No abstract

Contagious agalactia (CA), an infectious disease of small ruminants, caused by Mycoplasma agalactiae, is responsible for severe losses to dairy sheep production with substantial socioeconomic impacts on small-scale farmers. The diagnosis of CA is still problematic, time-consuming and requires well-equipped labs for confirmation of outbreaks. Therefore, rapid, accurate and cost-effective diagnostic tests are urgently needed. This work aims to validate a novel Loop-Mediated Isothermal Amplification (LAMP) test, based on the p40 target gene, for the detection of M. agalactiae in dairy sheep in order to confirm its potential practical use as a rapid and cheap field test. The LAMP system proposed in this study consists of a portable device composed of real-time fluorometer with the automatic interpretation of results displayed in a tablet. A total of 110 milk samples (90 positives and 20 negatives) were analysed to optimise the analysis procedure and to investigate the efficacy and robustness of the LAMP method. All samples were analysed using LAMP and conventional real-time PCR to compare the diagnostic sensitivity of the methods. The sensitivity of the LAMP was 10-fold higher than that of real-time PCR, with a detection limit up to 103 CFU/ml. The LAMP assay was able to detect M. agalactiae in 81 of 90 (90%, 95%CI 0.84–0.96) positive milk samples compared to 69 (77%, 95%CI 0.59–0.95) positive samples detected by real-time PCR; no positive signal occurred for any of the negative milk samples in either test. Therefore, the LAMP assay was found to be more sensitive than real-time PCR, low-cost, easy to perform, fast and not affected by contamination, indicating its potential as an effective diagnostic tool in the field level for the diagnosis of CA.

The aim of the work was to develop a new, robust, portable diagnostic system based on the latest development of loop-mediated isothermal amplification (LAMP) technology for the detection of the main causative agent of contagious agalactia, an important disease of small ruminants. The integrated diagnostic system consists of a portable instrument and a kit, specific for the pathogen. The kit is ready to use and provides both the reagents for a rapid nucleic acid extraction from milk, and the amplification reagents using LAMP technology, The test showed a high sensitivity identifying only M.agalactiae DNA with a detection limit of less than 100 CFU / ml in milk samples, exceeding the traditional laboratory diagnostics for speed and handling The test also proved to be more tolerant to the inhibitory effect of milk components that affect amplification in the PCR. These preliminary results show that the LAMP system can be a practical and effective diagnostic tool for the diagnosis in the field of M.agalactiae pathogen.

Sicily (Italy) hosts a "relict", endemic population of the birds Alectoris graeca whitakeri commonly known as Sicilian Rock Partridge. In the last decades, due to the risk of restocking with other European and Asiatic species for hunting purpose, a study was carried out to investigate the potential risk of hybridisation. The mtDNA control-region and nuclear microsatellites were genotyped. Due to the importance of the species, samples were mainly characterized by feather and stool samples, and rarely by carcasses found in the environment, from year 2011 to 2012. A panel of 7 microsatellite loci was validated. Three multiplexes that allowed the simultaneous amplification of 3 microsatellites, and 2 for other two microsatellites, for a total of 7 markers, were utilized. Results showed the occurrence of hybridization both towards the Middle Eastern species,A. chukar and the Northern European species, A. rufa. 18.5% of the samples were collected from the wild environment showed an high degree of hybridization. This fact, even if linked to a small number of samples, highlights a potential risk of hybridization 2 in 4 Sicilian provinces and underlines the importance of further investigations to understand the entity of the problem.

Sicily (Italy) hosts an ‘endangered’, endemic population of Alectoris graeca whitakeri, commonly known as Sicilian Rock Partridge. An EU-funded Life Natura 2000 project has been founded, involving Istituto Zooprofilattico Sperimentale of Sicily for veterinary aspects: a total of 15 Sicilian Rock Partridge found dead were collected, identified and processed by postmortem examination and laboratory investigations. The evidence of internal parasites was the most relevant finding, showing different types of infections by Nematoda, Cestoda and Coccidia. 60 per cent of these cases were infected with more than one parasite. In one single case, a pathogenic strain of Escherichia coli related to granulocytic lesions in liver was found and another cause of death was due to respiratory disease caused by Aspergillus fumigatus. The study represents the first veterinary report on this rare species and underlines the importance to monitor the health status of wild species in the Italian environment in order to preserve local biodiversity.