Technical note: molecular typing of Corynebacterium bovis isolates by pulsed-field gel electrophoresis.

A typing method for Corynebacterium bovis based on pulsed-field gel electrophoresis (PFGE) is described and applied to type 162 strains isolated from mastitic milk from 57 Friesian dairy herds in northern Spain. Isolates were identified to the species level using the API-Coryne system and a polymerase chain reaction-restriction fragment length polymorphism of the 16S rDNA amplicon. Cultures in logarithmic phase were harvested, mixed with agarose, and treated with lisozyme and proteinase K to release intact genomic DNA that was digested with XbaI and submitted to PFGE. Seven different pulsotypes were identified; none of them were similar to that of the reference strain (ATCC 7715). The most frequent pulsotype was pattern I (51.9%) followed by pattern II (29.6%). The majority of the herds showed infection with one single pulsotype, but 12 herds yielded 2 pulsotypes, and one herd had 3 pulsotypes. No relationship was found between the geographical location of the herd and the pulsotype. This is the first description of a typing method for C. bovis; the technique might prove useful for epidemiological studies on this species.

Differential expression of calretinin in the developing and regenerating zebrafish visual system.

Calretinin is a calcium-binding protein which participates in a variety of functions including calcium buffering and neuronal protection. It also serves as a developmental marker of retinal ganglion cells (RGCs). In order to study the role of calretinin in the development and regeneration of RGCs, we have studied its pattern of expression in the retina at different developmental stages, as well as during optic nerve regeneration by means of immunohistochemistry. During development, calretinin is found for the first time in RGCs when they connect with the optic tectum. Optic nerves from adult zebrafish were crushed and after different survival times, calretinin expression in the retina, optic nerve tract and optic tectum was studied. From the day of crushing to 10 days later, calretinin expression was found to be downregulated within RGCs and their axons, as was also observed during the early developmental stages of RGCs, when they are not committed to a definite cell phenotype. Moreover, 13 days after lesion, when the regenerating axons arrived at the optic tectum, a recovery of calretinin immunoreactivity within the RGCs was observed. These results indicate that calretinin may play an important role during optic nerve regeneration, Thus, the down-regulation of Calretinin during the growth of the RGC axons towards the target during development as well as during their regeneration after injury, indicates that an increase the availability of cytosolic calcium is integral to axon outgrowth thus recapitulating the pattern observed during development.

PrP polymorphisms in Basque sheep breeds determined by PCR-restriction fragment length polymorphism and real-time PCR.

Two new PCR-based methods were developed to decode prion protein (PrP) gene polymorphisms at codons 136, 154 and 171: a PCR-restriction fragment length polymorphism (RFLP) analysis consisting of two PCR reactions followed by three enzymatic digestions, and a real-time PCR consisting of four reactions with seven fluorogenic probes. Both methods were used to study the distribution of PrP gene polymorphisms in a representative sample (1297 animals) of the populations of the two native breeds of sheep of the Spanish Basque Country, Latxa and Carranzana. Fourteen genotypes were found in the Latxa breed, in which ARQ/ARQ was the genotype most frequently observed (49.3 per cent), followed by ARR/ARQ (32.6 per cent) and ARQ/ARH (5.8 per cent). The genotype associated with the highest resistance to scrapie (ARR/ARR) was present in 5 per cent of the animals analysed. Similar results were observed in the Carranzana sheep.