The 16S rRNA sequence of newly characterized haemotrophic bacteria in an opossum (Didelphis virginiana) and alpaca (Lama pacos) was determined. In addition, the 16S rRNA sequence of a haemotrophic parasite in the dog (Canis familiaris) was determined. Sequence alignment and evolutionary analysis as well as secondary structural similarity and signature nucleotide sequence motifs of their 16S rRNA genes, positioned these organisms in the genus Mycoplasma. The highest scoring sequence similarities were 16S rRNA genes from haemotrophic mycoplasma species (Haemobartonella and Eperythrozoon spp.). However, the lack of several higher-order structural idiosyncrasies used to define the pneumoniae group, suggests that these organisms and related haemotrophic mycoplasmas represent a new group of mycoplasmas. It is recommended that the organisms be named 'Candidatus Mycoplasma haemodidelphidis', 'Candidatus Mycoplasma haemolamae' and Mycoplasma haemocanis comb. nov., to provide some indication of the target cell and host species of these parasites, and to reflect their phylogenetic affiliation.

The 16S rRNA gene of Haemobartonella felis was amplified by using universal eubacterial primers and was subsequently cloned and sequenced. Based on this sequence data, we designed a set ofH. felis-specific primers. These primers selectively amplified a 1,316-bp DNA fragment of the 16S rRNA gene of H. felis from each of four experimentally infected cats at peak parasitemia. No PCR product was amplified from purified DNA ofEperythrozoon suis, Mycoplasma genitalium, andBartonella bacilliformis. Blood from the experimental cats prior to infection was negative for PCR products and was greatly diminished or absent 1 month after doxycycline treatment. The overall sequence identity of this fragment varied by less than 1.0% among experimentally infected cats. By taking into consideration the secondary structure of the 16S rRNA molecule, we were able to further verify the alignment of nucleotides and quality of our sequence data. In this PCR assay, the minimum detectable number of H. felis organisms was determined to be between 50 and 704. The potential usefulness of restriction enzymes DdeI andMnlI for distinguishing H. felis from closely related bacteria was examined. This is the first report of the utility of PCR-facilitated diagnosis and discrimination of H. felisinfection in cats.

The dogs receiving (177)Lu-DOTMP tolerated the administration and the effects of the compound without apparent clinical toxicity. The results of this experiment support the further evaluation in tumor-bearing dogs of (177)Lu-DOTMP as a potential therapy for metastatic bone cancer and primary bone tumors in humans and dogs.

Mycoplasma haemofelis is an uncultivable red-cell pathogen of cats. Isolated M. haemofelis DNA was used to create a bacterial artificial chromosome library and physical map. Random sequencing of this material revealed 75 genes that had not been previously reported for M. haemofelis or any other hemotrophic mycoplasma.Mycoplasma haemofelis is a member of a newly defined group of mycoplasmas that parasitize the red blood cells of animals and humans. M. haemofelis, which was formerly known as Haemobartonella felis strain OH (18), was chosen for use in these molecular studies because it is the highly pathogenic causative agent of the syndrome historically reported as feline infectious anemia (8,11). This syndrome is often associated with feline retroviral infections and may serve as a model for hemotrophic mycoplasmal infection in immunosuppressed human patients (13; M. I. Duarte, M. S. Oliveira, M. A. ShikanaiYasuda, O. N. Mariano, C. F. Takakura, C. Pagliari, and C. E. Corbett, Letter, J. Infect. Dis. 165:976-977, 1992). This paper describes the genome size of M. haemofelis, the creation of the first genome encyclopedia and physical map of an uncultured mycoplasma, and the collection of M. haemofelis genome survey sequences (GSSs) for the purpose of gene discovery.To acquire large quantities of M. haemofelis DNA for the following experiments, a feline leukemia virus-and feline immunodeficiency virus-seronegative cat was splenectomized and infected with M. haemofelis. When more than 60% of the red cells contained organisms, blood was aseptically drawn from the jugular vein into a syringe containing 1 ml of anticoagulantcitrate-dextrose solution per 5 ml of blood. The organisms were released from the red cells and embedded in agarose plugs, and the DNA was extracted by using previously described protocols (1,19). Related organisms Mycoplasma genitalium (ATCC 49895) and Mycoplasma haemosuis (15), as well as a commercial DNA marker, were used as size controls in subsequent experiments.Gamma radiation was used to linearize the bacterial DNA prior to pulsed-field gel electrophoresis (PFGE) to determine the full genome length of M. haemofelis (20,31). A 1.0% 0.5ϫ Tris-borate-EDTA gel was run for 24 h at 14°C and 6 V/cm, with 60-to 120-s switch times and a field angle of 120°. This PFGE was repeated four times with M. haemofelis plugs from separate blood collections. To confirm the identify of the bands seen, the Southern blotted DNA was probed by using a 393-bp digoxigenin-labeled fragment of the M. haemofelis 16S rRNA gene which spanned hypervariable regions 1 to 3 (1, 17). Membranes were prehybridized in 10 to 20 ml of PerfectHyb Plus (Sigma-Aldrich Corp., St. Louis, Mo.) at 68°C for at least 5 min. Labeled probe was boiled for 10 min and added to the prehybridization solution, and the probe was allowed to hybridize overnight at 68°C. Stringency washes and signal detection with CDP-star were performed according to the manufacturer's instructions (Boehringer Mannheim Biochemicals [Roche Molecular Biochemicals], Indianap...

The variability in cytologic findings and frequent presence of dysplastic spindloid cells suggest that cytology alone may not be a reliable tool to differentiate degenerate canine disk material from a mesenchymal neoplasm.