A novel haemoplasma species identified in archived primate blood smears.

In order to confirm a microscopic diagnosis of 'eperythrozoonosis' made over 40 years ago in a captive owl monkey (Aotus trivirgatus), DNA was extracted from archived fixed and stained blood smears and subjected to generic haemotropic mycoplasma (haemoplasma) quantitative real-time PCR (qPCR) and a human glyceraldehyde-3-phosphate dehydrogenase qPCR as an amplification control. The qPCRs confirmed the extraction of host DNA from the samples and the presence of a haemoplasma species. Partial 16S rRNA and ribonuclease P ribosomal gene fragments were amplified by PCR, cloned and sequenced. Sequence data and phylogeny showed the owl monkey haemoplasma to lie in the haemominutum clade of haemoplasmas, most closely related to 'Candidatus Mycoplasma kahaneii'. This study confirms the use of generic haemoplasma qPCRs to successfully amplify haemoplasma DNA from fixed, stained and archived blood smears from the early 1970s and provides molecular confirmation of the existence of a novel haemoplasma species in an owl monkey, for which the name 'Candidatus Mycoplasma aoti' sp. nov. is proposed.

Investigation of human haemotropic Mycoplasma infections using a novel generic haemoplasma qPCR assay on blood samples and blood smears.

The aim of this study was to develop quantitative real-time (q)PCR assays to detect all known haemoplasma species, and a human housekeeping gene in order to demonstrate both successful DNA extraction from clinical samples and to test for sample inhibition, and to apply these qPCRs to human blood samples and blood smears. Sensitive and specific generic haemoplasma qPCR assays were developed to amplify haemoplasma species, as well as human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal amplification control. An optimized technique for extracting DNA from stained blood smears was also developed. These methods were applied to anonymized blood samples obtained from 100 human immunodeficiency virus (HIV)-infected South Africans and 920 UK patients undergoing haematological examination, and to 15 blood smears recruited from previous studies describing human haemoplasmosis. Human GAPDH levels were acceptable in all but three of the blood samples and all but two of the blood smears. The latter could have arisen due to DNA degradation due to the old age (over 35 years) of these smears. Haemoplasma infection was found in one HIV-infected South African, but the species could not be characterized due to the very low levels of DNA present. This report describes novel extraction and qPCR methodologies for haemoplasma screening. Previously reported human haemoplasmosis based on cytological diagnosis alone should be viewed with caution.

RNase P RNA gene (rnpB) phylogeny of Hemoplasmas and other Mycoplasma species.

Partial sequences of the RNase P RNA gene (rnpB) were obtained from a number of hemoplasmas and other Mycoplasma species. Phylogenetic analysis of these sequences showed that all hemoplasmas were present within a single clade and were most closely related to Mycoplasma fastidiosum, similar to the results found with 16S rRNA gene phylogeny.

Real-time PCR investigation of potential vectors, reservoirs, and shedding patterns of feline hemotropic mycoplasmas.

Three hemotropic mycoplasmas have been identified in pet cats: Mycoplasma haemofelis, "Candidatus Mycoplasma haemominutum," and "Candidatus Mycoplasma turicensis." The way in which these agents are transmitted is largely unknown. Thus, this study aimed to investigate fleas, ticks, and rodents as well as saliva and feces from infected cats for the presence of hemotropic mycoplasmas, to gain insight into potential transmission routes for these agents. DNA was extracted from arthropods and from rodent blood or tissue samples from Switzerland and from salivary and fecal swabs from two experimentally infected and six naturally infected cats. All samples were analyzed with real-time PCR, and some positive samples were confirmed by sequencing. Feline hemotropic mycoplasmas were detected in cat fleas and in a few Ixodes sp. and Rhipicephalus sp. ticks collected from animals but not in ticks collected from vegetation or from rodent samples, although the latter were frequently Mycoplasma coccoides PCR positive. When shedding patterns of feline hemotropic mycoplasmas were investigated, "Ca. Mycoplasma turicensis" DNA was detected in saliva and feces at the early but not at the late phase of infection. M. haemofelis and "Ca. Mycoplasma haemominutum" DNA was not amplified from saliva and feces of naturally infected cats, despite high hemotropic mycoplasma blood loads. Our results suggest that besides an ostensibly indirect transmission by fleas, direct transmission through saliva and feces at the early phase of infection could play a role in the epizootiology of feline hemotropic mycoplasmas. Neither the investigated tick nor the rodent population seems to represent a major reservoir for feline hemotropic mycoplasmas in Switzerland.

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.

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.

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.

Revision of haemotrophic Mycoplasma species names.

The recently proposed transfer of four rickettsias from the genera Haemobartonella and Eperythrozoon to the genus Mycoplasma with the Candidatus status is herein revised. This is because the Candidatus designation is for new, incompletely described taxa, in order to give them a provisional status. Thus, 'Candidatus Mycoplasma haemofelis' is revised to Mycoplasma haemofelis comb. nov., nom. nov., 'Candidatus Mycoplasma haemomuris' is revised to Mycoplasma haemomuris comb. nov., nom. nov., 'Candidatus Mycoplasma haemosuis' is revised to Mycoplasma haemosuis comb. nov., nom. nov. and 'Candidatus Mycoplasma wenyonii' is revised to Mycoplasma wenyonii comb. nov.