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.

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 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.

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.

BackgroundThis study is a comparative epigenetic evaluation of the methylation status of the DLC1 tumor suppressor gene in naturally-occurring canine lymphoma. Canine non-Hodgkin's lymphoma (NHL) has been proposed to be a relevant preclinical model that occurs spontaneously and may share causative factors with human NHL due to a shared home environment. The canine DLC1 mRNA sequence was derived from normal tissue. Using lymphoid samples from 21 dogs with NHL and 7 normal dogs, the methylation status of the promoter CpG island of the gene was defined for each sample using combined bisulfite restriction analysis (COBRA), methylation-specific PCR (MSP), and bisulfite sequencing methods. Relative gene expression was determined using real-time PCR.ResultsThe mRNA sequence of canine DLC1 is highly similar to the human orthologue and contains all protein functional groups, with 97% or greater similarity in functional regions. Hypermethylation of the 5' and 3' flanking regions of the promoter was statistically significantly associated with the NHL phenotype, but was not associated with silencing of expression or differences in survival.ConclusionThe canine DLC1 is constructed highly similarly to the human gene, which has been shown to be an important tumor suppressor in many forms of cancer. As in human NHL, the promoter CpG island of DLC1 in canine NHL samples is abnormally hypermethylated, relative to normal lymphoid tissue. This study confirms that hypermethylation occurs in canine cancers, further supporting the use of companion dogs as comparative models of disease for evaluation of carcinogenesis, biomarker diagnosis, and therapy.

A 27 kg, 6-year-old, male castrated German shorthaired pointer presented to the University of Missouri, Veterinary Teaching Hospital with the complaint of progressive exophthalmia of 2 years duration optical density (OD). Lack of retropulsion OD was noted on physical examination. Anterior segment examination OU and fundic examination OS did not reveal any abnormalities. Examination of the fundus OD revealed focal scleral indentation of the inferior nasal globe. The indentation changed location with globe movement OD. MRI and CT scan revealed a well-circumscribed, approximately 2 cm in diameter mass located caudal and ventral to the affected globe that appeared to communicate with the nictitating membrane with absence of any bony involvement. A modified lateral orbitotomy was recommended and performed to remove the orbital mass and nictitating membrane en-bloc. Histopathology and immunohistochemistry of the mass confirmed a diagnosis of nodular granulomatous episcleritis (NGE). Postoperatively, the dog developed absolute keratoconjunctivitis sicca (KCS). Examples of primary episcleral inflammation in the dog include diffuse episcleritis, NGE, nodular fasciitis, fibrous histiocytoma, proliferative conjunctivitis/keratoconjunctivitis, pseudotumor, and Collie granuloma. The etiology of these episcleral inflammations is presumed to be immune mediated. To our knowledge, this is the first report of NGE affecting the orbital region of a dog. Development of absolute KCS resulting from excision of the nictitating membrane is also supported by this case.

Haemobartonella felis is an epierythrocytic bacterium suspected to be the causative agent of feline infectious anemia. Previous studies with a polymerase chain reaction assay have identified a mycoplasmal 16S rRNA gene sequence that coincides with clinical disease and the presence of organisms in the blood. Tissues from a cat experimentally infected with H. felis were used for in situ hybridization studies to physically link this 16S rRNA gene to the organisms on the red cells. A biotin-labeled probe was used in conjunction with tyramide signal amplification to visualize the hybridization signal. This study clearly demonstrates a specific hybridization signal on the red cells in the tissues of the H. felis-infected cat. This in situ hybridization study is the final step in fulfilling the molecular guidelines for disease causation and proves that H. felis, a mycoplasmal organism, is the causative agent of feline infectious anemia.