Improved Strategy for Molecular Characterization of African Swine Fever Viruses from Sardinia, Based on Analysis of p30, CD2V and I73R/I329L Variable Regions.

African swine fever virus (ASFV) is the aetiological agent of a highly lethal haemorrhagic disease affecting pigs that inflicts significant economic damage on the swine industry. ASF is present in many African countries, in several eastern and central European countries and in Sardinia (Italy). Sequence analyses of variable genomic regions have been extensively used for molecular epidemiological studies of ASFV isolates. Previous sequencing data of genes that codify for viral protein p54, p72 and the central variable region (CVR) within the B602L gene revealed that Sardinian isolates show a very low level of variability. To achieve a finer level of discrimination among such closely related viruses, in this study, we have chosen three different genome regions to investigate the within-genotype relationships and to provide a more accurate assessment of the origin of outbreaks. The analysis of p30 and I73R/I329L sequences obtained from ASFV collected in Sardinia over a 13-years period confirms a remarkable genetic stability in these regions. The sequence comparison of the protein encoded by the EP402R gene (CD2v), carried out on various strains from 1978 to 2014, revealed a temporal subdivision of Sardinian viruses into two subgroups: one group includes the historical isolates from 1978 to 1990, and the second one is comprised of the viruses collected from 1990 until 2014. These data, together with those obtained from CVR within the B602L gene analysis, demonstrated that the viruses circulating in Sardinia belong to p72 genotype I, but have undergone genetic variations in two different regions of the genome since 1990. We proposed the cytoplasmic region of CD2v protein as a new genetic marker that could be use to analyse ASFVs from different locations to track virus spread. Our study reaffirms the need to analyse other genome regions in order to improve the molecular characterization of ASFV.

Experimental vaccination against Mycoplasma agalactiae using different inactivated vaccines.

Five sets of vaccines were prepared using Mycoplasma agalactiae washed cultures inactivated with phenol (1), formalin (2), heat-treatment (3), sodium hypochlorite (4) and saponin (5). All sets, except for saponin-vaccine, were adjuvated with aluminium hydroxide. Five groups of six sarda ewes were inoculated twice before pregnancy, once during pregnancy and challenged during the lactation period. Monthly blood samples were taken from the vaccinated sheep and from 12 controls: sera were assayed by immunoblotting, ELISA and growth inhibition tests. Four control sheep were infected by intracanalicular route with pooled mycoplasmas at concentrations of 10(4), 10(5), 10(6) and 10(7) CCU. The challenge involved using infected milk to contaminate the remaining sheep. All the controls and some ewes from groups 2, 3 and 4 developed contagious agalactia. Ewes vaccinated with phenol- and saponin-inactivated mycoplasmas resisted experimental challenge. These results suggest that these two vaccines are effective and that their use could limit the diffusion of M. agalactiae infection.

Comparison of restriction pattern polymorphism of Mycoplasma agalactiae and Mycoplasma bovis by pulsed field gel electrophoresis.

Mycoplasma agalactiae and Mycoplasma bovis are closely related species in phylogenetic terms. Pulsed Field Gel Electrophoresis (PFGE) of SmaI, EclXI, Bsi WI, MluI, BssHII, SalI, XhoI, NruI and ApaI digested DNAs were used to analyse and to compare restriction fragment length polymorphism between M. agalactiae and M. bovis and to estimate their genome sizes. SmaI, EclXI and Bsi WI enzymes cleaved DNAs of both microrganisms. MluI, BssHII, SalI, XhoI and NruI digested only M. agalactiae DNA whereas ApaI cut only M. bovis DNA. The total DNA length was established to be 945 +/- 8.4 Kb for M. agalactiae and 961 +/- 18.9 Kb for M. bovis.