Presence of Mycoplasma haemofelis, Mycoplasma haemominutum and piroplasmids in cats from southern Europe: a molecular study

Abstract

Clinical symptoms produced by Mycoplasma spp. and piroplasmids in cats are sometimes similar. Diagnosis of these pathogens is difficult by microscopic procedures and molecular methods have been used as an alternative. We present in this work, the development of new molecular procedures for diagnosis of the aforementioned organisms, together with a molecular characterization of isolates found in southern European cats.

A single PCR-RFLP procedure was designed for diagnosis of Mycoplasma spp. and a seminested PCR-RFLP was designed for diagnosis of piroplasmids. The 16S or 18S rRNA genes of isolates found in clinical samples were partially sequenced in all positive cases.

Mycoplasma spp. was detected in 9 (30%) out of 30 symptomatic cats from Spain. Sequencing indicated that 66.6% of these isolates can be ascribed to Mycoplasma haemofelis and only 33.3% to Mycoplasma haemominutum. Partial 16S rRNA sequences obtained in Spanish isolates were very similar to those previously published from the UK and the USA.

The presence of piroplasmids (Babesia and Theileria spp.) was studied in 16 cats from Spain (n=13) and Portugal (n=3). Animals analyzed were 10 cats with immunosuppressive viral infection (either FeLV or FIV), 5 asymptomatic cats and 1 cat with Babesia-compatible symptoms. Asymptomatic cats were all PCR-negative. Partial sequencing of 18S rRNA gene demonstrated that the Babesia-symptomatic cat was infected with Babesia canis canis whereas 3 (30%) out of the 10 cats with immunosuppressive viral infection were coinfected with piroplasmids (1 with B. canis canis, 1 with Theileria annae, and 1 with B. canis canis and T. annae both).

Introduction

Mycoplasma haemofelis and Mycoplasma haemominutum are unculturable organisms related to mollicutes (Neimark et al., 2001, Neimark et al., 2002, Foley and Pedersen, 2001), previously ascribed to the genus Haemobartonella. These bacterial pathogens are sometimes present in blood from mammals such as cats, mice and dogs. They grow attached to red blood cells, and the only possible diagnosis procedure until the arrival of molecular diagnosis was microscopic examination of blood smears (Foreyt, 1989). This procedure has many drawbacks, since bacterial pathogens may be confused with artifacts or lost after EDTA treatment of collected blood (Berent et al., 1998). A number of PCR tests based on amplification of 16S RNA gene sequences have been published for Mycoplasma spp. (Berent et al., 1998, Messick et al., 1998, Jensen et al., 2001). Some of these PCR assays can discriminate between M. haemofelis (the so called “large form” of the bacteria) and M. haemominutum (small form). Correct species identification is an important issue as some authors have pointed out differences in pathogenicity between M. haemominutum and M. haemofelis (Foley et al., 1998). Previous reports on molecular epidemiology of Mycoplasma spp. are mainly from the USA, although some isolates have recently been sequenced in the UK. They were closely related to the small M. haemominutum (Tasker et al., 2001), although a large strain was also detected. No reports on molecular characterization of this pathogen are available in other European countries, so that the molecular epidemiology of Mycoplasma spp. infection remains poorly understood.

Feline babesiosis is an infection caused in cats by an intra-erythrocytic apicomplexan protozoa, Babesia felis. The disease is apparently absent in Spain (Navarrete and Nieto, 1999), but in certain countries such as South Africa, feline babesiosis is diagnosed quite frequently (Schoeman et al., 2001). Some pathological features of the infection are similar to those reported in other Babesia-infected mammals and consist of anemia, elevated hepatic cytosol enzyme activity and increased total bilirubin and globulin concentrations (Schoeman et al., 2001). “Classical” diagnosis of feline babesiosis relies mainly on microscopic identification of piroplasms in red blood cells (Foreyt, 1989). This is an insensitive procedure, particularly for animals in the carrier state. Significant steps towards improved molecular detection have been recently undertaken by sequencing 18S rRNA genes from some feline Babesia spp. in South Africa (Penzhorn et al., 2001). According to this study, feline Babesia spp. are a phylogenetic cluster separated from the three piroplasmid groups previously described, namely theilerids, Babesia sensu stricto and Babesia “microti type”. The knowledge of DNA sequences from feline Babesia spp. should prompt the design of reliable molecular diagnosis tests for these pathogens. Unfortunately, no data on molecular epidemiology and diagnosis of feline piroplasmids in other geographical locations have been published up to now.

We have used a combined approach for Mycoplasma and piroplasmid infections, since some authors have pointed out that differential diagnosis between these pathogens is difficult. Both diseases cause clinical signs of anorexia, depression, regenerative anemia, weakness, weight loss and occasional icterus during the acute phase of the disease (Schoeman et al., 2001). The present work is aimed to develop sensitive molecular diagnosis procedures for detection of both Mycoplasma spp. and piroplasmids, as well as to provide data on molecular characterization and epidemiology of these feline hemoparasites in southern Europe.