Entrance and survival of <i>Brucella pinnipedialis</i> hooded seal strain in human macrophages and epithelial cells

Marine mammal Brucella spp. have been isolated from pinnipeds (B. pinnipedialis) and cetaceans (B. ceti) from around the world. Although the zoonotic potential of marine mammal brucellae is largely unknown, reports of human disease exist. There are few studies of the mechanisms of bacterial intracellular invasion and multiplication involving the marine mammal Brucella spp. We examined the infective capacity of two genetically different B. pinnipedialis strains (reference strain; NTCT 12890 and a hooded seal isolate; B17) by measuring the ability of the bacteria to enter and replicate in cultured phagocytes and epithelial cells. Human macrophage-like cells (THP-1), two murine macrophage cell lines (RAW264.7 and J774A.1), and a human malignant epithelial cell line (HeLa S3) were challenged with bacteria in a gentamicin protection assay. Our results show that B. pinnipedialis is internalized, but is then gradually eliminated during the next 72 – 96 hours. Confocal microscopy revealed that intracellular B. pinnipedialis hooded seal strain colocalized with lysosomal compartments at 1.5 and 24 hours after infection. Intracellular presence of B. pinnipedialis hooded seal strain was verified by transmission electron microscopy. By using a cholesterol-scavenging lipid inhibitor, entrance of B. pinnipedialis hooded seal strain in human macrophages was significantly reduced by 65.8 % (± 17.3), suggesting involvement of lipid-rafts in intracellular entry. Murine macrophages invaded by B. pinnipedialis do not release nitric oxide (NO) and intracellular bacterial presence does not induce cell death. In summary, B. pinnipedialis hooded seal strain can enter human and murine macrophages, as well as human epithelial cells. Intracellular entry of B. pinnipedialis hooded seal strain involves, but seems not to be limited to, lipid-rafts in human macrophages. Brucella pinnipedialis does not multiply or survive for prolonged periods intracellulary

An evaluation of Irish cattle herds with inconclusive serological evidence of bovine brucellosis

Since 1998, there has been a steady decline in herd restrictions and de-populations in Ireland due to bovine brucellosis. There is concern that the interpretation of laboratory results may become increasingly problematic, as brucellosis prevalence falls in Ireland. Therefore, the purpose of the current study was to evaluate the infection status of Irish herds and animals with inconclusive serological evidence of bovine brucellosis. During 12 months from September 1, 2004, laboratory and observational epidemiological data were collected from all Irish herds where animal testing identified at least one animal with a complement fixation test (CFT) reading greater than zero and/or a positive result to the indirect enzyme-linked immunosorbent assay (iELISA). Due to the observational nature of the study, we have robust estimates of the relative, but not the absolute, performance of the CFT, iELISA and brucellin skin test (BST). Herds were divided into three categories (Group A, B or C) on the basis of test results at initial assessment. A total of 639 herds were enrolled into the study, and observed for at least two years following enrolment. A rising CFT titre, with a CFT reading of 111 International CFT Units (IU) or greater at the subsequent blood test, was generally associated with herds where other evidence of infection was also available. Knowledge of the CFT reading at the initial and a subsequent blood test proved useful in distinguishing false-positive and true-positive brucellosis results. There was poor correlation between the CFT and iELISA results, and between the CFT and BST results. As a result of this study, national policy has been modified to include re-sampling of all animals with CFT readings of 20 IU or greater. This project has also led to a reduction in the number of herds restricted, as well as restriction duration. It has also contributed to a reduction in the number of herds listed for contiguous tests, and therefore the potential for contiguity testing of false positive results

Highly specific and sensitive non-radioactive molecular identification of Phytophthora cinnamomi

In response to the need for a faster, more reliable method for identifying Phytophthora cinnamomi in cork oak soils in Portugal, a simple, fast, sensitive molecular identification method is described. It is based on a colorimetric assay which involves an oligonucleotide capture probe covalently immobilised on microtitration wells, a multi-biotinylated oligonucleotide detection probe and the PCR-amplified target DNA. The target DNA is a 349 bp DNA fragment partially covering the 3'-translated and 3'- untranslated regions of the cinnamomin gene. When the specificity of the PCR reaction was evaluated in vitro using isolates of P. cinnamomi and eight other Phytophthora species, including the related P. cambivora, it was specific to P. cinnamomi. When 30 isolates of P. cinnamomi from oak roots in southern Portugal were assayed, 26 gave a strong positive response. The assay has a sensitivity of about 2±5 genome equivalents of P. cinnamomi. The reason for the negative response of four isolates remains unclear

Modelling the control of bovine brucellosis in India.

Brucellosis imposes substantial impacts on livestock production and public health worldwide. A stochastic, age-structured model incorporating herd demographics was developed describing within- and between-herd transmission of Brucella abortus in dairy cattle herds. The model was fitted to data from a cross-sectional study conducted in Punjab State of India and used to evaluate the effectiveness of control strategies under consideration. Based on model results, stakeholder acceptance and constraints regarding vaccine supply, vaccination of replacement calves in large farms should be prioritized. Test and removal applied at early stages of the control programme where seroprevalence is high would not constitute an effective or acceptable use of resources because significant numbers of animals would be 'removed' (culled or not used for breeding) based on false positive results. To achieve sustained reductions in brucellosis, policymakers must commit to maintaining vaccination in the long term, which may eventually reduce frequency of infection in the livestock reservoir to a low enough level for elimination to be a realistic objective. This work provides key strategic insights into the control of brucellosis in India, which has the largest cattle population globally, and a general modelling framework for evaluating control strategies in endemic settings

Antitumor Effect by Either FLASH or Conventional Dose Rate Irradiation Involves Equivalent Immune Responses.

The capability of ultrahigh dose rate FLASH radiation therapy to generate the FLASH effect has opened the possibility to enhance the therapeutic index of radiation therapy. The contribution of the immune response has frequently been hypothesized to account for a certain fraction of the antitumor efficacy and tumor kill of FLASH but has yet to be rigorously evaluated. To investigate the immune response as a potentially important mechanism of the antitumor effect of FLASH, various murine tumor models were grafted either subcutaneously or orthotopically into immunocompetent mice or in moderately and severely immunocompromised mice. Mice were locally irradiated with single dose (20 Gy) or hypofractionated regimens (3 × 8 or 2 × 6 Gy) using FLASH (≥2000 Gy/s) and conventional (CONV) dose rates (0.1 Gy/s), with/without anti-CTLA-4. Tumor growth was monitored over time and immune profiling performed. FLASH and CONV 20 Gy were isoeffective in delaying tumor growth in immunocompetent and moderately immunodeficient hosts and increased tumor doubling time to &gt;14 days versus &gt;7 days in control animals. Similar observations were obtained with a hypofractionated scheme, regardless of the microenvironment (subcutaneous flank vs ortho lungs). Interestingly, in profoundly immunocompromised mice, 20 Gy FLASH retained antitumor activity and significantly increased tumor doubling time to &gt;14 days versus &gt;8 days in control animals, suggesting a possible antitumor mechanism independent of the immune response. Analysis of the tumor microenvironment showed similar immune profiles after both irradiation modalities with significant decrease of lymphoid cells by ∼40% and a corresponding increase of myeloid cells. In addition, FLASH and CONV did not increase transforming growth factor-β1 levels in tumors compared with unirradiated control animals. Furthermore, when a complete and long-lasting antitumor response was obtained (&gt;140 days), both modalities of irradiation were able to generate a long-term immunologic memory response. The present results clearly document that the tumor responses across multiple immunocompetent and immunodeficient mouse models are largely dose rate independent and simultaneously contradict a major role of the immune response in the antitumor efficacy of FLASH. Therefore, our study indicates that FLASH is as potent as CONV in modulating antitumor immune response and can be used as an immunomodulatory agent

Design and synthesis of imidazoline derivatives active on glucose homeostasis in a rat model of type II diabetes.2. Syntheses and biological activities of 1,4-dialkyl-,1,4-dibenzyl and 1-benzyl-4-alkyl-2-(4',5'-dihydro-1' H-imidazol-2'yl)piperazines and isosteric analogues of imidazoline

International audienc

Brucellosis remains a neglected disease inthe developing world: a call forinterdisciplinary action

Brucellosis places significant burdens on the human healthcare system and limits the economic growth of individuals, communities, and nations where such development is especially important to diminish the prevalence of poverty. The implementation of public policy focused on mitigating the socioeconomic effects of brucellosis in human and animal populations is desperately needed. When developing a plan to mitigate the associated consequences, it is vital to consider both the abstract and quantifiable effects. This requires an interdisciplinary and collaborative, or One Health, approach that consists of public education, the development of an infrastructure for disease surveillance and reporting in both veterinary and medical fields, and campaigns for control in livestock and wildlife species

MLVA-16 typing of 295 marine mammal <i>Brucella</i> isolates from different animal and geographic origins identifies 7 major groups within <i>Brucella ceti</i> and <i>Brucella pinnipedialis</i>

BackgroundSince 1994, Brucella strains have been isolated from a wide range of marine mammals. They are currently recognized as two new Brucella species, B. pinnipedialis for the pinniped isolates and B. ceti for the cetacean isolates in agreement with host preference and specific phenotypic and molecular markers. In order to investigate the genetic relationships within the marine mammal Brucella isolates and with reference to terrestrial mammal Brucella isolates, we applied in this study the Multiple Loci VNTR (Variable Number of Tandem Repeats) Analysis (MLVA) approach. A previously published assay comprising 16 loci (MLVA-16) that has been shown to be highly relevant and efficient for typing and clustering Brucella strains from animal and human origin was used.Results294 marine mammal Brucella strains collected in European waters from 173 animals and a human isolate from New Zealand presumably from marine origin were investigated by MLVA-16. Marine mammal Brucella isolates were shown to be different from the recognized terrestrial mammal Brucella species and biovars and corresponded to 3 major related groups, one specific of the B. ceti strains, one of the B. pinnipedialis strains and the last composed of the human isolate. In the B. ceti group, 3 subclusters were identified, distinguishing a cluster of dolphin, minke whale and porpoise isolates and two clusters mostly composed of dolphin isolates. These results were in accordance with published analyses using other phenotypic or molecular approaches, or different panels of VNTR loci. The B. pinnipedialis group could be similarly subdivided in 3 subclusters, one composed exclusively of isolates from hooded seals (Cystophora cristata) and the two others comprising other seal species isolates.ConclusionThe clustering analysis of a large collection of marine mammal Brucella isolates from European waters significantly strengthens the current view of the population structure of these two species, and their relative position with respect to the rest of the Brucella genus. MLVA-16 is confirmed as being a rapid, highly discriminatory and reproducible method to classify Brucella strains including the marine mammal isolates. The Brucella2009 MLVA-16 genotyping database available at http://mlva.u-psud.fr is providing a detailed coverage of all 9 currently recognized Brucella species

OspA heterogeneity of Borrelia valaisiana confirmed by phenotypic and genotypic analyses

BACKGROUND: Although European Borrelia burgdorferi sensu lato isolates have been divided into five genospecies, specific tools for the serotype characterization of only three genospecies are available. Monoclonals antibodies (mAbs) H3TS, D6 and I17.3 identify B. burgdorferi sensu stricto (ss.), B. garinii and B. afzelii respectively, but no mAbs are available to identify B. valaisiana. In the same way, specific primers exist to amplify the OspA gene of B. burgdorferi ss., B. garinii and B. afzelii. The aim of the study was to develop species-specific mAb and PCR primers for the phenotypic and genetic identification of B. valaisiana. RESULTS: This study describes a mAb that targets OspA of B. valaisiana and primers targeting the OspA gene of this species. As the monoclonal antibody A116k did not react with strains NE231, M7, M53 and Frank and no amplification was observed with strains NE231, M7 and M53, the existence of two subgroups among European B. valaisiana species was confirmed. CONCLUSIONS: The association of both monoclonal antibody A116k and primers Bval 1F and Bval 1R allows to specific identification of the B. valaisiana isolates belonging to subgroup 1