Chlamydiose abortive en Tunisie (tests de diagnostic, caractérisation moléculaire, phylogénétique de souches de Chlamydophila et étude de la protection du vaccin vivant 1B)

Les avortements d'origine infectieuse représentent une pathologie des plus importantes pour les élevages des petits ruminants en Tunisie. La chlamydiose constitue par ailleurs, une des principales causes d'avortements infectieux. Le but ce travail était d'isoler et de caractériser des souches pour définir dans le contexte tunisien, les conditions optimales de diagnostic et de prévention. Huit souches de Chlamydophila ont été isolées. Sept souches appartenaient à l'espèce C. abortus et une à l'espèce C. pecorum d'après leur réponse antigénique en MIF et le profil de digestion de l'espace intergénique 16S-23S par PCR-RFLP. Par la suite, la prospection plus précise du génome a été entreprise pour étudier le degré de parenté entre les souches françaises et tunisiennes et éventuellement mettre en évidence des marqueurs épidémiologiques. L'étude de protection du vaccin vivant 1B vis à vis des souches tunisiennes a été étudiée. Nous avons également vérifié dans un modèle murin que l'efficacité du vaccin vivant 1B n'était pas modifiée par la vaccination contre la fièvre Q.L'efficacité du vaccin n'ayant jamais été étudiée vis à vis de C. pecorum, nous avons utilisé un modèle murin plus sensible, en dénombrant les chlamydia par la technique des plages de lyse 5 jours après l'épreuve virulente dans les rates de souris gravides ou non ainsi que dans les placentas et fœtus des souris gravides.The abortions of infectious origin represent a most important pathology in small ruminant livestock in Tunisia. Chlamydiosis constitute a main cause of infectious abortions. The goal of this work was the isolation and the characterisation of some Chlamydophila strains in order to determine in the Tunisian context, the optimal conditions of diagnosis and of prevention. Eight strains of Chlamydophila were isolated. Seven of them belonged to C. abortus and one to C. pecorum according to their antigenic and genomic profiles in MIF and PCR-RFLP of the 16S-23S intergenic spaces, respectively. Thereafter, a more precise prospecting of the genome was enterprise in order to study the degree of relationship between French and Tunisian strains. The protection of the live vaccine strain 1B against two-selected Tunisian Chlamydophila abortus strains was studied. We have verified also in a mouse model that the efficiency of living vaccine 1B had not modified by the vaccination against the fever Q.The efficiency of vaccine having never been studied against C. pecorum strains. We used a more sensitive mouse model, by comparing the reduction in number of bacteria 5 days after the challenge in the spleen of pregnant mice or not as well as the placentas and the foetuses of pregnant mice.TOURS-BU Sciences Pharmacie (372612104) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Simultaneous differential detection of <it>Chlamydophila abortus, Chlamydophila pecorum </it>and <it>Coxiella burnetii </it>from aborted ruminant's clinical samples using multiplex PCR

<p>Abstract</p> <p>Background</p> <p>Chlamydiosis and Q fever, two zoonosis, are important causes of ruminants' abortion around the world. They are caused respectively by strictly intracellular and Gram negative bacterium <it>Chlamydophila abortus (Cp. abortus</it>) and <it>Coxiella burnetii (C. burnetii). Chlamydophila pecorum (Cp. pecorum) </it>is commonly isolated from the digestive tract of clinically inconspicuous ruminants but the abortive and zoonotic impact of this bacterium is still unknown because <it>Cp. pecorum </it>is rarely suspected in abortion cases of small ruminants. We have developed a multiplex PCR (m-PCR) for rapid simultaneous differential detection of <it>Cp. abortus</it>, <it>Cp. pecorum </it>and <it>C. burnetii </it>in clinical samples taken from infected animals.</p> <p>Results</p> <p>Specific PCR primers were designed and a sensitive and specific m-PCR was developed to detect simultaneously, in one tube reaction, three specific fragments of 821, 526 and 687-bp long for <it>Cp. abortus, Cp. pecorum </it>and <it>C</it>. <it>burnetii </it>respectively. This m-PCR assay was performed on 253 clinical samples taken from infected ruminant's flocks that have showed problems of abortion diseases. Thus, 67 samples were infected by either one of the three pathogens: 16 (13 vaginal swabs and 3 placentas) were positive for <it>Cp. abortus</it>, 2 were positive for <it>Cp. pecorum </it>(1 vaginal swab and 1 placenta) and 49 samples (33 vaginal swabs, 11 raw milks, 4 faeces and 1 placenta) were positive for <it>C. burnetii</it>. Two vaginal swabs were m-PCR positive of both <it>Cp. abortus </it>and <it>C. burnetii </it>and none of the tested samples was shown to be infected simultaneously with the three pathogens.</p> <p>Conclusion</p> <p>We have successfully developed a rapid multiplex PCR that can detect and differentiate <it>Cp. abortus</it>, <it>Cp. pecorum </it>and <it>C. burnetii; </it>with a good sensitivity and specificity. The diagnosis of chlamydiosis and Q fever may be greatly simplified and performed at low cost. In addition, the improvement in diagnostic techniques will enhance our knowledge regarding the prevalence and the pathogenetic significance of Q fever and chlamydiosis.</p

Isolation and characterisation of local strains of Chlamydophila abortus (Chlamydia psittaci serotype 1) from Tunisia

Chlamydiosis is one of the major diseases that can lead to abortion in ewes. Since 1997, in 5 regions of Tunisia, Chlamydia-related abortions have been reported in 15 sheep and goat flocks. One hundred and sixty-six sera and 50 vaginal swab samples were collected from adult ewes. Chlamydial antigens were detected in 29 (58%) of the vaginal swabs using Enzyme Linked Immunsorbent Assay (ELISA) while 9 (18%) were positive by cell culture. Five strains were recovered from 4 different sheep flocks. Monoclonal antibody profiles and restriction fragment length polymorphism (RFLP) analysis of the 16S-23S rRNA spacer region showed that these isolates were C. abortus. Using amplified fragment length polymorphism (AFLP), these Tunisian strains were shown to exhibit the same pattern as strains isolated in France.Isolement et typage de souches locales de Chlamydophila abortus (Chlamydia psittaci sérotype 1). La chlamydiose est une des principales cause d'avortements infectieux en Tunisie. Lors de la période d'agnelage de 1997, 166 prises de sang et 50 écouvillons vaginaux ont été prélevés dans 15 troupeaux répartis sur 5 gouvernorats et ayant eu des problèmes d'avortements. Des chlamydia ont été mises en évidence dans 29 (58 %) écouvillons vaginaux appartenant à 13 troupeaux différents par ELISA directement sur l'écouvillon vaginal et 9 (18 %) après multiplication sur cellules. Cinq souches tunisiennes appartenant à 4 troupeaux différents ont ainsi pu être isolées. Leur caractérisation par une panoplie d'anticorps monoclonaux et par étude du profil de restriction de l'espace intergénique 16S-23S a démontré qu'elles appartenaient toutes à l'espèce Chlamydophila abortus. Par amplification sélective de fragment de restriction les souches tunisiennes présentaient le profil caractéristique des souches françaises

Acinetobacter baumannii: Setup and characterization of pneumonia and septicemia preclinical models with longitudinal monitoring using bioluminescent strains

International audienceBacterial multidrug resistance (MDR) is responsible of up to 4.9 million deaths,with the MDR ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus,Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, andEnterobacter spp) being the leading cause of nosocomial infections worldwide.Treating these MDR bacteria represents one of the greatest challenges in clinicalpractice.Among ESKAPE pathogens, the emergence of rapidly spreading carbapenem-resistant Acinetobacter baumannii, combined to the lack of available active antibioticshas led the WHO to raise the threat level to "Urgent" and classify it in the criticalgroup of its Bacterial Priority Pathogens List (2024). These observations call for anurgent need for new anti-microbials.The goal of this study was to setup preclinical models mimicking the mainclinical infections caused by A. baumannii (bacteraemia and pneumonia) usingbioluminescent strains that allow to perform longitudinal monitoring of the bacterialdissemination and assess antibacterial candidate’s efficac

In vitro characterization of fluorescence by unbound excitation from luminescence: Broadening the scope of energy transfer

International audienceEnergy transfer mechanisms represent the basis for an array of valuable tools to infer interactions in vitro and in vivo, enhance detection or resolve interspecies distances such as with resonance. Based upon our own previously published studies and new results shown here we present a novel framework describing for the first time a model giving a view of the biophysical relationship between Fluorescence by Unbound Excitation from Luminescence (FUEL), a conventional radiative excitation-emission process, and bioluminescence resonance energy transfer. We show here that in homogeneous solutions and in fluorophore-targeted bacteria, FUEL is the dominant mechanism responsible for the production of red-shifted photons. The minor resonance contribution was ascertained by comparing the intensity of the experimental signal to its theoretical resonance counterpart. Distinctive features of the in vitro FUEL signal include a macroscopic depth dependency, a lack of enhancement upon targeting at a constant fluorophore concentration cf and a non-square dependency on cf. Significantly, FUEL is an important, so far overlooked, component of all resonance phenomena which should guide the design of appropriate controls when elucidating interactions. Last, our results highlight the potential for FUEL as a means to enhance in vivo and in vitro detection through complex media while alleviating the need for targeting

The chemokine receptor CXCR6 controls the functional topography of interleukin-22 producing intestinal innate lymphoid cells

Interleukin-22 (IL-22) plays a critical role in mucosal defense, although the molecular mechanisms that ensure IL-22 tissue distribution remain poorly understood. We show that the CXCL16-CXCR6 chemokine-chemokine receptor axis regulated group 3 innate lymphoid cell (ILC3) diversity and function. CXCL16 was constitutively expressed by CX3CR1+ intestinal dendritic cells (DCs) and coexpressed with IL-23 after Citrobacter rodentium infection. Intestinal ILC3s expressed CXCR6 and its ablation generated a selective loss of the NKp46+ ILC3 subset, a depletion of intestinal IL-22, and the inability to control C. rodentium infection. CD4+ ILC3s were unaffected by CXCR6 deficiency and remained clustered within lymphoid follicles. In contrast, the lamina propria of Cxcr6−/− mice was devoid of ILC3s. The loss of ILC3-dependent IL-22 epithelial stimulation reduced antimicrobial peptide expression that explained the sensitivity of Cxcr6−/− mice to C. rodentium. Our results delineate a critical CXCL16-CXCR6 crosstalk that coordinates the intestinal topography of IL-22 secretion required for mucosal defense

In vivo excitation of nanoparticles using luminescent bacteria

The lux operon derived from Photorhabdus luminescens incorporated into bacterial genomes, elicits the production of biological chemiluminescence typically centered on 490 nm. The light-producing bacteria are widely used for in vivo bioluminescence imaging. However, in living samples, a common difficulty is the presence of blue-green absorbers such as hemoglobin. Here we report a characterization of fluorescence by unbound excitation from luminescence, a phenomenon that exploits radiating luminescence to excite nearby fluorophores by epifluorescence. We show that photons from bioluminescent bacteria radiate over mesoscopic distances and induce a red-shifted fluorescent emission from appropriate fluorophores in a manner distinct from bioluminescence resonance energy transfer. Our results characterizing fluorescence by unbound excitation from luminescence, both in vitro and in vivo, demonstrate how the resulting blue-to-red wavelength shift is both necessary and sufficient to yield contrast enhancement revealing mesoscopic proximity of luminescent and fluorescent probes in the context of living biological tissues