The Hsp90 isoforms from S. cerevisiae differ in structure, function and client range.

The molecular chaperone Hsp90 is an important regulator of proteostasis. It has remained unclear why S. cerevisiae possesses two Hsp90 isoforms, the constitutively expressed Hsc82 and the stress-inducible Hsp82. Here, we report distinct differences despite a sequence identity of 97%. Consistent with its function under stress conditions, Hsp82 is more stable and refolds more efficiently than Hsc82. The two isoforms also differ in their ATPases and conformational cycles. Hsc82 is more processive and populates closed states to a greater extent. Variations in the N-terminal ATP-binding domain modulate its dynamics and conformational cycle. Despite these differences, the client interactomes are largely identical, but isoform-specific interactors exist both under physiological and heat shock conditions. Taken together, changes mainly in the N-domain create a stress-specific, more resilient protein with a shifted activity profile. Thus, the precise tuning of the Hsp90 isoforms preserves the basic mechanism but adapts it to specific needs.

Quantitative real-time PCR and phase specific serology are mutually supportive in Q fever diagnostics in goats.

This study presents results of quantitative pathogen detection by real-time PCR (qPCR) and phase-specific serology for complete Q fever diagnostics. For this, samples of 42 goats in total were taken during a Q fever outbreak. In the early phase of the Q-fever infection, 10(4)-10(8)Coxiella (C.) burnetii pathogens per vaginal swab and 10(2)-10(6)C. burnetii per ml milk were detected using quantitative real-time PCR (qPCR). Pathogen excretion decreased continuously within two months to less than 10(4) (vaginal swab) and 10(2) (milk) C. burnetii. At the end of the study there was a shift toward a 10 fold higher excretion of the pathogen via the genital tract and milk. At the start of the study, serological tests showed a dominance of the phase-2 antibody in 76% (22/29) of the goats in the MONA- (Multiple of Normal Activity) ELISA and 79% (23/29) in the IDEXX-ELISA, which was replaced by a phase-1 dominance in 85% (29/34) and 62% (21/34), of the animals respectively at the end of the study. Serum samples from 13 goats before lambing that excreted C. burnetii after lambing showed antibodies against phase 2 of 100% using MONA-ELISA and 77% in the IDEXX-ELISA. The most important diagnostic instrument for Q-fever infection in goats following birth is testing of vaginal swabs using qPCR. Phase-specific serology allows an estimation of possible pathogen excretion even before birth, as well as achieving valuable results for determination of the infection phase.

Differences in the antigen structures of Corynebacterium pseudotuberculosis and the induced humoral immune response in sheep and goats.

Caseous lymphadenitis (CLA), a disease affecting sheep and goats, is caused by Corynebacterium pseudotuberculosis (Cp). Eradication programs are based on the serological identification of Cp infected animals. However, available diagnostic ELISAs are not similarly suitable for sheep and goats. In the present study the comparison of antigens revealed major species specific differences between sheep and goat derived Cp field isolates as well as between field isolates and the Cp ATCC reference strains. Furthermore, we found species-specific differences in the anti-Cp humoral immune response between sheep and goats. The analysis of band frequency was able to distinguish between immunodominant and non-immunodominant protein bands. The 150 kDa, 74 kDa, 48 kDa, and 30 kDa antigens were immunodominant in both, sheep and goats. Interestingly, the most commonly used diagnostic antigen, i.e. the 30 kDa phospholipase D (PLD), was recognized by 100% of the Cp positive goats but only by 70% of the Cp positive sheep. Furthermore, analysis of field sera revealed that there were a particular percentage of Cp positive sera which reacted negative with the PLD. In conclusion our results clearly showed that (1) the application of a combination of further defined immunodominant Cp antigens - in addition to the PLD antigen - and (2) consideration of species-specific differences in the anti-Cp immune response will substantially contribute to the improvement of Cp serological diagnostics and to effective eradication programs in both sheep and goats.

Evaluating leaf litter beetle data sampled by Winkler extraction from Atlantic forest sites in southern Brazil

Evaluating leaf litter beetle data sampled by Winkler extraction from Atlantic forest sites in southern Brazil. To evaluate the reliability of data obtained by Winkler extraction in Atlantic forest sites in southern Brazil, we studied litter beetle assemblages in secondary forests (5 to 55 years after abandonment) and old-growth forests at two seasonally different points in time. For all regeneration stages, species density and abundance were lower in April compared to August; but, assemblage composition of the corresponding forest stages was similar in both months. We suggest that sampling of small litter inhabiting beetles at different points in time using the Winkler technique reveals identical ecological patterns, which are more likely to be influenced by sample incompleteness than by differences in their assemblage composition. A strong relationship between litter quantity and beetle occurrences indicates the importance of this variable for the temporal species density pattern. Additionally, the sampled beetle material was compared with beetle data obtained with pitfall traps in one old-growth forest. Over 60 percent of the focal species captured with pitfall traps were also sampled by Winkler extraction in different forest stages. Few beetles with a body size too large to be sampled by Winkler extraction were only sampled with pitfall traps. This indicates that the local litter beetle fauna is dominated by small species. Hence, being aware of the exclusion of large beetles and beetle species occurring during the wet season, the Winkler method reveals a reliable picture of the local leaf litter beetle community.

Avaliação dos besouros da liteira amostrados por extração Winkler na Floresta Atlântica do Sul do Brasil. Para avaliar a confiabilidade dos dados obtidos pela extração Winkler em coletas na Floresta Atlântica do Sul do Brasil, nós estudamos as assembléias de besouros da liteira em florestas secundárias (5 a 55 anos após abandono) e no estágio avançado em dois pontos no tempo sazonalmente diferentes. Para todos os estágios de renegeração, a densidade e abundância das espécies foram menores em abril comparado a agosto; porém, a composição das assembléias dos estágios florestais correspondentes foi similar em ambos os meses. Nós sugerimos que amostragens de pequenos besouros habitantes de liteira em diferentes pontos no tempo usando o método Winkler revelam padrões ecológicos idênticos. Um forte relacionamento entre a quantidade da liteira e a ocorrência de besouros indica a importância dessa variável no padrão temporal de densidade das espécies. Adicionalmente, o material amostrado foi comparado com dados de besouros obtidos utilizando armadilhas do tipo pitfall em um estágio avançado de regeneração. Cerca de 60 por cento das espécies de interesse capturadas em pitfall foram também amostradas pela extração Winkler. Poucos besouros com tamanho corporal grande para ser amostrado pela extração Winkler foram capturados com a armadilha pitfall. Isso indica que a fauna local de besouros da liteira é dominada por espécies pequenas. Portanto, sabendo da exclusão das espécies grandes e das espécies que ocorrem durante a estação chuvosa, o método Winkler revela um cenário confiável da comunidade local de besouros da literia.

Application of phage typing and pulsed-field gel electrophoresis to analyse Salmonella enterica isolates from a suspected outbreak in Lagos, Nigeria.

INTRODUCTION: Inadequate potable water supply and poor sanitation predispose to food- and water-borne diseases associated with Salmonella enterica serovars in developing countries. In this study the possible source of an unprecedented upsurge of Salmonella-associated community gastroenteritis was traced using both phage-typing and pulsed-field gel electrophoresis (PFGE). METHODOLOGY: Nineteen Salmonella Typhimurium (three sporadic isolates included) and 13 Salmonella Enteritidis isolates from clinical, animal, and environmental samples were subjected to antimicrobial susceptibility testing, phage-typing, and PFGE analysis using standard procedures. RESULTS: Eleven (68.8%) of the 16 outbreak-related multidrug resistant S. Typhimurium belonged to DT 71 phage type with cluster PFGE type X3, representing the most prevalent strain identified among human, animal, and environmental isolates. The remaining five (31.2%) outbreak-related strains  reacted but did not conform with clear phage types (RDNC) with cluster PFGE types X1 and X2 (96.8% similarity). Sporadic strains were untypable and belonged to X4 PFGE type. However, the evaluated S. Enteritidis strains that were multidrug resistant without a definite phage type belonged to PFGE cluster type X1e and were identified among the water and human strains. None of the Typhimurium and Enteritdis isolates was resistant to the fluoroquinolone antibiotics that were evaluated. CONCLUSION: This study emphasizes the epidemiological usefulness of PFGE typing in the detection of emerging strains of multipledrug resistant Salmonella, particularly S. Typhimurium DT71, that pose serious health implications in our environment. The study provides epidemiological links between environmental reservoirs and human infection in this community.