IS711-based real-time PCR assay as a tool for detection of Brucella spp. in wild boars and comparison with bacterial isolation and serology

BACKGROUND: Control of brucellosis in livestock, wildlife and humans depends on the reliability of the methods used for detection and identification of bacteria. In the present study, we describe the evaluation of the recently established real-time PCR assay based on the Brucella-specific insertion sequence IS711 with blood samples from 199 wild boars (first group of animals) and tissue samples from 53 wild boars (second group of animals) collected in Switzerland. Results from IS711 real-time PCR were compared to those obtained by bacterial isolation, Rose Bengal Test (RBT), competitive ELISA (c-ELISA) and indirect ELISA (i-ELISA). RESULTS: In the first group of animals, IS711 real-time PCR detected infection in 11.1% (16/144) of wild boars that were serologically negative. Serological tests showed different sensitivities [RBT 15.6%, c-ELISA 7.5% and i-ELISA 5.5%] and only 2% of blood samples were positive with all three tests, which makes interpretation of the serological results very difficult. Regarding the second group of animals, the IS711 real-time PCR detected infection in 26% of animals, while Brucella spp. could be isolated from tissues of only 9.4% of the animals. CONCLUSION: The results presented here indicate that IS711 real-time PCR assay is a specific and sensitive tool for detection of Brucella spp. infections in wild boars. For this reason, we propose the employment of IS711 real-time PCR as a complementary tool in brucellosis screening programs and for confirmation of diagnosis in doubtful cases.

Isolation and molecular characterization of recombinant Echinococcus granulosus P29 protein (recP29) and its assessment for the post-surgical serological follow-up of human cystic echinococcosis in young patients

We synthesized recombinant Echinococcus granulosus protoscolex recP29 antigen to be preliminarily assessed by ELISA and immunoblotting. RecP29-serology was carried out on 54 young patients with cystic echinococcosis (CE). Patients were classified into either cured (CCE) (n=40) or non-cured (NCCE) (n=14) CE patients. RecP29 ELISA showed a gradual decrease of antibody concentrations in all CCE cases that were initially (before treatment) seropositive to this antigen (25 out of 40) or that seroconverted following treatment. A complete seronegativity was reached within 3 years post-surgery in all of these cases. Conventional HCF ELISA yielded seronegativity in only 10% of initially recP29-seropositive CCE patients (P=0.086). Likewise, recP29 immunoblotting yielded seronegativity in 93% of 29 out of 40 initially recP29-immunoblot-positive CCE patients after 3 years follow-up, compared with 72% in the HCF immunoblotting (P=0.060). Eleven out of 14 NCCE patients were initially positive by recP29 ELISA, and 10 out of these maintained a marked anti-recP29 antibody reactivity until the endpoint of the follow-up period. All 14 NCCE cases were initially seropositive by recP29 immunoblotting, and 13 cases remained seropositive until the end of the study. Thus, recombinant P29 protein appears prognostically useful for monitoring those post-surgical CE cases with an initial seropositivity to this marker.

Isolation of myeloid dendritic cells and epithelial cells from human thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c(+)), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45(hi)) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

Trigger activity more than three years after left atrial linear ablation without pulmonary vein isolation in patients with atrial fibrillation.

OBJECTIVES The aim of this study was to analyze trigger activity in the long-term follow-up after left atrial (LA) linear ablation. BACKGROUND Interventional strategies for curative treatment of atrial fibrillation (AF) are targeted at the triggers and/or the maintaining substrate. After substrate modification using nonisolating linear lesions, the activity of triggers is unknown. METHODS With the LA linear lesion concept, 129 patients were treated using intraoperative ablation with minimal invasive surgical techniques. Contiguous radiofrequency energy-induced lesion lines involving the mitral annulus and the orifices of the pulmonary veins without isolation were placed under direct vision. RESULTS After a mean follow-up of 3.6 +/- 0.4 years, atrial ectopy, atrial runs, and reoccurrence of AF episodes were analyzed by digital 7-day electrocardiograms in 30 patients. Atrial ectopy was present in all patients. Atrial runs were present in 25 of 30 patients (83%), with a median number of 9 runs per patient/week (range 1 to 321) and a median duration of 1.2 s/run (range 0.7 to 25), without a significant difference in atrial ectopy and atrial runs between patients with former paroxysmal (n = 17) or persistent AF (n = 13). Overall, 87% of all patients were completely free from AF without antiarrhythmic drugs. CONCLUSIONS A detailed rhythm analysis late after specific LA linear lesion ablation shows that trigger activity remains relatively frequent but short and does not induce AF episodes in most patients. The long-term success rate of this concept is high in patients with paroxysmal or persistent AF.

Benefit of pulmonary vein isolation guided by loss of pace capture on the ablation line: results from a prospective 2-center randomized trial.

OBJECTIVES This study was conducted to determine if an additional procedural endpoint of unexcitability (UE) to pacing along the ablation line reduces recurrence of atrial fibrillation (AF) or atrial tachycardia (AT) after radiofrequency catheter ablation. BACKGROUND AF/AT recurrence is common after pulmonary vein isolation (PVI). METHODS We included 102 patients from 2 centers (age 63 ± 10 years; 33 women; left atrium 38 ± 7 mm; left ventricular ejection fraction 61 ± 6%) with symptomatic paroxysmal AF. A 3-dimensional mapping system and circumferential mapping catheter were used in all patients for PVI. In group 1 (n = 50), the procedural endpoint was bidirectional block across the ablation line. In group 2 (n = 52), additional UE to bipolar pacing at an output of 10 mA and 2-ms pulse width was required. The primary endpoint was freedom from any AF/AT (>30 s) after discontinuation of antiarrhythmic drugs. RESULTS Procedural endpoints were successfully achieved in all patients. Procedure duration was significantly longer in group 2 (185 ± 58 min vs. 139 ± 57 min; p < 0.001); however, fluoroscopy times were not different (23 ± 9 min vs. 23 ± 9 min; p = 0.49). After a follow-up of 12 months in all patients, 26 patients (52%) in group 1 versus 43 (82.7%) in group 2 were free from any AF/AT (p = 0.001) after a single procedure. No major complications occurred. CONCLUSIONS The use of pacing to ensure UE along the PVI line markedly improved near-term single-procedure success, compared with demonstration of bidirectional block alone. This additional endpoint significantly improved patient outcomes after PVI. (Unexcitability Along the Ablation as an Endpoint for Atrial Fibrillation Ablation; NCT01724437).

Isolation of Nicoletella semolina from Equine Tracheal Washes

The aim of the present study was to describe the prevalence of Nicoletella semolina in equine airways and its relationships with cytological evaluation of tracheal wash (TW). Samples were collected in the framework of routine bacteriological diagnostics of equine TW between May 2010 and June 2011. N semolina has been isolated, along with either common pathogens or contaminants, from 19 (1.8%) of the 1,054 TW samples. Median TW neutrophil counts (87.0%) in specimens from N semolina-positive horses were significantly different from those from N semolina-“negative” horses (52.0%). The data presented in this report may suggest considering such bacteria in horses clinically suffering from airway inflammation.

Behavioural isolation may facilitate homoploid hybrid speciation in cichlid fish

Hybrid speciation is constrained by the homogenizing effects of gene flow from the parental species. In the absence of post-mating isolation due to structural changes in the genome, or temporal or spatial premating isolation, another form of reproductive isolation would be needed for homoploid hybrid speciation to occur. Here, we investigate the potential of behavioural mate choice to generate assortative mating among hybrids and parental species. We made three-first-generation hybrid crosses between different species of African cichlid fish. In three-way mate-choice experiments, we allowed hybrid and nonhybrid females to mate with either hybrid or nonhybrid males. We found that hybrids generally mated nonrandomly and that hybridization can lead to the expression of new combinations of traits and preferences that behaviourally isolate hybrids from both parental species. Specifically, we find that the phenotypic distinctiveness of hybrids predicts the symmetry and extent of their reproductive isolation. Our data suggest that behavioural mate choice among hybrids may facilitate the establishment of isolated hybrid populations, even in proximity to one or both parental species.

Identification and isolation of small CD44-negative mesenchymal stem/progenitor cells from human bone marrow using elutriation and polychromatic flow cytometry

The method of isolation of bone marrow (BM) mesenchymal stem/stromal cells (MSCs) is a limiting factor in their study and therapeutic use. MSCs are typically expanded from BM cells selected on the basis of their adherence to plastic, which results in a heterogeneous population of cells. Prospective identification of the antigenic profile of the MSC population(s) in BM that gives rise to cells with MSC activity in vitro would allow the preparation of very pure populations of MSCs for research or clinical use. To address this issue, we used polychromatic flow cytometry and counterflow centrifugal elutriation to identify a phenotypically distinct population of mesenchymal stem/progenitor cells (MSPCs) within human BM. The MSPC activity resided within a population of rare, small CD45⁻CD73⁺CD90⁺CD105⁺ cells that lack CD44, an antigen that is highly expressed on culture-expanded MSCs. In culture, these MSPCs adhere to plastic, rapidly proliferate, and acquire CD44 expression. They form colony forming units-fibroblast and are able to differentiate into osteoblasts, chondrocytes, and adipocytes under defined in vitro conditions. Their acquired expression of CD44 can be partially downregulated by treatment with recombinant human granulocyte-colony stimulating factor, a response not found in BM-MSCs derived from conventional plastic adherence methods. These observations indicate that MSPCs within human BM are rare, small CD45⁻CD73⁺CD90⁺CD105⁺ cells that lack expression of CD44. These MSPCs give rise to MSCs that have phenotypic and functional properties that are distinct from those of BM-MSCs purified by plastic adherence.

Isolation and characterization of nine polymorphic microsatellite markers for the deep-sea shrimp Nematocarcinus lanceopes (Crustacea: Decapoda: Caridea)

Background: The shrimp Nematocarcinus lanceopes Bate, 1888 is found in the deep sea around Antarctica and sub-Antarctic islands. Previous studies on mitochondrial data and species distribution models provided evidence for a homogenous circum-Antarctic population of N. lanceopes. However, to analyze the fine-scale population genetic structure and to examine influences of abiotic environmental conditions on population composition and genetic diversity, a set of fast evolving nuclear microsatellite markers is required. Findings: We report the isolation and characterization of nine polymorphic microsatellite markers from the Antarctic deep-sea shrimp species Nematocarcinus lanceopes (Crustacea: Decapoda: Caridea). Microsatellite markers were screened in 55 individuals from different locations around the Antarctic continent. All markers were polymorphic with 9 to 25 alleles per locus. The observed heterozygosity ranged from 0.545 to 0.927 and the expected heterozygosity from 0.549 to 0.934. Conclusions: The reported markers provide a novel tool to study genetic structure and diversity in Nematocarcinus lanceopes populations in the Southern Ocean and monitor effects of ongoing climate change in the region on the populations inhabiting these.

Impact of cardiac computed tomography of the interatrial septum before pulmonary vein isolation

BACKGROUND Multidetector computed tomography (MDCT) may be useful to identify patients with patent foramen ovale (PFO). The aim of this study was to analyze whether a MDCT performed before pulmonary vein isolation reliably detects a PFO that may be used for access to the left atrium. METHODS AND RESULTS In 79 consecutive patients, who were referred for catheter ablation of symptomatic paroxysmal or persistent atrial fibrillation (AF), the presence of a PFO was explored by MDCT and transesophageal echocardiography (TEE). TEE was considered as the gold standard, and quality of TEE was good in all patients. In 16 patients (20.3%), MDCT could not be used for analysis because of artifacts, mainly because of AF. On TEE, a PFO was found in 15 (23.8%) of the 63 patients with usable MDCT. MDCT detected six PFO of which four were present on TEE. This corresponded to a sensitivity of 26.7%, a specificity of 95.8%, a negative predictive value of 80.7%, and a positive predictive value of 66.7%. The receiver operating characteristics curve of MDCT for the detection of PFO was 0.613 (95% confidence interval 0.493-0.732). CONCLUSIONS MDCT may detect a PFO before pulmonary isolation. However, presence of AF may lead to artifacts on MDCT impeding a meaningful analysis. Furthermore, in this study sensitivity and positive predictive value of MDCT were low and therefore MDCT was not a reliable screening tool for detection of PFO.

Isolation of Lautropia mirabilis from oral cavities of human immunodeficiency virus-infected children.

Lautropia mirabilis, a pleomorphic, motile, gram-negative coccus, has been isolated from the oral cavities of 32 of 60 (53.3%) children infected with human immunodeficiency virus (HIV) and 3 of 25 (12.0%) HIV-uninfected controls; the association of L. mirabilis isolation with HIV infection is significant (P < 0.001). All children in the study, both HIV-infected children and controls, were born to HIV-infected mothers. The presence of this bacterium was not associated with clinical disease in these children. The HIV-infected children with L. mirabilis did not differ from the HIV-infected children without L. mirabilis in immunological status, clinical status, or systemic medications. The role of HIV infection itself or concomitant factors in the establishment of L. mirabilis in the oral cavity remains to be elucidated.

DEVELOPMENTAL AND CELLULAR FUNCTIONS OF DELTA-CATENIN

Catenins have diverse and powerful roles in embryogenesis, homeostasis or disease progression, as best exemplified by the well-known beta-catenin. The less studied delta-catenin likewise contains a central Armadillo-domain. In common with other p120 sub-class members, it acts in a variety of intracellular compartments and modulates cadherin stability, small GTPase activities and gene transcription. In mammals, delta-catenin exhibits neural specific expression, with its knock-out in mice correspondingly producing cognitive defects and synaptic dysfunctions. My work instead employed the amphibian, Xenopus laevis, to explore delta-catenin’s physiological functions in a distinct vertebrate system. Initial isolation and characterization indicated delta-catenin’s expression in Xenopus. Unlike the pattern observed for mammals, delta-catenin was detected in most adult Xenopus tissues, although enriched in embryonic structures of neural fate as visualized using RNA in-situ hybridization. To determine delta-catenin’s requirement in amphibian development, I employed anti-sense morpholinos to knock-down gene products, finding that delta-catenin depletion results in developmental defects in gastrulation, neural crest migration and kidney tubulogenesis, phenotypes that were specific based upon rescue experiments. In biochemical and cellular assays, delta-catenin knock-down reduced cadherin levels and cell adhesion, and impaired activation of RhoA and Rac1, small GTPases that regulate actin dynamics and morphogenetic movements. Indeed, exogenous C-cadherin, or dominant-negative RhoA or dominant-active Rac1, significantly rescued delta-catenin depletion. Thus, my results indicate delta-catenin’s essential roles in Xenopus development, with contributing functional links to cadherins and Rho family small G proteins. In examining delta-catenin’s nuclear roles, I identified delta-catenin as an interacting partner and substrate of the caspase-3 protease, which plays critical roles in apoptotic as well as non-apoptotic processes. Delta-catenin’s interaction with and sensitivity to caspase-3 was confirmed using assays involving its cleavage in vitro, as well as within Xenopus apoptotic extracts or mammalian cell lines. The cleavage site, a highly conserved caspase consensus motif (DELD) within Armadillo-repeat 6 of delta-catenin, was identified through peptide sequencing. Cleavage thus generates an amino- (1-816) and carboxyl-terminal (817-1314) fragment each containing about half of the central Armadillo-domain. I found that cleavage of delta-catenin both abolishes its association with cadherins, and impairs its ability to modulate small GTPases. Interestingly, the carboxyl-terminal fragment (817-1314) possesses a conserved putative nuclear localization signal that I found is needed to facilitate delta-catenin’s nuclear targeting. To probe for novel nuclear roles of delta-catenin, I performed yeast two-hybrid screening of a mouse brain cDNA library, resolving and then validating its interaction with an uncharacterized KRAB family zinc finger protein I named ZIFCAT. My results indicate that ZIFCAT is nuclear, and suggest that it may associate with DNA as a transcriptional repressor. I further determined that other p120 sub-class catenins are similarly cleaved by caspase-3, and likewise bind ZIFCAT. These findings potentially reveal a simple yet novel signaling pathway based upon caspase-3 cleavage of p120 sub-family members, facilitating the coordinate modulation of cadherins, small GTPases and nuclear functions. Together, my work suggested delta-catenin’s essential roles in Xenopus development, and has revealed its novel contributions to cell junctions (via cadherins), cytoskeleton (via small G proteins), and nucleus (via ZIFCAT). Future questions include the larger role and gene targets of delta-catenin in nucleus, and identification of upstream signaling events controlling delta-catenin’s activities in development or disease progression.