Hemotrophic Mycoplasmas Research Articles

First LightCycler real-time PCR assay for the quantitative detection of Mycoplasma suis in clinical samples

Mycoplasma suis cannot be cultivated in vitro. Therefore, PCR-based methods are irreplaceable for the diagnosis of M. suis infections especially when clinical symptoms are not evident. Currently, no easy and reliable method allowing the quantitative detection of M. suis is available. This report describes the development of a quantitative LightCycler™ PCR assay based on the msg1 gene of M. suis (LC MSG1 PCR). No PCR signals were obtained with closely related haemotrophic and non-haemotrophic mycoplasmas, with other bacteria, and with M. suis-free blood and tissue arguing for a high analytical specificity. Test sensitivity was found to be 100%, and test specificity 96.7%. To test the diagnostic suitability of the LC MSG1 PCR, 25 pigs with clinical porcine eperythrozoonosis and 25 healthy pigs were investigated. All ill pigs revealed a positive real-time PCR result whereas only one healthy pig was detected to be M. suis-infected. M. suis was quantitatively detected in 19 blood specimens of 100 sows from Switzerland and in 17 of 160 post-weaning piglets from Germany. In conclusion, this new LC MSG1 PCR assay represents a powerful tool for the improvement of the current M. suis diagnosis and for prevalence and pathogenesis studies.

First identification and functional characterization of an immunogenic protein in unculturable haemotrophic Mycoplasmas (Mycoplasma suisHspA1)

The antigenic structures of the haemotrophic Mycoplasma suis, an epicellular parasite of porcine erythrocytes, are largely unknown due to its unculturability. In this study, serological proteome and mass spectrometry analyses allowed the characterization of M. suis proteins targeted by the porcine antibody response: two proteins with characteristics of heat shock proteins, two proteins with characteristics of glycolytic enzymes, a RNA helicase- and an actin-like protein. The DnaK-like protein of M. suis (HspA1) was further analysed genetically and functionally. Its encoding gene (M. suis a1 gene) is 1.830 bp in size and corresponds to a 67 kDa protein. Immunoelectron microscopy verified the surface accessibility of HspA1 in M. suis. Recombinant HspA1 expressed in Escherichia coli demonstrated ATPase activity and antigenicity in experimentally infected pigs. In conclusion, this first identification and recombinant expression of an antigenic protein of M. suis provides the basis for the development of vaccines and new in vitro diagnostic assays.

Significance of haemotrophic mycoplasmas in veterinary medicine with particular regard to the Mycoplasma suis infection in swine

Phylogenetic investigations led to the definition of a new group of bacteria called haemotrophic mycoplasmas (haemoplasmas). The life cycle of said globally spread bacteria is dependent on their intimate contact with their host erythrocytes. Illnesses showing symptoms of a haemolytic anaemia are usually found in cats and swine. Haemoplasmas are small pleomorphic bacteria (0.3-3 microm) with a very small genome (745-1245 kbp). To date there is very limited knowledge on their biology and the host-bacteria-interactions (immune response, pathogenesis) since these bacteria are yet not culturable. Applying modern molecular biological techniques we succeeded during the last few years in gaining new facts on the antigen structure of M.suis as well as on the immunology and pathogenesis of the porcine eperythrozoonosis. Thus we detected three main antigens two of which we expressed recombinant in our laboratory. These surface-exposed antigens serve as a basis for establishing serologic assays, and are vaccine candidates, too. Preliminary studies allowed us to find the function of an adhesin of M. suis for one of the two proteins.

Phylogenetic analysis and description of Eperythrozoon coccoides, proposal to transfer to the genus Mycoplasma as Mycoplasma coccoides comb. nov. and Request for an Opinion

Eperythrozoon coccoides, an epierythrocytic organism that causes a mild haemolytic anaemia in laboratory and wild mice, currently is thought to be a rickettsia. To determine the relationship of this agent to other haemotrophic bacterial parasites, the 16S rRNA gene of this organism has been sequenced and it is shown by phylogenetic analysis that this wall-less bacterium is not a rickettsia but actually is a mycoplasma. This mycoplasma shares properties with and is closely related to the other uncultivated mycoplasmas that comprise a recently identified group, the haemotrophic mycoplasmas (haemoplasmas). The haemoplasma group is composed of former Eperythrozoon and Haemobartonella species as well as newly identified haemotrophic mycoplasmas. Haemoplasmas parasitize the surface of erythrocytes of a wide variety of vertebrate animal hosts and are transmitted mainly by blood-feeding arthropod vectors. Because both primary infections and chronic latent infections caused by this bacterium have been observed in many laboratories and this bacterium has been the subject of much experimental work, considerable information exists about this haemotrophic mycoplasma that may be applicable to other haemoplasmas. It is proposed that Eperythrozoon coccoides be reclassified as Mycoplasma coccoides comb. nov. A Request for an Opinion is submitted to the Judicial Commission of the International Committee on Systematics of Prokaryotes regarding this reclassification.

Artifactual hypoglycemia associated with hemotrophic mycoplasma infection in a lamb

A 35-day-old male lamb with Mycoplasma ovis infection (previously Eperythrozoon ovis) was evaluated because of severe hypoglycemia (serum glucose 4 mg/dL, Hitachi 704 automated chemistry analyzer) inconsistent with the animal's condition. Whole blood glucose concentration measured with a glucometer was 74 mg/dL. The purpose of this study was to investigate this discrepancy through in vitro evaluation of the patient's blood. Blood was incubated alone, with increasing concentrations of plasma, or with equine serum of known glucose concentration for 0, 15, 30, and 60 minutes at room temperature; end-point glucose concentrations were compared with blood from a control sheep handled similarly. A rapid decline in glucose concentration was observed in heparinized or EDTA anticoagulated whole blood from the infected lamb incubated alone or with the equine serum. Glucose concentrations in incubated samples from a control sheep remained stable. Incubation of increasing concentrations of heparinized blood with autologous plasma resulted in decreased glucose concentrations in patient, but not control, blood. As parasitemia decreased after treatment, serum glucose concentration increased, serum lactate concentration decreased, and in vitro glucose concentration stabilized. These findings are consistent with parasite-associated in vitro glucose consumption. An increase in the lamb's plasma glucose concentration associated with reduction of parasite load suggested excess glucose consumption also may have occurred in vivo.

Hemotrophic mycoplasmas (hemoplasmas): a review and new insights into pathogenic potential.

The red cell parasites formerly known as Haemobartonella and Eperythrozoon spp have been reclassified as hemotrophic mycoplasmas (hemoplasmas) based on strong phylogenetic evidence and 16S ribosomal RNA gene sequences. The latter form the basis for polymerase chain reaction assays used to detect infection. Candidatus designation was given to incompletely characterized species. Like other mycoplasmas, hemoplasmas are small epicellular parasites that lack a cell wall and are susceptible to tetracyclines; their circular, double-stranded DNA encodes only those gene products essential for life. Diseases caused by infection with hemoplasmas range from overt life-threatening hemolytic anemia to subtle chronic anemia, ill-thrift, and infertility. In addition, the organisms may act as cofactors in the progression of retroviral, neoplastic, and immune-mediated diseases. Intimate contact of hemoplasma organisms with RBCs leads to cell injury through immune-mediated and other mechanisms that have not yet been defined. Despite an intense immune response and even with antibiotic treatment, infected animals probably remain chronic carriers after clinical signs have resolved.

Mycoplasma ovis comb. nov. (formerly Eperythrozoon ovis), an epierythrocytic agent of haemolytic anaemia in sheep and goats.

Eperythrozoon ovis, an erythrocytic agent that causes haemolytic anaemia in sheep and goats, occurs worldwide and is currently thought to be a rickettsia. To determine the relationship between this agent and other haemotrophic bacterial parasites, the 16S rRNA gene of this organism was sequenced. Phylogenetic analysis revealed that this wall-less bacterium is not a rickettsia, but a mycoplasma. This mycoplasma is related closely to several other uncultivated, epierythrocytic mycoplasmas that comprise a recently identified group, the haemotrophic mycoplasmas (haemoplasmas). The haemoplasma group is composed of former Eperythrozoon and Haemobartonella species, as well as newly identified epierythrocytic mycoplasmas. Haemoplasmas parasitize the surface of erythrocytes of a wide variety of vertebrate animal hosts and are transmitted mainly by blood-feeding arthropod vectors. Recognition that E. ovis is a mycoplasma provides a new approach to dealing with this bacterium. It is proposed that E. ovis should be reclassified as Mycoplasma ovis comb. nov.

Physical map and genome sequencing survey of Mycoplasma haemofelis (Haemobartonella felis).

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.

The putative haemobartonella that influences Plasmodium falciparum parasitaemia in squirrel monkeys is a haemotrophic mycoplasma

Splenectomised squirrel monkeys ( Saimiri sciureus) are increasingly being used as an experimental host for human malaria studies, notably for the assessment of candidate vaccines against Plasmodium falciparum blood-stage infection. Recently, S. sciureus monkeys in our primate-breeding colony were reported to be asymptomatic carriers of a putative Haemobartonella species. Patent haemobartonella infection is frequently activated following splenectomy, and may interfere with studies on the course of P. falciparum parasitaemia in these animals. Here, we show by 16S rRNA gene sequence analysis that this wall-less bacterium is not a rickettsia but, instead, is a haemotrophic mycoplasma. Haemotrophic mycoplasmas are a newly identified group of mycoplasmas that parasitise the surfaces of erythrocytes of a wide variety of vertebrate hosts.

'Candidatus mycoplasma haemodidelphidis' sp. nov., 'Candidatus mycoplasma haemolamae' sp. nov. and Mycoplasma haemocanis comb. nov., haemotrophic parasites from a naturally infected opossum (Didelphis virginiana), alpaca (Lama pacos) and dog (Canis familiaris): phylogenetic and secondary structural relatedness of their 16S rRNA genes to other mycoplasmas.

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.