First detection of Waddlia chondrophila in Africa using SYBR Green real-time PCR on veterinary samples.

Waddlia chondrophila is a strict intracellular microorganism belonging to the order Chlamydiales that has been isolated twice from aborted bovine fetuses, once in USA and once in Germany. This bacterium is now considered as an abortigenic agent in cattle. However, no information is available regarding the presence of this bacterium in Africa. Given the low sensitivity of cell culture to recover such an obligate intracellular bacterium, molecular-based diagnostic approaches are warranted. This report describes the development of a quantitative SYBR Green real-time PCR assay targeting the recA gene of W. chondrophila. Analytical sensitivity was 10 copies of control plasmid DNA per reaction. No cross-amplification was observed when testing pathogens that can cause abortion in cattle. The PCR exhibited a good intra-run and inter-run reproducibility. This real-time PCR was then applied to 150 vaginal swabs taken from Tunisian cows that have aborted. Twelve samples revealed to be Waddlia positive, suggesting a possible role of this bacterium in this setting. This new real-time PCR assay represents a diagnostic tool that may be used to further study the prevalence of Waddlia infection

Molecular prevalence of Chlamydia and Chlamydia-like bacteria in Tunisian domestic ruminant farms and their influencing risk factors

Chlamydia and Chlamydia-like bacteria are well known to infect several organisms and may cause a wide range of diseases, particularly in ruminants. To gain insight into the prevalence and diversity of these intracellular bacteria, we applied a pan-Chlamydiales real-time PCR to 1,134 veterinary samples taken from 130 Tunisian ruminant herds. The true adjusted animal population-level prevalence was 12.9% in cattle, against 8.7% in sheep. In addition, the true adjusted herd-level prevalence of Chlamydiae was 80% in cattle and 25.5% in sheep. Chlamydiales from three familylevel lineages were detected indicating a high biodiversity of Chlamydiales in ruminant herds. Our results showed that Parachlamydia acanthamoebae could be responsiblefor bovine and ovine chlamydiosis in central-eastern Tunisia. Multivariable logistic regression analysis at the animal population level indicated that strata and digestive disorders variables were the important risk factors of bovine and ovine chlamydiosis. However, origin and age variables were found to be associated withbovine and ovine chlamydiosis, respectively. At the herd level, risk factors for Chlamydia positivity were as follows: abortion and herd size for cattle against breeding system, cleaning frequency, quarantine, use of disinfectant and floor type for sheep. Paying attention to these risk factors will help improvement of control programs against this harmful zoonotic disease

Protoplasting impact on polyketide activity and characterization of the interspecific fusants from Streptomyces

The influence of protoplasting and protoplast regeneration on antibiotic activity, transfer of biosynthesis encoding genes in local Streptomyces spp. CN207 was studied. The frequency of regenerated protoplasts in the lag phase was 1.7x103 CFU/ml, in the beginning of the exponential phase 0.4x102 CFU/ml, in the exponential growth phase 2.5x103 CFU/ml, and 1.0x105 CFU/ml in stationary phase. The protoplast formation and regeneration technique resulted in a new isolate strain ofStreptomyces spp.PR01 that produced approximately 5 fold more Streptomyces spp. CN207 antibiotic. The protoplast fusion resulted in increased isolation of variants with higher antibiotic activity.Recombinant Streptomyces coelicolor PF04 was increased 10 times more than the wild strain. Theprocesses also affected on the strain resistance to some antibiotics but had no effect on the components of the antibiotic. The characteristics of this recombinant product were similar considerably to Streptomyces spp. CN207 product. Our data, in principal, indicate the possibility of transferring antibiotics cluster genes by fusion and provide a starting point for genetic and biochemicalinvestigations of CN207 biosynthesis

Detection of WWE2-related Lentisphaerae by 16S rRNA gene sequencing and fluorescence in situ hybridization in landfill leachate

International audienceWe collected samples of anaerobic landfill leachate from municipal solid waste landfill (Vert-le-Grand, France) and constructed 16S rRNA clone libraries using primers targeting Planctomycetes and relatives (Pla46F and 1390R). Analyses of 16S rRNA gene sequences resulted in the abundant representation of WWE2-related Lentisphaerae, members of the phylum Lentisphaerae, in the clone library (98% of the retrieved sequences). Although the sequences that are phylogenetically affiliated with the cultured isolate Victivallis vadensis were identified (WWE2 subgroup II), the majority of the sequences were affiliated with an uncultured Lentisphaerae lineage (WWE2 subgroup I). We designed oligonucleotides probes targeting the specific 16S rRNA gene regions of those 2 subgroups. Fluorescence in situ hybridization confirmed the abundance of the uncultivated WWE2 subgroup I in our leachate samples.Nous avons récolté des échantillons anaérobies du lixiviat d'une décharge municipale de déchets solides (Vert-le-Grand, France), et nous avons construit des banques de clones d'ARNr 16S à l'aide d'amorces ciblant Planctomycetes et les bactéries apparentées (Pla46F et 1390R). Les analyses des séquences géniques de l'ARNr 16S ont révélé que les séquences du groupe WWE2 relié aux Lentisphaerae, appartenant au phylum Lentisphaerae étaient abondamment représentées dans la banque de clones (98 % des séquences retrouvées). Même si des séquences affiliées d'un point de vue phylogénique à l'isolat cultivé Victivallis ont été identifiées (sous-groupe II de WWE2), la majorité des séquences étaient affiliées à un lignage non cultivé de Lentisphaerae (sous groupe I de WWE2). Nous avons conçu des sondes d'oligonucléotides qui ciblaient des régions spécifiques du gène de l'ARNr 16S de ces 2 sous-groupes. L'hybridation in situ fluorescente a confirmé l'abondance du sous-groupe I de WWE2 non cultivé dans les échantillons de lixiviat

Prévalence et identification d’isolats humains et alimentaires de Listeria spp. par puce à ADN en Tunisie

International audienceButs de l’étudeL’objectif de ce travail est d’évaluer la prévalence de Listeria spp. dans différentes matrices alimentaires et de caractériser des isolats alimentaires et humains.Matériel et méthodesEntre 2005 et 2007, 100 échantillons alimentaires prélevés dans les marchés de Tunis ont été analysés ; cinq souches humaines de Listeria monocytogenes ont été étudiées. La caractérisation des isolats de L. monocytogenes a été réalisée par multiplex PCR sérogroupage et par électrophorèse sur gel en champ pulsé (PFGE) appliquant l’enzyme Ascl et Apal. Nous avons développé une puce à ADN afin de différencier les espèces au sein du genre Listeria.RésultatsLa prévalence de Listeria spp. dans les échantillons alimentaires a été estimée à 14 %. Deux isolats ont été identifiés L. monocytogenes et 12 L. innocua. La méthode de puce à ADN par sa précision a permis de distinguer entre les différentes espèces de Listeria spp. Les résultats étaient conformes à l’identification biochimique. Les isolats alimentaires de L. monocytogenes ont été assignés au sérogroupe IIa (sérovar 1/2a). Cependant, les isolats humains ont été assignés au sérogroupe IVb (sérovars 4b). Ces isolats ont présenté une similarité importante par PFGE. Les isolats alimentaires de L. monocytogenes ont été classés en deux pulsotypes différents. Ces pulsotypes étaient différents de celui des souches humaines.ConclusionNos résultats confirment la présence de Listeria spp. dans différentes matrices alimentaires collectées à Tunis. Des efforts supplémentaires devraient être déployés afin de prendre en considération le risque de toxi-infections alimentaires liées à L. monocytogenes et d’identifier les sources potentielles d’infection