Co-infection with Mycoplasma haemofelis and ‘Candidatus Mycoplasma haemominutum’ in three cats from Brazil

Abstract

The two most common haemotropic Mycoplasma of cats, Mycoplasma haemofelis and ‘Candidatus Mycoplasma haemominutum’ have been identified using molecular techniques in all continents, except Antarctica. We report the first molecular characterization in South America of a dual infection with M haemofelis and ‘Candidatus Mycoplasma haemominutum’ in three domestic cats. The 16S ribosomal RNA gene was amplified in three anaemic cats in which haemoplasma organisms were seen attached to the erythrocytes in the peripheral blood smear. Bands of the expected size for M haemofelis and ‘Candidatus Mycoplasma haemominutum’ were observed in all three cats. The 393 bp segment of one of the amplicons had a similarity value of 100% to M haemofelis, whereas the other amplicon, a 192 bp segment, was 100% similar to ‘Candidatus Mycoplasma haemominutum’. After diagnosis, two cats received blood transfusion and they were all treated with doxycycline. All three cats recovered uneventfully.

Mycoplasma haemofelis is the causative agent of feline infectious anaemia (FIA). This red cell parasite of cats was originally named, Eperythrozoon felis, and subsequently renamed, Haemobartonella felis (Neimark et al 2001). Its description in domestic cats in Brazil was first reported in 1976 (Massard et al 1976). More recently, sequence analysis has shown that Haemobartonella felis and related haemotrophic bacteria belong to the genus Mycoplasmas (Neimark et al 2001). The trivial name, haemoplasmas was given to these red cell parasites, which form two subclusters within the pneumoniae group of Mycoplasma. Mycoplasma haemofelis, M haemocanis, and M haemomuris have a characteristic truncation of about 10 bp in a segment corresponding to positions 453–481 of the 16S rRNA gene sequence of Escherichia coli (Johansson et al 1999). This deletion is not present in the other subcluster of haemoplasmas that includes ‘Candidatus M haemominutum’, another haemoplasma that infects the cat (Messick et al 2002). ‘Candidatus M haemominutum’ is smaller than M haemofelis and was previously called H felis California (Foley and Pedersen 2001). A third feline haemoplasma, ‘Candidatus Mycoplasma turicensis’ was also recently described in a cat with haemolytic anaemia in Switzerland (Willi et al 2005). Haemoplasmosis caused by M haemofelis causes life-threatening haemolytic anaemia in cats, whereas clinical signs in haemoplasmosis caused by ‘Candidatus M haemominutum’ are minor or absent (Foley and Pedersen 2001). We report the first molecular characterization in South America of a dual infection with M haemofelis and ‘Candidatus Mycoplasma haemominutum’ in three domestic cats.

Three adult domestic longhair cats, two females and one male were presented with lethargy, anorexia, and increased body temperature. One cat had pale mucous membranes, whereas the remaining two were icteric. All cats were anaemic (packed cell volume: 21%, 9%, and 6%) and haemoplasma organisms were seen attached to the erythrocytes in the peripheral blood smear. Blood from the cats was lysed and DNA was extracted using a commercially available kit (Gentra Systems, Minneapolis, USA). Reaction mixtures for amplification were prepared under a hood, which was subsequently irradiated by ultraviolet light. To avoid contamination, a separate set of pipettes and aerosol-guarded tips were used exclusively for the preparation of reaction mixtures. Using a universal primer set, nearly complete 16S ribosomal RNA genes were amplified (Messick et al 1998) and produced a band of the expected size (approximately 1400 bp), which identified a bacterial infection in the blood of all three cats. A fragment of 16S ribosomal RNA gene sequence of M haemofelis (Berent et al 1998) and ‘Candidatus Mycoplasma haemominutum’ (Foley et al 1998, Foley and Pedersen 2001) was amplified by polymerase chain reaction (PCR) using species-specific primers as previously described. Bands of the expected size for M haemofelis (393 bp) and ‘Candidatus Mycoplasma haemominutum’ (192 bp) were observed in all three cats. All products were separated by electrophoresis in a 1% agarose gel containing 5 μg/ml of ethidium bromide, and photographed under ultraviolet light with an Alpha Imager 2200 imaging system (Alpha Innotech, San Leandro, CA, USA). Following gel-purification of the 393 bp and 192 bp fragments of the 16S ribosomal RNA gene of M haemofelis and M haemominutum, respectively, were cloned and sequenced in the sense and antisense directions by use of a dideoxy terminator method as previously described (Messick et al 1998).

Gene sequence analysis (GenBank) for the larger amplicon (393 bp) was identified as M haemofelis. A sequence similarity score of 100% was observed with the Oklahoma isolate of M haemofelis (AF178677). Similarly, the smaller amplicon (192 bp) was identified by sequence analysis as ‘Candidatus Mycoplasma haemominutum’. This fragment showed 100% identity with ‘Candidatus Mycoplasma haemominutum’ strains from California (U88564), Australia (AY150978), South Africa (AY150979), United Kingdom (AY150980), with two mutations when compared to the Israeli isolate (AY150974) and to a small haemoplasma observed in a dog in Brazil (AY297712). This small variation was not unexpected. Despite amplifying a conserved region of the 16S ribosomal RNA gene, haemoplasma isolates from different continents have shown minor variations in these regions (Tasker et al 2003).

After diagnosis, two cats received blood transfusion and they were all treated with doxycycline (2.5–5 mg/kg of body weight, PO, q 12 h, for 21 days). All three cats recovered uneventfully. Our results confirm that the haemotropic mycoplasmal species previously identified morphologically in cats from South America are M haemofelis and ‘Candidatus Mycoplasma haemominutum’. Clinical signs and laboratory abnormalities were similar to the ones observed in other parts of the world. It has been previously shown that the cat flea Ctenocephalides felis is a potential vector for both haemoplasma of cats (Woods et al 2005). This flea is common in cats in Brazil (Pereira and Santos 1998) and may serve as the vector in South America.