Göran Bölske

Survey of mycoplasma infections in cell cultures and a comparison of detection methods

A total of 1424 cell cultures was assayed for mycoplasmas by microbiological culture and fluorescent DNA staining. Of these cultures, 412 (29%) were infected with mycoplasmas. The most frequently occurring mycoplasma species were Mycoplasma orale (34%), M. hyorhinis (26%), M. arginini (21%), M. fermentans (13%) and Acholeplasma laidlawii (5%). A few isolates each of M. hominis, M. pulmonis and M. bovis were also detected. When detection methods were compared, microbiological culture produced false-negative results for 0.7% (3 of 412) of the infected cell cultures. DNA staining performed directly on the cells was falsely negative in 2.4% (5/207) of the mycoplasma-infected cultures that were compared, DNA staining performed on indicator cells was falsely negative in 3.1% (7/226). False positives appeared in direct DNA-staining in 1.8% (7/386) of the mycoplasma-free cultures and with DNA staining on indicator cells in 0.5% (3/620). For 11% of the cell cultures, the reading of the DNA staining was ambiguous. With DNA staining on indicator cells, 10% of the test results were ambiguous, but by further passage and staining on new indicator cells it was possible to get a definite diagnosis.

In vitro amplification of the 16S rRNA genes from Mycoplasma bovis and Mycoplasma agalactiae by PCR.

Mycoplasma bovis and Mycoplasma agalactiae are two very closely related species which cause mastitis in cows and goats, respectively. M. bovis can also cause arthritis and respiratory disease in cattle. It has recently been shown that the 16S rRNA sequences differ only in 8 nucleotide positions between the two species [J.G. Mattsson, B. Guss and K.-E. Johansson (1994) FEMS Microbiol. Lett., 115: 325-328]. These nucleotide differences are distributed over the molecule in such a way that it is difficult to design specific identification systems, based on PCR only, for M. bovis and M. agalactiae. Two different PCR systems based on 16S rRNA sequence data have, however, been designed for these two species. The forward primers were identical in the two systems and complementary to a segment of the evolutionarily variable region V2. The reverse primers were complementary to the variable region V6, in which there are two nucleotide differences between M. bovis and M. agalactiae. The size of the PCR products, generated with these primers, was 360 bp. Cross-amplification was obtained with the two species in the heterologous PCR systems, but with approximately a 100-fold lower efficiency. Cross-amplification was not obtained with any other bovine or caprine mycoplasma except for Mycoplasma sp. strain A1343 of the caprine group 7. The detection limit of the PCR system for M. bovis with a reference culture was 4 x 10(2) CFU/ml and of the PCR system for M. agalactiae 2 x 10(2) CFU/ml. The M. bovis-PCR system was used to analyze nasal samples of calves from a herd where an outbreak of pneumonia had occured and it proved possible to detect M. bovis in these samples.

Single PCR and nested PCR with a mimic molecule for detection of Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis is the causative agent of Johnes disease in ruminants. The current methods for detection of M. avium subsp. paratuberculosis are slow and insensitive. We report the use of a polymerase chain reaction (PCR) based on IS900 to confirm growth of M. avium subsp. paratuberculosis in primary bacterial cultures from bovine tissue and fecal samples. The use of PCR on single colonies reduced the time for analysis by 2 months compared with conventional methods. We also report the development of a nested PCR based on IS900 and the development of a positive internal control molecule, a so-called mimic. The system was tested with spiked tissue samples, and the sensitivity was estimated to 10 CFU per sample. Seventeen tissue samples, previously found M. avium subsp. paratuberculosis positive by microbiological culture, were analyzed by nested PCR and the efficiency of the PCR was checked by co-amplification of the mimic. Absence of the mimic amplicon indicated inhibition of the amplification. Ten of the samples were positive and five were negative, as judged from the presence or absence of the IS900 PCR product. Two negative samples could not be judged because of inhibition revealed by mimic molecules. It was concluded that the nested PCR, together with the mimic, could be a useful tool in screening tissue materials.

Updated phylogenetic description of the Mycoplasma hominis cluster (Weisburg et al. 1989) based on 16S rDNA sequences

The fastidious nature of the mollicutes (mycoplasmas), their lack of a classic bacterial cell wall, and their very small genome, make phylogenetic placements of new species in this enlarging group of prokaryotes an important and valuable aid in their classification. In this report we have determined the phylogeny of the Mycoplasma hominis cluster of the hominis group. The 16S rDNA sequences from several previously described Mycoplasma species were determined and ten species were found to belong to the M. hominis cluster. With almost complete sequences available, the phylogenetic analysis revealed that the M. hominis cluster currently comprises 19 species, forming a distinct clade as judged from branch lengths, bootstrap percentage values, nucleotide signature analysis, and structural elements in the 16S rRNA molecule. The 16S rRNA gene sequences of species in the M. hominis cluster were found to be > or = 94% similar and the range within which similarities can be used in the classification of new species is discussed. Members of the M. hominis cluster all share a major biochemical property of M. hominis, in that they hydrolyse arginine and are incapable of fermenting glucose. This consistency in phenotypic pattern has not been found in any of the other phylogenetic clusters of the hominis group. Two species, the non-cultivable agent of Grey Lung disease in rodents (tentatively named Candidatus Mycoplasma ravipulmonis) and the avian species Mycoplasma gypis strain B1/T1T, were regarded as close relatives to the M. hominis cluster, but are clearly separated from the species of this cluster. Both species formed early branches of the M. hominis cluster and should be regarded as individual lines containing one species.

New PCR systems to confirm real-time PCR detection of Mycobacterium avium subsp. paratuberculosis

BackgroundJohnes disease, a serious chronic form of enteritis in ruminants, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). As the organism is very slow-growing and fastidious, several PCR-based methods for detection have been developed, based mainly on the MAP-specific gene IS900. However, because this gene is similar to genes in other mycobacteria, there is a need for sensitive and reliable methods to confirm the presence of MAP. As described here, two new real-time PCR systems on the IS900 gene and one on the F57 gene were developed and carefully validated on 267 strains and 56 positive clinical faecal samples.ResultsOur confirmatory PCR systems on IS900 were found sensitive and specific, only yielding weak false positive reactions in one strain for each system. The PCR system on F57 did not elicit any false positives and was only slightly less sensitive than our primary IS900-system. DNA from both naturally infected and spiked faeces that tested positive with our primary system could be confirmed with all new systems, except one low-level infected sample that tested negative with the F57 system.ConclusionWe recommend using the newly constructed DH3 PCR system on the F57 gene as the primary confirmatory test for PCR positives, but should it fail due to its lower sensitivity, the DH1 and DH2 PCR systems should be used.

Novel deoxynucleoside-phosphorylating enzymes in mycoplasmas: evidence for efficient utilization of deoxynucleosides

Mycoplasmas are unable to synthesize purine and pyrimidine bases de novo. Therefore, salvage of existing nucleosides and bases is essential for their survival. Four mycoplasma species were studied with regard to their ability to phosphorylate deoxynucleosides. High levels of thymidine kinase (TK), deoxycytidine kinase (dCK), deoxyguanosine kinase (dGK) and deoxyadenosine kinase (dAK) activities were detected in extracts from Mycoplasma pneumoniae, Mycoplasma mycoides subsp. mycoides SC (M. mymySC), Acholeplasma laidlawii (A. laidlawii) and Mycoplasma arginini (M. arginini). Nucleoside phosphotransferase activities were found at high levels in A. laidlawii and low levels in M. arginini. Pyrophosphate‐dependent deoxynucleoside kinase activities were detected mainly in A. laidlawii and M. mymySC extracts. Two open reading frames were identified in the M. mymySC genome; one showed 25% sequence identity to human dGK and the other one had about 26% sequence identity to human TK1. The M. mymySC dGK‐like enzyme was cloned, expressed in Escherichia coli and affinity‐purified. This enzyme phosphorylated dAdo, dGuo and dCyd, and the highest catalytic rate was with dAdo as substrate. Therefore, we suggest that this enzyme should be named deoxyadenosine kinase. The physiological role of mycoplasma dAK and TK may be to support the unusually large dATP and dTTP pools required for replication of mycoplasma genomes.

The use of monoclonal antibodies in the diagnosis of contagious caprine pleuropneumonia (CCPP)

Contagious caprine pleuropneumonia is a severe disease affecting goats in Eastern Africa and the Middle East, caused by Mycoplasma sp. type F38. Its exact geographical distribution is however not exactly known due to the lack of specificity of the available serological tests and the difficulty in cultivating M. sp. F38. A panel of monoclonal antibodies (mAbs) was produced, using crude or membrane proteins antigens from type F38 strains to immunize mice. The reactivity of the mAbs was tested by an immunobinding assay with crude mycoplasma antigens spotted on nitrocellulose filters. One hundred and twelve antigens, standardized at 0.5 mg protein/ml, were used. Mycoplasma strains were chosen among closely related species of the mycoides cluster, M. capricolum, Group 7 of Leach, M. mycoides mycoides LC, M. mycoides mycoides SC, M. mycoides capri, as well as among species that are isolated from goat lungs, M. arginini, M. ovipneumoniae, M. putrefaciens, M. agalactiae. Out of 60 mAbs, 4 were chosen to build an identification test for mycoplasmas of the mycoides cluster. Controls showed that accurate identification could be hampered by antigenic heterogeneity within the M. capricolum species. One mAb was used for the direct detection of M. sp. F38 antigen in pleural fluid from goats suspected of CCPP. The sensitivity of the test can be estimated at 0.5 micrograms protein/ml. Comparison with isolation results show a 74% agreement between the two methods. The same mAb was used to build a blocking ELISA. This serological test was strictly specific for CCPP. It detects antibodies in sera of naturally infected or artificially immunized animals while it remained negative with hyperimmune sera to related strains such as PG 50. Direct antigen detection and blocking ELISA are tools that may enable a better assessment of CCPP distribution.

Intranasal inoculation of Mycoplasma pulmonis in mice with severe combined immunodeficiency (SCID) causes a wasting disease with grave arthritis

Mycoplasma pulmonis or Myc. pneumoniae were inoculated intranasally to C.B‐17 scid/scid mice (severe combined immunodeficient (SCID) mice). Immunocompetent C.B‐17 mice were inoculated as controls. During the observation period of 5 weeks the mice were killed and necropsied. Mycoplasma pulmonis was recovered from all of the inoculated mice, and dissemination to various tissues increased with time. SCID mice, unlike immunocompetent mice, did not show lung lesions but exhibited severe inflammatory changes of the joints. Mycoplasma pulmonis, however, was isolated both from the lungs and the articular lesions. In addition, SCID mice infected for more than 3 weeks suffered from a pronounced loss of weight and emaciation. In the experiment with Myc. pneumoniae the agent could be reisolated, but lesions were not found in any of the infected mice. Mycoplasma pulmonis infection in SCID mice may be useful as a model of arthritis in immunodeficient humans.

Molecular characterization of thymidine kinase from Ureaplasma urealyticum: nucleoside analogues as potent inhibitors of mycoplasma growth.

Ureaplasma urealyticum (U. urealyticum), belonging to the class Mollicutes, is a human pathogen colonizing the urogenital tract and causes among other things respiratory diseases in premature infants. We have studied the salvage of pyrimidine deoxynucleosides in U. urealyticum and cloned a key salvage enzyme, thymidine kinase (TK) from U. urealyticum. Recombinant Uu‐TK was expressed in E. coli, purified and characterized with regards to substrate specificity and feedback inhibition. Uu‐TK efficiently phosphorylated thymidine (dThd) and deoxyuridine (dUrd) as well as a number of pyrimidine nucleoside analogues. All natural ribonucleoside/deoxyribonucleoside triphosphates, except dTTP, served as phosphate donors, while dTTP was a feedback inhibitor. The level of Uu‐TK activity in U. urealyticum extracts increased upon addition of dUrd to the growth medium. Fluoropyrimidine nucleosides inhibited U. urealyticum and M. pneumoniae growth and this inhibitory effect could be reversed by addition of dThd, dUrd or deoxytetrahydrouridine to the growth medium. Thus, the mechanism of inhibition was most likely the depletion of dTTP, either via a blocked thymidine kinase reaction and/or thymidylate synthesis step and these metabolic reactions should be suitable targets for antimycoplasma chemotherapy.

Humoral immune responses to Mycoplasma hyopneumoniae in sows and offspring following an outbreak of mycoplasmosis

Previously healthy sows, seropositive to Mycoplasma hyopneumoniae, developed clinical signs of mycoplasmosis, as well as increasing amounts of antibodies to M. hyopneumoniae during an outbreak of the disease in a herd. During the early phase of the outbreak, young piglets (2 weeks) with maternal antibodies remained healthy while older seronegative piglets (4-7 weeks) developed the disease. The duration of the maternal antibodies to M. hyopneumoniae varied between litters and was related to the amount of antibodies in the serum of the dam. In sows, the level of serum antibodies decreased continuously from 4 weeks ante partum to partus, and the level of antibodies in the whey of colostrum was comparable to that in serum 4 weeks ante partum. After loss of maternal antibodies to M. hyopneumoniae, seropositive animals were not found among piglets younger than 9 weeks. Therefore peripheral blood mononuclear cells (PBMC) were collected from various age categories of piglets in order to measure the ability to produce antibodies to M. hyopneumoniae in vitro. PBMC obtained from piglets aged 1 and 3 weeks produced few antibodies to M. hyopneumoniae. Significantly higher levels of antibodies to M. hyopneumoniae were produced by PBMC obtained from pigs aged 5-9 weeks. Thus, the ability of PBMC to produce antibodies to M. hyopneumoniae in vitro seemed to be age-dependent.