Georgi Radoslavov

Genetic diversity of liver flukes (Fasciola hepatica) from Eastern Europe

The genetic diversity of liver fluke populations in three different countries from Eastern Europe (Greece, Bulgaria, and Poland) was determined and compared with available data from other countries. Specifically, SNPs from regions of two nuclear genes, 28S rDNA, β-tubulin 3 and an informative region of the mitochondrial genome were examined. Two major lineages for the 28S rDNA gene based on the highly polymorphic 105th nucleotide position were found. These lineages were widely and almost equally spread not only through the countries studied but also in other investigated geographical areas. Two basic lineages and additional haplotypes were defined for the mtDNA gene region which consisted of the cytochrome c oxidase subunit III gene, transfer RNA histidine gene and cytochome b gene. The basic lineages were observed within Greek, Bulgarian, and Polish Fasciola hepatica populations but the distribution of additional haplotypes differed between the populations from the three countries. For the β-tubulin 3 gene multiple polymorphic sites were revealed but no explicit clades. The SNPs were spread unequally in all studied geographical regions with an evident distinction between the Greek and Polish specimens. Additional genotypes for the 28S rDNA region as well as haplotypes of the mtDNA region that were typical for the Greek or Polish populations were observed. Significant polymorphisms for β-tubulin 3 gene were displayed with decreasing percentage of presence within populations from Greece to Poland. There was an amino acid substitution in β-tubulin 3 protein found only among Polish specimens. It is hypothesized that genotypic differences between Greek, Bulgarian, and Polish liver fluke populations are due to territorial division and genetic drift in past epochs.

Conformational and functional analysis of the lipid binding protein Ag-NPA-1 from the parasitic nematode Ascaridia galli

Ag‐NPA‐1 (AgFABP), a 15 kDa lipid binding protein (LBP) from Ascaridia galli, is a member of the nematode polyprotein allergen/antigen (NPA) family. Spectroscopic analysis shows that Ag‐NPA‐1 is a highly ordered, α‐helical protein and that ligand binding slightly increases the ordered secondary structure content. The conserved, single Trp residue (Trp17) and three Tyr residues determine the fluorescence properties of Ag‐NPA‐1. Analysis of the efficiency of the energy transfer between these chromophores shows a high degree of Tyr‐Trp dipole‐dipole coupling. Binding of fatty acids and retinol was accompanied by enhancement of the Trp emission, which allowed calculation of the affinity constants of the binary complexes. The distance between the single Trp of Ag‐NPA‐1 and the fluorescent fatty acid analogue 11‐[(5‐dimethylaminonaphthalene‐1‐ sulfonyl)amino]undecanoic acid (DAUDA) from the protein binding site is 1.41 nm as estimated by fluorescence resonance energy transfer. A chemical modification of the Cys residues of Ag‐NPA‐1 (Cys66 and Cys122) with the thiol reactive probes 5‐({[(2‐iodoacetyl)amino]ethyl}amino) naphthalene‐1‐sulfonic acid (IAEDANS) and N,N′‐dimethyl‐N‐(iodoacetyl)‐N′‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)ethylenediamine (IANBD), followed by MALDI‐TOF analysis showed that only Cys66 was labeled. The observed similar affinities for fatty acids of the modified and native Ag‐NPA‐1 suggest that Cys66 is not a part of the protein binding pocket but is located close to it. Ag‐NPA‐1 is one of the most abundant proteins in A. galli and it is distributed extracellularly mainly as shown by immunohistology and immunogold electron microscopy. This suggests that Ag‐NPA‐1 plays an important role in the transport of fatty acids and retinoids.

The highly abundant protein Ag-Ibp55 from Ascaridia galli represents a novel type of lipid-binding proteins

Lipid-binding proteins exhibit important functions in lipid transport, cellular signaling, gene transcription, and cytoprotection. Their functional analogues in nematodes are nematode polyprotein allergens/antigens and fatty acid and retinoid-binding proteins. This work describes a novel 55-kDa protein, Ag-lbp55, purified from the parasitic nematode Ascaridia galli. By direct N-terminal sequencing, a partial amino acid sequence was obtained that allowed the design of oligonucleotide primers to obtain the full-length cDNA sequence. Sequence analysis revealed the presence of an N-terminal signal peptide of 25 amino acid residues and a FAR domain at the C terminus. Data base searches showed almost no significant homologies to other described proteins. The secondary structure of Ag-lbp55 was predominantly α-helical (65%) as shown by CD spectroscopy. It was found to bind with high affinity fatty acids (caprylic, oleic, and palmitic acid) and their fluorescent analogue dansylaminoundecanic acid. Immunolocalization showed that Ag-lbp55 is a highly abundant protein, mainly distributed in the inner hypodermis and extracellularly in the pseudocoelomatic fluid. A similar staining pattern was observed in other pathogenic nematodes, indicating the existence of similar proteins in these species.

Redescription of Aphalloides coelomicola Dollfus, Chabaud & Golvan, 1957 (Digenea, Opisthorchioidea) based on specimens from Knipowitschia caucasica (Berg) (Actinopterygii, Gobionellidae) from a Black Sea lagoon, with comments on the systematic position of the genus

The genus Aphalloides Dollfus, Chabaud & Golvan, 1957 consists of two species parasitic in the body cavity of sand gobies. Its systematic position in the superfamily Opisthorchioidea Looss, 1899 is unresolved and it has been placed by various authors in three families, i.e. Cryptogonimidae Ward, 1917, Heterophyidae Leiper, 1909 and Opisthorchiidae Looss, 1899. Its type-species, Aphalloidescoelomicola Dollfus, Chabaud & Golvan, 1957, is here reported from the Caucasian dwarf goby Knipowitschia caucasica (Berg) in the lagoon Atanasovsko Lake, Black Sea coast of Bulgaria (new geographical record). The species is redescribed based on light and scanning electron microscopy demonstrating some characters typical for the Cryptogonimidae but also characters distinguishing it from the other genera of the family such as the lack of tegumental spines and the presence of a short excretory vesicle, which does not extend into the forebody. Phylogenetic analysis of the D2-D3 expansion segments of the 28S rRNA gene suggests phylogenetic relationships of Aphalloidescoelomicola with the cryptogonimid Centrovarium lobotes (MacCallum, 1895). These data support the affiliation of the genus Aphalloides to the family Cryptogonimidae. The peculiar morphology of the species in the genus is explained by their unusual life-cycles characterised by progenetic development; sand gobies being simultaneously second intermediate and definitive hosts.

Calcaridorylaimus castaneae sp. n. (Nematoda, Dorylaimidae) from Bulgaria with an identification key to the species of the genus

Abstract An unknown species belonging to the genusCalcaridorylaimus Andrássy, 1986 was collected from the litter of broadleaf forests dominated by Castanea sativa Mill. and mixed with Quercus daleshampii Ten. and Fagus sylvatica L. on Belasitsa Mountain, south-western Bulgaria. Calcaridorylaimus castaneae sp. n. is characterised by its long body (1.4–2.1 mm), lip region practically not offset, vulva transverse, short odontostyle (14.5–16 μm) and tail (75.5–110.5 μm, c=14.7–23.6; c’=2.9–4.4) in females and 38–46 μm long spicules with small spur before their distant end in males. It is most similar to C. andrassyi Ahmad & Shaheen, 2004, but differs in having transverse vs pore-like vulva and shorter spicules (38–46 μm vs 52–57 μm). An identification key to the species of the genus Calcaridorylaimus is proposed. Phylogenetic analyses were performed on 18S and D2-D3 expansion domains of 28S rRNA genes by Neighbor-Joining, Maximum Likelihood and Bayesian Inference methods. The phylograms inferred from 18S sequences showed closest relationships of the new species with some species belonging to the genus Mesodorylaimus. However, insufficient molecular data for members of both genera do not allow the phylogenetic relationships of Calcaridorylaimus and the new species described herein to be elucidated.

Haplotype diversity in autochthonous Balkan cattle breeds

The Bulgarian Grey cattle (BGC) and theShorthorn Rhodopean cattle (SRC) are Balkan indigenousbreeds that are part of the Podolian and Brachicerous(Busha) cattle groups respectively. Concerning the mtDNAdiversity of Balkan cattle breeds, previous studies focusedon Busha cattle and Grey cattle populations.

A Novel Secretory Poly-Cysteine and Histidine-Tailed Metalloprotein (Ts-PCHTP) from Trichinella spiralis (Nematoda)

Background Trichinella spiralis is an unusual parasitic intracellular nematode causing dedifferentiation of the host myofiber. Trichinella proteomic analyses have identified proteins that act at the interface between the parasite and the host and are probably important for the infection and pathogenesis. Many parasitic proteins, including a number of metalloproteins are unique for the nematodes and trichinellids and therefore present good targets for future therapeutic developments. Furthermore, detailed information on such proteins and their function in the nematode organism would provide better understanding of the parasite - host interactions. Methodology/Principal Findings In this study we report the identification, biochemical characterization and localization of a novel poly-cysteine and histidine-tailed metalloprotein (Ts-PCHTP). The native Ts-PCHTP was purified from T. spiralis muscle larvae that were isolated from infected rats as a model system. The sequence analysis showed no homology with other proteins. Two unique poly-cysteine domains were found in the amino acid sequence of Ts-PCHTP. This protein is also the first reported natural histidine tailed protein. It was suggested that Ts-PCHTP has metal binding properties. Total Reflection X-ray Fluorescence (TXRF) assay revealed that it binds significant concentrations of iron, nickel and zinc at protein:metal ratio of about 1∶2. Immunohistochemical analysis showed that the Ts-PCHTP is localized in the cuticle and in all tissues of the larvae, but that it is not excreted outside the parasite. Conclusions/Significance Our data suggest that Ts-PCHTP is the first described member of a novel nematode poly-cysteine protein family and its function could be metal storage and/or transport. Since this protein family is unique for parasites from Superfamily Trichinelloidea its potential applications in diagnostics and treatment could be exploited in future.

An independent event of Neolithic cattle domestication on the South-eastern Balkans: evidence from prehistoric aurochs and cattle populations.

Abstract Neolithic/Chalcolithic livestock domestication is an important issue for understanding the mode of life and economics of ancient human communities. The Balkans appears to be a crucial point for clarifying the socio-economical interrelations between the Oldest Middle Eastern/Anatolian and newly formed cultures in Europe. Two main hypotheses regarding the early history of cattle domestication, from their ancestor – the aurochs, have been discussed: multipoint domestication centers or single point origin and subsequent worldwide dissemination. In this study, we provide molecular data about the Balkan aurochs for the first time as well as additional information for the Neolithic/Chalcolithic cattle populations in this geographic location. A total of seventeen samples from different ancient settlements were analyzed according to D-loop control region. The results did not show different genetic profile of wild and domestic populations. All haplotypes were found to belong to the basic macro-haplogroup T. The majority of specimens (n = 14) were defined to form a new Balkan-specific T6 haplogroup. Only two of the ancient samples analyzed were assigned to the T3 haplotype predominating in Europe. We attempt to throw new light on the earliest cattle domestication events in Europe, thus, the results presented are discussed in two directions: (a) The possibility of local independent domestication processes in Neolithic South-Eastern Europe; (b) The single point domestication in the Middle East and subsequent cattle dissemination in Europe. Our data does not exclude the possibility for independent domestication events followed by a second wave of parallel dissemination of cattle herds via the Mediterranean route.

SIGNIFICANCE OF MILK PROTEIN GENES POLYMORPHISM FOR BULGARIAN RHODOPEAN CATTLE: COMPARATIVE STUDIES

ABSTRACT The aim of the present research was to reveal the genotype profile of the local Bulgarian Rhodopean cattle population with respect to αs1-casein, kappa-casein andβ-lactoglobulin genes, by PCR-RFLP assay and conformational 2D PAGE. According to these profiles, it is possible to determine the association between each genotype and milk qualitative and quantitative traits and to establish the position of the breed with regard to the genetic diversity other European cattle breeds. The investigation also revealed the high significance of the genetic variants of the three above-mentioned milk protein genes for: Clarification of the influence of other breeds on the Bulgarian Rhodopean cattle population; Obtaining data for genetic drift among the Bulgarian Rhodopean cattle population, the indigenous Shorthorn Rhodopean cattle population as a predecessor breed and the Jersey cow as a main improvement breed; Gathering the necessary information about preservation of this local breed, which is important for the Bulgarian biodiversity gene fund.

Application of Milk Proteins Genetic Polymorphism for Selection and Breeding of Dairy Cows in Bulgaria

© 2012 Hristov et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Application of Milk Proteins Genetic Polymorphism for Selection and Breeding of Dairy Cows in Bulgaria