Counting of viable C. burnetii cells by quantitative reverse transcription PCR using a recombinant plasmid (pCB-dotA) as a standard.

Coxiella burnetii is an intracellular pathogenic bacterium and etiological agent of Q fever in humans. Recently, the bacterium has been set free from the strictly intracellular condition by successful cultivation in acidified citrate cysteine medium. Here, we report a bacterial cell counting method that allows rapid quantification of the absolute or relative number of live cells of C. burnetii in a high throughput manner. The method utilizes TaqMan-based quantitative polymerase chain reaction (qPCR) targeting a single dotA gene for determination of genome equivalent (GE) presented either as DNA or complementary DNA (cDNA) synthesized via reverse transcription. The assay was shown to be specific, sensitive and efficiently reproducible. The quantification was linear over a range of 30 to 3x108 copies. Since there is only one copy of the dotA gene per Coxiella chromosome, the calculated dotA copy numbers can be compared to the number of bacterial cells. Finally, we demonstrated the potential of the method to assess effects of antibiotic on cell viability and to determine the antibiotic-tolerant fraction within a cell population. Keywords: Coxiella burnetii; Q fever; real-time polymerase chain reaction; copy number; antibiotic; axenic media; dotA gene.

New way of purification of pathogenic rickettsiae reducing health risks.

In general, cultivation and purification of intracellular pathogenic rickettsiae represents a risk for laboratory personnel due to exposure to highly infectious aerosol or accidental inoculation during these procedures. In this study, we describe an alternative, effective and time saving technique for rickettsial purification using digitonin to release intracellular bacteria from host cell without physical disruption. No significant differences were noted in yield and infectivity between digitonin treated rickettsiae and rickettsiae purified by sonication. This is the first report of using digitonin in purification of pathogenic rickettsiae and this approach might be effective for other intracellular pathogenic bacteria.

Isolation of rickettsia helvetica from ticks in Slovakia.

UNLABELLED: To date, only three rickettsial species have been found in ticks in Slovakia by serological and/or molecular-biological techniques, namely Rickettsia slovaca, Candidatus rickettsia IRS, and Rickettsia raoultii. Recently, we succeeded in isolation of the forth species, Rickettsia helvetica from Ixodes ricinus, the most frequent tick in Slovakia. The isolation, positive for 10% of tested ticks, was performed on XTC cells by the shell-vial technique, Gimenez staining and light microscopy. The infected cell cultures contained rod-shaped particles morphologically identical to rickettsiae. The isolation was confirmed by direct detection of a fragment of the R. helvetica gene for citrate synthase in the positive ticks by PCR and its subsequent cloning, sequencing and comparison with the database. KEYWORDS: Rickettsia helvetica; isolation; Ixodes ricinus; Slovakia.

Caracterización del binomio adulto mayor-familiar de convivencia / Characterization of binomial older adult-relative of coexistence

Introducción: El incremento de población adulto mayor exige responsabilidad sanitaria y familiar para que el aumento en años de vida sea de vida saludable. Objetivo: Identificar las características del adulto mayor y del familiar de convivencia como base para las acciones. Metodología: Estudio descriptivo; transversal con 96 binomios adulto mayor-familiar de convivencia. Cuestionario de datos biográficos y de salud para el adulto mayor y datos socios demográficos para el familiar; aplicados mediante entrevista en los hogares. Se utilizó el Programa Estadístico SPSS Versión 17. Resultados: Relación significativa en edad y sexo entre adultos mayores y familiares de convivencia. Media de edad 70 y 45; sexo; mujeres 59.4% y 67.7%; casados o en unión libre 56.3% y 72.9%; respectivamente. Familiar de convivencia hijo/a (54 %) y esposo/a (32%) mayoría mujeres con bajo nivel de escolaridad (65%) y ocupación labores del hogar (64%). La convivencia es favorable para la salud porque hay un mayor control de las conductas de riesgo. Relevancia de las características del binomio adulto mayor-familiar de convivencia para las acciones de promoción y prevención en los hogares; orientadas a fortalecer la relación y desarrollar habilidad en la atención al adulto mayor.

Abstract Nurses have sensitivity, knowledge and experience to attend the binomial older adult-relative of coexistence in family context. The purpose of the study was to identify characteristics of the older adult and the relative of coexistence as base to plan actions. The study was descriptive, cross-sectional with 96 binomials adult major-relative of greater coexistence. Biographical data and health questionnaire for the older adult and demographic data for the relative applied by means of interview in the homes. We used the statisti-cal program SPSS version 17. Age and sex had significant relation between older adults and relatives of coexistence. Age average, 70 and 45; woman, 59. 4% and 67.7%; ma-rried, 56.3% and 72.9, respectively. Relative of coexistence was child (54%) and wife or husband (32%), majority were woman with low level of education (65%) and occupation of the household work (64%).The coexistence is favorable for the health because there is a greater control of the risk conducts. Relevance of the characteristics of the binomial older adult-relative of coex-istence for promotion and prevention actions in the homes, oriented to fortify the relation and to develop ability in the at-tention to the older adult.

Detergent extract from chlamydial elementary bodies used as antigen for enzyme-linked immunosorbent assay.

Soluble antigen (SA) from chlamydial elementary bodies (EBs) was extracted with N-lauroylsarcosine. The extracted SA composed of lipopolysaccharide (LPS) and proteins was compared with EBs using an enzyme-linked immunosorbent assay (ELISA). Patient sera from natural chlamydial infections exhibited ELISA mean absorbance (A(492) and A(405/650)) values 2-5 times higher with SA than with EBs, resulting in a better discrimination between positive and negative human sera.

Green fluorescent protein as a detection marker for Coxiella burnetii transformation.

The molecular biological study of the obligate intracellular bacterium Coxiella burnetii is hampered because of the lack of an efficient DNA transformation system. We used expression of the green fluorescent protein (GFP) in addition to ampicillin resistance as a selection marker for detection of transformed C. burnetii cells. Fluorescent microscopy studies revealed that transformed C. burnetii cells can be detected easily inside the host cell line. A high level of GFP expression was reached with the strong Escherichia coli trc (trp/lac) promoter. The use of GFP not only provides a convenient marker for transformation of C. burnetii, but also allows detection of this obligate intracellular pathogen inside host eukaryotic cells. Possible applications for GFP in the study of host-pathogen interactions are discussed.

Immunological consequences of Coxiella burnetii phase variation.

The influence of the number of passages in chick embryo yolk sac (EPs) on the properties of the lipopolysaccharide (LPS) and other antigens of Coxiella burnetii Priscilla strain in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE), immunoblot analysis, enzyme-linked immunosorbent assay (ELISA) and complement-fixation reaction (CFR) test has been studied. Three phases in the phase variation of Coxiella burnetii could be distinguished by these methods: phase I lasting up to the 20th passage (EP 20), intermediate phase corresponding to EP 20-EP 70, and phase II beginning at EP 80. The changes in LPS were more marked than those in proteins which conserved their immunoblot profile up to EP 80. The phase II was clearly demonstrated by all the methods used.