Dirofilaria Immitis in Bulgaria: The First Genetic Baseline Data and an Overview of the Current Status.

Dirofilaria immitis, the agent of canine dirofilariosis, is a common parasite of domestic and wild carnivores with zoonotic potential and worldwide distribution, being endemic in many countries. Bulgaria is among European countries recognized as endemic for this heartworm parasite. In the present study, D. immitis adults recovered from pulmonary arteries of domestic dog and golden jackal originating from the Pazardzhik region in southern Bulgaria, and from red fox originating from the Plovdiv region in central-southern Bulgaria, were genetically analyzed in nuclear targets. The first PCR amplification of the internal transcribed region 2 (ITS2) of the ribosomal DNA with previously published D. immitis-specific primers yielded single fragments in size of 302 bp that is characteristic for these heartworms. PCR products of three isolates, resulted from the second amplification of the 5.8S-ITS2 region (235 bp) with pan-filarioid primers, were subjected to direct DNA sequencing. Identical nucleotide composition was detected across the screened target region for these Bulgarian isolates. When the 5.8S-ITS2 sequences were phylogenetically compared to the GenBank-retrieved D. immitis sequences in a worldwide context, the neighbor-joining analysis has shown three discrete clades. The first clade was composed of D. immitis isolates from Europe (including the studied Bulgarian samples), Asia and South America, in the second clade samples from Asia and South America were placed, whereas the third clade was formed by two Brazilian dog isolates originated from the north and southeast part of the country. The purpose of the present study was to verify the taxonomic characterization of D. immitis nematodes from Bulgaria based on morphology and compare their genetic structure with filariae obtained from the different world regions using molecular assays. It also summarizes previous epidemiological and ecological studies on the parasite distribution and prevalences in different hosts and regions undertaken so far in Bulgaria.

The structural link between polymerization and sickle cell disease.

Sickle hemoglobin molecules assemble into polymers composed of seven helically twisted double strands. Intermolecular contacts involving the mutation sites within the double strands are well established. We show that the same contact sites are present at the polymer surface on four of the ten exterior molecules in each layer, and demonstrate that the identical contact geometry can be achieved between polymers as found within the double strands. This provides a structural rationale for the exponential rate of polymer growth that characterizes the kinetics of gelation. This also gives a structural basis for the cross-linking which solidifies the polymer gel. In the absence of these surface contact regions sickle cell disease would be a much milder syndrome.

Nucleation and polymerization of sickle hemoglobin with Leu beta 88 substituted by Ala.

We have measured the solubility, and the rates of homogeneous and heterogeneous nucleation on sickle hemoglobin (HbS beta 6 Glu-->Val) additionally modified by site-directed mutagenesis to possess Ala rather than Leu at beta 88, which forms part of the receptor site for beta 6 Val in the sickle polymer. The solubility of the hemoglobin is increased at all temperatures, and is about 29 g/dl at 25 degrees C. Polymerization kinetics, induced by laser photolysis and observed by light-scattering intensity, showed exponential growth with rates about 300 times slower than experiments done on similar concentrations of HbS. When polymerization is carried out in small volumes, the time of measurable light-scattering signal to reach one-tenth of its final value (denoted as the tenth time) showed stochastic fluctuations, as is seen in pure HbS. Homogeneous nucleation rates were measured by observing distributions of tenth times and these rates were slowed by the mutation by almost 1000-fold relative to pure HbS. The kinetics, including the exponential progress curves and shape of the tenth time distributions, are well described by the double nucleation mechanism for polymerization. Analysis of the homogeneous nucleation rates leads to the surprising conclusion that the mutation has scarcely changed the energy of the intermolecular contacts despite the increase in solubility of the double mutant. This conclusion is supported by the stereochemistry of the modified contact site, in which the amount of exposed hydrophobic surface appears to be unchanged by the mutation. The increased solubility must therefore result from decreased motional freedom of molecules within the polymer, which could arise from tighter packing into the enlarged receptor pocket. This points up the ability of kinetic analysis to reveal important thermodynamic properties of assembly, and underlines the importance of the vibrational degrees of freedom in setting the final equilibrium constant. Chemical modifications to restrict vibrations and enhance the cost of polymerization may prove useful in constructing compounds to act as inhibitors of sickle cell gelation.