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1.  A novel haemoplasma species identified in archived primate blood smears 
Veterinary Microbiology  2011;149(3-4):478-481.
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.
doi:10.1016/j.vetmic.2010.11.016
PMCID: PMC3075546  PMID: 21145673
Haemoplasma; Quantitative polymerase chain reaction; Primate; Aotus trivirgatus; RNase P RNA gene; Phylogeny
2.  Is Mycoplasma pneumoniae Adherence to Erythrocytes a Factor in Extrapulmonary Dissemination? 
PLoS Pathogens  2010;6(12):e1001219.
doi:10.1371/journal.ppat.1001219
PMCID: PMC3009594  PMID: 21203479
3.  Investigation of human haemotropic Mycoplasma infections using a novel generic haemoplasma qPCR assay on blood samples and blood smears 
Journal of Medical Microbiology  2010;59(Pt 11):1285-1292.
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.
doi:10.1099/jmm.0.021691-0
PMCID: PMC3090618  PMID: 20651038
4.  RNase P RNA Gene (rnpB) Phylogeny of Hemoplasmas and Other Mycoplasma Species▿ † 
Journal of Clinical Microbiology  2008;46(5):1873-1877.
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.
doi:10.1128/JCM.01859-07
PMCID: PMC2395117  PMID: 18337389
5.  Real-Time PCR Investigation of Potential Vectors, Reservoirs, and Shedding Patterns of Feline Hemotropic Mycoplasmas▿  
Applied and Environmental Microbiology  2007;73(12):3798-3802.
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.
doi:10.1128/AEM.02977-06
PMCID: PMC1932730  PMID: 17468284
6.  Properties of a Fructose-1, 6-Diphosphate-Activated Lactate Dehydrogenase from Acholeplasma laidlawii Type A 
Journal of Bacteriology  1973;114(3):1025-1033.
Acholeplasma laidlawii A possesses a nicotinamide adenine dinucleotide (NAD)-dependent l(+)-lactate dehydrogenase (LDH) which is activated specifically by low concentrations of fructose-1, 6-diphosphate (FDP). Studies with partially purified enzyme show that the kinetic response to FDP is hyperbolic. The enzyme is inhibited by inorganic phosphate, adenosine triphosphate, and high concentrations of reduced NAD (NADH). Low activity is demonstrable in the absence of FDP at pH 6.0 to 7.2, but FDP is absolutely required in the region of pH 8. FDP causes an upward shift in the optimum pH of the enzyme, which is near 7.2 in tris (hydroxymethyl)aminomethane buffer. Activation of the enzyme by FDP is markedly affected by substrate concentration; FDP lowers the apparent Km for pyruvate and NADH. The affinity of the enzyme for pyruvate is also influenced by H+ concentration. The pyruvate analogue α-ketobutyrate serves as an effective substrate for the enzyme; when it is utilized, the enzyme is still activated by FDP. Reversal of the pyruvate reduction reaction catalyzed by the enzyme can be demonstrated with the 3-acetylpyridine analogue of NAD. The catalytic properties of the A. laidlawii enzyme and the known FDP-activated LDHs which occur among lactic acid bacteria are discussed.
PMCID: PMC285360  PMID: 4712565
7.  Occurrence and Properties of Lactic Dehydrogenases of Fermentative Mycoplasmas 
Journal of Bacteriology  1972;111(3):633-640.
Eight fermentative mycoplasmas differing in genome size, deoxyribonucleic acid (DNA) base composition, or sterol dependence were examined for lactic dehydrogenase composition by spectrophotometric assay and polyacrylamide gel electrophoresis. Three completely different patterns of lactic dehydrogenase composition were found. (i) A nicotinamide adenine dinucleotide (NAD)-dependent l(+)-lactic dehydrogenase was found in Mycoplasma pneumoniae, M. gallisepticum, M. mycoides var. mycoides, mycoplasma UM 30847, M. neurolyticum, and Acholeplasma axanthum. Electrophoresis of cell-free extracts of each of these mycoplasmas produced, with the exception of M. mycoides var. mycoides and UM 30847, single, different enzyme bands. M. mycoides var. mycoides and UM 30847 were similar and formed multiple bands of enzyme activity. We were unable to establish whether these multiple bands were due to lactic dehydrogenase isoenzymes or artifacts. (ii) An NAD-dependent d(−)-lactic dehydrogenase which could not be reversed to oxidize lactate was found in M. fermentans. (iii) A. laidlawii A possessed an NAD-independent d(−)-lactic dehydrogenase capable of reducing dichlorophenol-indophenol, and an NAD-dependent l(+)-lactic dehydrogenase which is specifically activated by fructose-1,6-diphosphate. Heretofore, this enzyme regulatory mechanism was known to occur only among the Lactobacillaceae. No yeast-type lactic dehydrogenase activity was found in any of the mycoplasmas examined. The stereoisomer of lactic acid accumulated during growth correlated perfectly with the type of NAD-dependent lactic dehydrogenase found in each mycoplasma. The types of lactic dehydrogenase activity found in these mycoplasmas were not related to genome size, DNA base composition, or sterol dependence.
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PMCID: PMC251334  PMID: 4115465

Results 1-7 (7)