Physical Map and Genome Sequencing Survey of Mycoplasma haemofelis ( Haemobartonella felis )

Abstract

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...

Human Coinfection with Bartonella henselae and Two Hemotropic Mycoplasma Variants Resembling Mycoplasma ovis

“…The only hemotropic mycoplasma species for which 16S rRNA gene copy number has been ascertained is Mycoplasma haemofelis, which has a single copy of the 16S rRNA gene (2). Furthermore, a similar large deletion in the 16S rRNA gene helps discriminate Mycoplasma haemofelis from "Candidatus Mycoplasma haemominutum.…”

Identification, Bioinformatics Analyses, and Expression of Immunoreactive Antigens of Mycoplasma haemofelis

“…Within the 21 reactive inserts, a total of 60 putative proteins were identified. Sequence analysis showed that 26/60 discovered proteins matched with M. haemofelis sequences deposited in the GSS database (5). Further, all 60 of these genes were found in the genomic sequence of M. haemofelis that was recently completed by our laboratory (27).…”

Haemotrophic mycoplasma: review of aetiology and prevalence

“…Three years later, the same group constructed the physical map of another haemotrophic mycoplasma species (Mycoplasma haemofelis) which was confirmed as comprising 1.2 Megabases, almost twice as large as E. suis. A total of 280 kb were sequenced and of these, 18.6% were homologous with Mycoplasma and 74% failed to yield a significant match, suggesting that the M. haemofelis genome encoded a large number of unique proteins [12]. The partially sequenced genome could provide information for comparative genomic and functional studies of other haemoplasma species.…”