Genetic diversity and evolution of Mycoplasma capricolum subsp. capripneumoniae strains from eastern Africa assessed by 16S rDNA sequence analysis.

Mycoplasma capricolum subsp. capripneumoniae (M. capripneumoniae), the causal agent of contagious caprine pleuropneumonia (CCPP), is a member of the so-called Mycoplasma mycoides cluster. These mycoplasmas have two rRNA operons in which intraspecific variations have been demonstrated. The sequences of the 16S rRNA genes of both operons from 13 field strains of M. capripneumoniae from three neighbouring African countries (Kenya, Ethiopia, and Tanzania) were determined. Four new and unique polymorphism patterns reflecting the intraspecific variations were found. Two of these patterns included length differences between the rrnA and rrnB operons. The length difference in one of the patterns was caused by a two-nucleotide insert (TG) in the rrnB operon and the length difference in the other pattern was due to a three-nucleotide deletion, also in the rrnB operon. Another pattern was characterised by a polymorphic position caused by a mutation that is known to cause streptomycin resistance in other bacterial species. The strain with this pattern was also found to be resistant to streptomycin. Streptomycin resistant clones were selected from four M. capripneumoniae strains to further investigate the correlation of this mutation to streptomycin resistance. Mutations in the 16S rRNA genes had occurred in two of these strains. The fourth pattern included a new polymorphism in position 1059. The results show that polymorphisms in M. capripneumoniae strains can be used as epidemiological markers for CCPP in smaller geographical areas and to study the molecular evolution of this species.

Detection of granulocytic Ehrlichia species DNA by PCR in persistently infected dogs.

Three female beagle dogs inoculated with granulocytic Ehrlichia species were monitored for four to six months to determine whether there was evidence that the organisms persisted. The dogs were inoculated intravenously with blood containing an Ehrlichia species closely related to Ehrlichia equi and Ehrlichia phagocytophila, and identical to the human granulocytic ehrlichiosis agent with respect to its 16S rRNA gene sequence. The clinical signs were evaluated, and blood samples were collected for haematology, serum biochemistry and serology. Ehrlichial inclusions in the blood were monitored by microscopy, and ehrlichial DNA was detected by the polymerase chain reaction (PCR). Two of the dogs were injected with prednisolone on days 54 to 56 and days 152 to 154 after infection, and the other was injected with prednisolone on days 95 to 97 after infection. The dogs were euthanased and examined postmortem. Ehrlichial inclusions were demonstrated in the neutrophils and seroconversion occurred shortly after inoculation. Two of the dogs developed acute disease with rectal temperatures above 39.0 degrees C, after which no further clinical signs were observed. The administration of corticosteroids seemed to facilitate the detection of ehrlichial inclusions. Ehrlichial DNA was detected intermittently by PCR in blood samples from two of the dogs throughout the study. Persistent infection was demonstrated up to five-and-a-half months after inoculation.

Genetic basis of macrolide and lincosamide resistance in Brachyspira (Serpulina) hyodysenteriae.

Macrolide antibiotic resistance is widespread among Brachyspira hyodysenteriae (formerly Serpulina hyodysenteriae) isolates. The genetic basis of macrolide and lincosamide resistance in B. hyodysenteriae was elucidated. Resistance to tylosin, erythromycin and clindamycin in B. hyodysenteriae was associated with an A-->T transversion mutation in the nucleotide position homologous with position 2058 of the Escherichia coli 23S rRNA gene. The nucleotide sequences of the peptidyl transferase region of the 23S rDNA from seven macrolide and lincosamide resistant and seven susceptible strains of Brachyspira spp. were determined. None of the susceptible strains were mutated whereas all the resistant strains had a mutation in position 2058. Susceptible strains became resistant in vitro after subculturing on agar containing 4 micrograms ml-1 of tylosin. Sequencing of these strains revealed an A-->G transition mutation in position 2058.

Sequences of the 16S rRNA genes and phylogeny of the goat mycoplasmas Mycoplasma adleri, Mycoplasma auris, Mycoplasma cottewii and Mycoplasma yeatsii.

The nucleotide sequences of the 16S rRNA genes from the type strains of four goat mycoplasmas, Mycoplasma adleri, Mycoplasma auris, Mycoplasma cottewii and Mycoplasma yeatsii, were determined by direct solid-phase DNA sequencing. Polymorphisms were found in two of the 16S rRNA gene sequences, showing the existence of two different rRNA operons. Three polymorphisms were found in M. adleri, and one was found in M. yeatsii. The sequence information was used for the construction of phylogenetic trees. M. adleri was included in the Mycoplasma lipophilum cluster within the hominis group. M. auris was comprised in the Mycoplasma hominis cluster of the hominis group. M. cottewii and M. yeatsii were found to be very closely related with only four nucleotide differences, and they grouped with Mycoplasma putrefaciens in the Mycoplasma mycoides cluster within the spiroplasma group. Sequencing of two field isolates of M. cottewii and M. yeatsii, geographically distant from the type strains, showed that the 16S rRNA gene from the field isolate of M. cottewii was identical to the one from the type strain. The field isolate of M. yeatsii had only two nucleotide differences to the type strain and these were present in only one of the two rRNA operons. Sequencing of the 16S rRNA genes from two unidentified mycoplasma isolates from Nepal indicated that they should both be regarded as M. auris strains.

Detection of HGE agent-like Ehrlichia in Ixodes ricinus ticks in northern Italy by PCR.

Little is known about the distribution of Human Granulocytic Ehrlichiosis (HGE) in Europe and even less is known in Italy, where no case of clinically documented HGE has been reported. In a previous study we reported the presence of Ehrlichia DNA in Ixodes ricinus ticks from Central Italy. By the use of an Ehrlichia-specific PCR we found that 24% of the ticks were positive. Furthermore, we demonstrated a simultaneous coinfection of the same tick by both, Borrelia burgdorferi and Ehrlichia phagocytophila. Since the Friuli-Venezia Giulia region (North-east of Italy) is endemic for Lyme borreliosis (LB) and the geographic distribution of HGE usually overlaps that of LB, we decided to carry out a survey concerning the presence of Ehrlichia spp. in a recreational area near Trieste where the presence of Lyme borreliosis and Borrelia burgdorferi sensu lato in ticks is well demonstrated. Ticks were analyzed in pools (because a low infection rate was expected): eleven samples out of 93 were found positive by Ehrlichia-PCR. Subsequent sequence analysis of some of the positive PCR products revealed a high homology with the HGE agent Ehrlichia (only one base substitution in almost 450 bp sequenced). These findings add new and interesting data on the Ehrlichia epidemiology in Italy. By now we have demonstrated the presence of two distinct granulocytic ehrlichiae in Italiyn ticks by the aid of a PCR-based analysis: Ehrlichia phagocytophila in Central Italy and an HGE-like Ehrlichia in the north-eastern Italy, in a region close to Slovenia where the first reported case of HGE in Europe occurred.