Determination of an efficient and reliable method for DNA extraction from ticks

Molecular detection of pathogenic microorganisms in ticks is based on DNA amplification of the target pathogen; therefore, extraction of DNA from the tick is a major step. In this study, we compared three different tick DNA extraction protocols based on an enzymatic digestion by proteinase K followed by DNA extraction by a commercial kit (method 1), or on mortar crushing, proteinase K digestion and phenol/chloroform DNA extraction (method 2) and fine crushing with a beads beater, proteinase K digestion and DNA extraction using a commercial kit (method 3). The absence of PCR inhibitors and the DNA quality were evaluated by PCR amplification of the tick mitochondrial 16S rRNA gene using tick-specific primers. With method 1, 23/30 (77%) of the samples were extracted; with method 2, 30/31 (97%) of the samples were extracted and with method 3, 30/30 (100%) of the samples were extracted. DNA extraction efficiency using method 3 is significantly higher than DNA extraction efficiency using method 1 (100% versus 77%, P < 0.05). There was no significant difference between methods 2 and 3. Method 3 was however more adapted to cohort studies than method 2. This technique was validated for cohort tick DNA extraction and applicable to the treatment of small samples such as nymphs and soft ticks with 100% efficiency.     

Modeling spatial and temporal transmission of foot-and-mouth disease in France: identification of high-risk areas

Foot-and-mouth disease is one of the most contagious diseases of animal livestock. We used statistical tools to explore the dynamics of epidemics and to evaluate the consequences of virus reintroduction in France. We developed a stochastic farm-based model adapted to the French farm structure from previous modeling works following the 2001 epidemic in the United Kingdom. This model depends upon the distance between the 280,000 French farms and on species type (e.g. cows and sheep) and it tracks each animal's farm status at any given day. Since data were only available at the town scale, the farm location and the number of animals in each farm were simulated over the surface area of each French town, as well as the number of mixed farms. Based on 200 simulations of the model, our results allowed for the study of local disease transmission, since it begins simulations once limitation of movement is put into place. On average, the same 50 randomly chosen initially infected farms would lead to 1,110 infected farms (610; 1,590) when two control strategies (culling within 0.5 km from an infected farm and vaccination within 3 km) are put into place. Regions with high densities of cows and sheep (e.g. Pays-de-la-Loire) are high-risk zones, confirming that the epidemic process depends upon the location and the type of initially infected farms (size, species type). The results of this model highlight the importance of Geographical Information Systems (GIS) to obtain more precise data concerning herds.     

Intracranial,extraneural ectopic lymph node in a bovine (Bos taurus)

The brain from a field necropsied 8‐month‐old feedlot heifer presenting with an acute history of depression, lethargy, dyspnoea and anorexia was evaluated grossly and by histopathology. The meninges overlying the left cerebral hemisphere contained a 12 × 26 × 32 mm, dark red, soft, ovoid mass. Histologic examination of this tissue revealed a well‐organized lymph node with normal architecture. Organization of reactive lymphoid tissue resembling normal lymph node architecture may occur under chronic stimulation. However, there are no known aggregates of lymphoid tissue present within the cranial vault in any veterinary species. This is the first reported case of an intracranial ectopic lymph node in any species.     

Molecular analysis of the sex chromosomes of the platyfish Xiphophorus maculatus: Towards the identification of a new type of master sexual regulator in vertebrates

In contrast to mammals and birds, fish display an amazing diversity of genetic sex determination systems, with frequent changes during evolution possibly associated with the emergence of new sex chromosomes and sex‐determining genes. To better understand the molecular and evolutionary mechanisms driving this diversity, several fish models are studied in parallel. Besides the medaka (Oryzias latipes Temminck and Schlegel, 1846) for which the master sex‐determination gene has been identified, one of the most advanced models for studying sex determination is the Southern platyfish (Xiphophorus maculatus, Günther 1966). Xiphophorus maculatus belongs to the Poeciliids, a family of live‐bearing freshwater fish, including platyfish, swordtails and guppies that perfectly illustrates the diversity of genetic sex‐determination mechanisms observed in teleosts. For X. maculatus, bacterial artificial chromosome contigs covering the sex‐determination region of the X and Y sex chromosomes have been constructed. Initial molecular analysis demonstrated that the sex‐determination region is very unstable and frequently undergoes duplications, deletions, inversions and other rearrangements. Eleven gene candidates linked to the master sex‐determining gene have been identified, some of them corresponding to pseudogenes. All putative genes are present on both the X and the Y chromosomes, suggesting a poor degree of differentiation and a young evolutionary age for platyfish sex chromosomes. When compared with other fish and tetrapod genomes, syntenies were detected only with autosomes. This observation supports an independent origin of sex chromosomes, not only in different vertebrate lineages but also between different fish species.     

Quantification of Common Wheat Adulteration of Durum Wheat Pasta Using Real‐Time Quantitative Polymerase Chain Reaction (PCR)

Common wheat adulteration of durum wheat pasta was quantified using real‐time duplex polymerase chain reaction (PCR). The total DNA content of pasta was determined by amplifying part of a wheat gene encoding a lipid transfer protein, and common wheat DNA was quantified by amplifying part of the puroindoline‐b gene. Under the conditions defined by this study, for pasta with a theoretical adulteration of 3%, the experimentally determined mean value was 2.6–3.4%, depending on drying temperature. Pure durum wheat pastas were distinguished from adulterated pastas without ambiguity. This study demonstrates the feasibility of using real‐time duplex PCR to quantify common wheat adulteration of pasta dried at high temperature, quantification that was impossible with the French official peroxidase‐marker method.