of the in-house mycoplasma library using 335 independent clinical isolates of 32 mycoplasma species confirmed the robustness of the established database by achieving reliable species identification with log scores of ≥1.80. In summary, MALDI-TOF MS proved to be an excellent method for the identification and differentiation of animal mycoplasmas, combining convenience, ease, speed, precision, and low running costs. Furthermore, this method is a powerful and supportive tool for the taxonomic resolution of animal mycoplasmas.

Identification of a novel Hemoplasma species from pigs in Zhejiang province, China.

Hemoplasmas belong to Mycoplasmataceae (Mollicutes: Mycoplasmatales) and are able to infect a broad range of mammalian species. We investigated prevalence of hemotropic mycoplasma species in pig farms in the region of Zhejiang by a PCR scheme using universal primers targeting 16S rRNA and RNase P RNA gene (rnpB). Representative positive samples from different farms were selected for sequencing of 16S rRNA and the 219bp rnpB gene fragments for phylogenetic analysis. Sequencing analysis of PCR products from first samples identified a novel hemoplasma species present in several pig farms in the region with highest nucleotide identity of 92% to Candidatus Mycoplasma turicensis. A duplex PCR assay was then designed for differential detection of the novel hemoplasma from Mycoplasma parvum/M. suis in field samples. Of 324 blood samples from clinically healthy pigs, 26.5% was positive for this novel hemoplasma species and 50% positive for M. suis/M. parvum, indicating that the novel hemotropic mycoplasma species were of considerably high prevalence in Zhejiang province, China.

The nasal mycoplasmal flora of healthy calves and cows.

The nasal mycoplasmal flora of 270 healthy cows from 27 herds in the Netherlands and 35 healthy calves from 7 of these herds was examined. Various methods for isolating mycoplasmas were compared. The prevalence of the various species was as follows: Ureaplasma diversum in 3 (9%) calves; Mycoplasma dispar in 14 (40%) calves; M. bovis in 1 (3%) calf; M. bovirhinis in 23 (66%) calves and 16 (6%) cows; M. bovoculi in 8 (23%) calves and 53 (20%) cows; M. canis in 1 (3%) calf; M. equirhinis in 2 (1%) cows; M. conjunctivae in 2 (1%) cows; Acholeplasma laidlawii in 1 (3%) calf and 3 (1%) cows; and A. axanthum in 7 (3%) cows. The noses of healthy calves were less frequently colonized by the pathogenic species U. diversum and contained fewer U. diversum and M. dispar organisms than the noses of pneumonic calves. We concluded that the mycoplasmal flora of calves and healthy cows was quite different and also that cows play only a minor role in the epidemiology of pathogenic mycoplasma species of calves in the Netherlands.

Survival of feline mycoplasmas in urine.

The effects of length of incubation and urine osmolality on the survival of feline mycoplasmas and ureaplasmas and representative gram-positive and gram-negative bacteria in synthetic urine which approximated the osmolality of normal cat urine were investigated. Both Escherichia coli and Staphylococcus aureus withstood the effects of increasing osmotic pressure. In the most concentrated urine, significant decreases (P less than 0.001) in CFU were observed for E. coli at exposure times of 30 min and longer. S. aureus was not affected by longer exposure or increased osmotic strength. Both Mycoplasma felis and Mycoplasma gateae were affected adversely by longer exposure times and high osmotic strength (P less than 0.001). A Ureaplasma sp. was not adversely affected except at very high (greater than or equal to 2,980 mosM) osmotic strengths or after prolonged incubation (120 min) at relatively high (1,976 mosM) osmotic strengths (P less than 0.001). The failure of both M. felis and M. gateae to survive under osmotic conditions present in normal feline urine suggests that it is unlikely that these mycoplasmas are involved in urinary disorders in cats.

[Contamination of fish tissue cell cultures by Mycoplasma]. / Mikoplazmennaia kontaminatsiia kul'tur kletok iz tkanei ryb.

The inoculated and primary cell cultures of fish (carp, salmon, and sturgeon) have been studied. Acholeplasma typed as A. laidlawii in terms of its biochemical properties shown in inhibited metabolism has been isolated from 19 samples. The authors consider the source of Acholeplasma-induced contamination of the cell cultures under study.

Mycoplasmas and acholeplasmas isolated from ducks and their possible association with pasteurellas.

Two hundred and sixty-three cases of clinically diseased ducks of all ages were examined for the presence of mycoplasmas. Mycoplasmas and acholeplasmas belonging to more than eight serogroups were cultured from 68 of them, and comprised 12 M anatis, one M columbinasale, two M gallinaceum, two M gallinarum, nine M synoviae, three unidentified Mycoplasma species, 37 Acholeplasma laidlawii and one unclassified acholeplasma belonging to each of serogroups 7 and 8. They were identified by biochemical characterisation, disc growth inhibition and agar gel diffusion tests. Fifty-three (78 per cent) of the isolates occurred with species of Pasteurella: 33.8 per cent with Pasteurella anatipestifer, 32.4 per cent with P multocida and 11.8 per cent with both P anatipestifer and P multocida. Nine of the isolates (13.2 per cent) were in pure culture and six (8.8 per cent) with other agents. Of the ducks negative for mycoplasmas 33.3 per cent were infected with P anatipestifer, 25.1 per cent with P multocida and 14.4 per cent with both P anatipestifer and P multocida. There was no correlation between the infections with mycoplasmas and P anatipestifer but there was a weak association between the infections with mycoplasmas, especially M anatis and P multocida.

Serological survey of the Iriomote cat (Felis iriomotensis) in Japan.

The Iriomote cat (Felis iriomotensis) was first discovered on Iriomote Island in the Yaeyama Islands of Japan in 1965. Ten male and 11 female adult cats were captured during the 6 yr period from 1983 to 1988. These were examined for evidence of viral and mycoplasmal infections. Neither Mycoplasma sp. nor Ureaplasma sp. were detected in swab samples of oropharyngeal and urogenital regions. A foamy virus was isolated from the oropharyngeal swab of a female cat examined in 1988. Feline leukemia virus was not detected in any of the cats. All cats were negative for serum antibodies to feline panleukopenia virus, feline herpesvirus, feline immunodeficiency virus and rotavirus. Eleven of 19 (58%), 14 of 17 (82%) and 6 of 17 cats (35%) had serum antibodies against feline calicivirus, coronavirus and feline syncytium forming virus, respectively.

Urethral infection of male monkeys by Mycoplasma hominis and Ureaplasma urealyticum.

Seven male Macaca nemestrina, eight male Macaca mulatta and three male Papio hamadrias were infected with M. hominis or U. urealyticum separately or simultaneously into the urethra. Animals of the first two species exhibited moderate infection and mild clinical urethritis characterized by increased number of PMNL in the urethral discharge and colonization and lymphocytic infiltration of the mucous membrane of the urethra and urinary bladder. Animals of the third species proved to be less sensitive.

Genomic heterogeneity of canine ureaplasmas revealed by restriction endonuclease analysis.

Genomic DNAs of ureaplasma strains isolated from dogs were analyzed by restriction endonucleases. The cleavage patterns revealed by agarose gel electrophoresis, which were characteristic for each strain examined, may provide a direct indication for the genomic index among ureaplasma strains, and can be used as an epidemiological marker in ureaplasma infections.

Biochemical and serological study of two mycoplasma strains isolated from geese.

2 mycoplasma strains were isolated, one from the phallic lymph of a gander and the other from a cloacal swab of a laying goose. The strains proved to be different from mycoplasma species isolated from geese before. Strain No. 1223 is a glucose-negative and arginine-negative species belonging to the genus Mycoplasma. In the growth inhibition test, it fails to react with hyperimmune sera raised in rabbits against the presently known mycoplasma species of avian origin nor with sera produced against mammalian mycoplasma species sharing its biochemical properties. Strain No. 1225 belongs to the digitonin resistant Acholeplasmataceae family. It is glucose-positive and aesculin-positive. It is negative by all the other tests and fails to react with sera produced against the presently known acholeplasma species.

Affinity of microorganisms of the genus ureaplasma to the reproductive organs of cattle.

The purpose of this work was to define more precisely the role of Ureaplasma organisms in the aetiology of granular vulvovaginitis and balanoposthitis (GVVBP) of cattle. To contribute to this question the frequency and degree of infection with Ureaplasmas in two main groups of cattle was taken into account: (a) in cattle with symptoms of the mentioned disease, (b) in cattle without clinical symptoms. The samples of semen from 301 sires with symptoms of GVVBP and from 43 healthy sires as also vaginal mucus swabs from 96 cows with GVVBP and from 40 cows mated by the sire infected with Ureaplasma organisms and from 50 cows inseminated with semen which contained Ureaplasma organisms were taken for bacteriological examinations. The control group in relation to the above mentioned cows constituted of 22 heifers free from symptoms of GVVBP and neither inseminated nor mated naturally. It has been shown that on an average 78.1% of sires with pathological changes in the mucosa of the penis or prepuce and only 25.6% of healthy sires were infected with Ureaplasma organisms. The concentration of Ureaplasma organisms was also significantly higher in material obtained from sires with symptoms of the disease than in that from healthy animals. Ureaplasma organisms were demonstrated more frequently (72.7%) in cows with GVVBP than in cows without these symptoms (13.3%). Similarly, as in the material obtained from sires, in the material taken from cows with symptoms of the disease the concentration of Ureaplasma organisms was significantly higher than that in the material originating from the healthy cows. The obtained findings may indicate that Ureaplasma organisms play a role in the aetiology of GVVBP.

The effects of two Ureaplasma diversum strains on early pregnancy in heifers.

Two field isolates of Ureaplasma diversum spp. were used to infect heifers at the time of insemination in a preliminary study to observe the effect of infection on early pregnancy. M84-14c-1 was a field isolate from a bull's prepuce typed by immunofluorescence to be similar to U. diversum strain T-44 (Group C). M84-477c-4 was a field isolate from bovine semen typed by immunofluorescence to be similar to U. diversum strain T-288 (Group A). All three heifers infected with M84-477c-4 had a mild granular vulvitis at some time during the trial. None was pregnant when slaughtered 27 days after infection. The result of infection with M84-14c-1, a preputial isolate, was not consistent. One heifer had no infection and a normal pregnancy, one heifer was infected with an abnormal pregnancy, and one heifer was open but ureaplasmas were not detected until day 17 of the trial.

The effect of intrauterine inoculation with Ureaplasma diversum on bovine fertility.

To determine the influence of Ureaplasma diversum on bovine fertility 11 uninfected virgin heifers with normal ovarian cyclic activity were randomly allocated to test or control groups. At a synchronized estrus, five test heifers were given an intrauterine broth inoculum containing 1.09 x 10(8) to 1.4 x 10(9) colony forming units of U. diversum and six control animals were infused with sterile ureaplasma broth medium. All animals were artificially inseminated within one hour of infusion. Pregnancy was diagnosed in one of five test heifers and all of six controls by serum progesterone concentrations measured to 25 days postinsemination. The difference in pregnancy rates between the two groups was statistically significant (p = 0.0152). It was concluded that under the conditions of this experiment U. diversum is capable of causing infertility in cattle.

Isolation of mycoplasmas from bovine semen in Northern Ireland.

In a survey of 332 fresh and 137 processed bovine semen samples and 25 preputial washes, mycoplasmas and, or, ureaplasmas were isolated from 46 per cent, 31 per cent and 80 per cent, respectively. Intermittent isolation from different semen collections from the same bull indicated that at least three collections per bull were necessary to determine whether infection was present. When stored processed samples were examined Mycoplasma canadense and M bovigenitalium were isolated from straws taken as long ago as 1975. Addition of lincomycin and spectinomycin to the semen extender eliminated the isolation of mycoplasmas and reduced the rate of isolation of ureaplasmas.

Pathology of experimental ureaplasma mastitis in ewes.

One mammary gland of each of nine primum partum suckling ewes was inoculated with ureaplasma 8 weeks post-partum. Infected glands were swollen, hot, and painful from 16 hours post-infection. Subsequently there was agalactia and reduction in gland size. Histopathologic and ultrastructural examination indicated that the acute phase of the induced mastitis was characterized by necrosis of secretory epithelium with a neutrophil, eosinophil, and macrophage response. Subsequently there was lymphoid infiltration, acinar involution, and fibrosis. This report confirms the pathogenicity of ureaplasma for the ovine mammary gland, and is the first description of the pathologic features of ureaplasmal mastitis in sheep.