Examination of taxonomic uncertainties surrounding Brucella abortus bv. 7 by phenotypic and molecular approaches.

Brucella taxonomy is perpetually being reshuffled, at both the species and intraspecies levels. Biovar 7 of Brucella abortus was suspended from the Approved Lists of Bacterial Names Brucella classification in 1988, because of unpublished evidence that the reference strain 63/75 was a mixture of B. abortus biovars 3 and 5. To formally clarify the situation, all isolates previously identified as B. abortus bv. 7 in the AHVLA and ANSES strain collections were characterized by classical microbiological and multiple molecular approaches. Among the 14 investigated strains, including strain 63/75, only four strains, isolated in Kenya, Turkey, and Mongolia, were pure and showed a phenotypic profile in agreement with the former biovar 7, particularly agglutination with both anti-A/anti-M monospecific sera. These results were strengthened by molecular strategies. Indeed, genus- and species-specific methods allowed confirmation that the four pure strains belonged to the B. abortus species. The combination of most approaches excluded their affiliation with the recognized biovars (biovars 1 to 6 and 9), while some suggested that they were close to biovar 3.These assays were complemented by phylogenetic and/or epidemiological methods, such as multilocus sequence analysis (MLSA) and variable-number tandem repeat (VNTR) analysis. The results of this polyphasic investigation allow us to propose the reintroduction of biovar 7 into the Brucella classification, with at least three representative strains. Interestingly, the Kenyan strain, sharing the same biovar 7 phenotype, was genetically divergent from other three isolates. These discrepancies illustrate the complexity of Brucella taxonomy. This study suggests that worldwide collections could include strains misidentified as B. abortus bv. 7, and it highlights the need to verify their real taxonomic position.

Harmonization of European laboratory response networks by implementing CWA 15793: use of a gap analysis and an "insider" exercise as tools.

Laboratory response networks (LRNs) have been established for security reasons in several countries including the Netherlands, France, and Sweden. LRNs function in these countries as a preparedness measure for a coordinated diagnostic response capability in case of a bioterrorism incident or other biocrimes. Generally, these LRNs are organized on a national level. The EU project AniBioThreat has identified the need for an integrated European LRN to strengthen preparedness against animal bioterrorism. One task of the AniBioThreat project is to suggest a plan to implement laboratory biorisk management CWA 15793:2011 (CWA 15793), a management system built on the principle of continual improvement through the Plan-Do-Check-Act (PDCA) cycle. The implementation of CWA 15793 can facilitate trust and credibility in a future European LRN and is an assurance that the work done at the laboratories is performed in a structured way with continuous improvements. As a first step, a gap analysis was performed to establish the current compliance status of biosafety and laboratory biosecurity management with CWA 15793 in 5 AniBioThreat partner institutes in France (ANSES), the Netherlands (CVI and RIVM), and Sweden (SMI and SVA). All 5 partners are national and/or international laboratory reference institutes in the field of public or animal health and possess high-containment laboratories and animal facilities. The gap analysis showed that the participating institutes already have robust biorisk management programs in place, but several gaps were identified that need to be addressed. Despite differences between the participating institutes in their compliance status, these variations are not significant. Biorisk management exercises also have been identified as a useful tool to control compliance status and thereby implementation of CWA 15793. An exercise concerning an insider threat and loss of a biological agent was performed at SVA in the AniBioThreat project to evaluate implementation of the contingency plans and as an activity in the implementation process of CWA 15793. The outcome of the exercise was perceived as very useful, and improvements to enhance biorisk preparedness were identified. Gap analyses and exercises are important, useful activities to facilitate implementation of CWA 15793. The PDCA cycle will enforce a structured way to work, with continual improvements concerning biorisk management activities. Based on the activities in the AniBioThreat project, the following requirements are suggested to promote implementation: support from the top management of the organizations, knowledge about CWA 15793, a compliance audit checklist and gap analysis, training and exercises, networking in LRNs and other networks, and interinstitutional audits. Implementation of CWA 15793 at each institute would strengthen the European animal bioterrorism response capabilities by establishing a well-prepared LRN.

Uterine contractions depend on KIT-positive interstitial cells in the mouse: genetic and pharmacological evidence.

In the gastrointestinal tract, interstitial cells of Cajal (ICCs) generate a pacemaker activity. They produce electric slow waves that trigger and coordinate gut smooth muscle contractions. Interstitial cells of Cajal's slender shape is revealed by KIT immunostaining. Based on several features, including KIT expression and KIT dependence, ICC-like cells were identified in nongastrointestinal tissues. Here, we investigated in the mouse whether uterine contractions depend on ICC-like cells' activity. By labeling KIT-expressing cells, we found putative ICC-like cells in the uterus, observed as KIT-positive interstitial, long spindle-shaped cells with fine branched cytoplasm processes, distributed in muscular layers and in subepithelial connective tissue. We then checked the potential KIT dependence of ex vivo contractile activity of the uterus by combining genetic and pharmacological approaches, using the Kit W-v hypomorphic mutation, and imatinib as a KIT noncompetitive inhibitor. We found a significant reduction in frequency of longitudinal uterine contractions in Kit W-v/Kit W-v compared with Kit+/+ mice, whereas amplitude was unaffected. There was no difference in frequency or amplitude of circular uterine contractions between Kit W-v/Kit W-v and Kit+/+ mice. Ex vivo treatment of Kit+/+ uterine horns with imatinib resulted in a dose-dependent reduction of the frequency and amplitude of longitudinal myometrial contractions. Amplitude and frequency of circular contractions were unaffected in presence of imatinib. These concurrent results suggest that longitudinal contractions of the uterus depend on a KIT signaling pathway of ICC-like cells. The existence of ICC-like cells in the myometrium may enhance our understanding of uterine spontaneous contractile activity and suggest new approaches for treatment of uterine contractility disorders.

Influence of temperature on urethra to bladder micturition reflex in the awake ewe.

AIMS: The flow of fluid along the urethra is known to facilitate detrusor contraction during micturition. This reflex, previously described in awake ewes, helps to achieve complete bladder emptying. In anesthetized cats, another urethra to bladder reflex involving urethral cold receptors has been described. The aim of this study was to investigate whether the urethral reflex first described in awake ewes could also be temperature-dependent. METHODS: Experiments were performed on 10 healthy ewes. Urethral flows were performed by injecting 10 ml saline (ranging from 17 to 43 degrees C) at the level of the proximal urethra. Catheterization of the bladder was performed so that detrusor pressure was continually recorded during the experiments. RESULTS: Urethral flows using body temperature saline (37-39 degrees C) consistently evoked detrusor contraction. Urethral flows using saline at temperatures between 40 and 43 degrees C induced detrusor contractions that were not significantly different from those observed at 37-39 degrees C. Urethral flows using saline at temperatures below 37-39 degrees C (17-36 degrees C) resulted in a weaker or absent detrusor contraction. CONCLUSIONS: In ewes, we have shown that urethral to bladder micturition reflex involving mechanoreceptors is decreased at temperatures below the physiological range. It is suggested that transient receptor potential vanilloid cation channels (e.g., TRPV4 which is activated by sheer/stress flows at near-body temperature) could be involved in this urethra to bladder reflex. In humans, this reflex has hardly been described and is still a matter of debate. Our results reinforce that its full investigation may require systematic use of a range of saline flows at different temperatures.