Ralstonia solanacearum as a potato pathogen in Serbia: Characterization of strains and influence on peroxidase activity in tubers

Since 2011, the outbreaks of brown rot caused by Ralstonia solanacearum race 3, biovar 2, phylotype IIB-1 (R3/B2/PIIB-1) have significantly compromised potato production in Serbia. During 6 years of monitoring (2013–2018) among 3,524 potato tuber samples, 344 were found positive for brown rot disease. R. solanacearum R3/B2/PIIB-1 was isolated from seven cultivars among 12 monitored, and in five localities among 17 monitored. Cultivar Lady Claire was found to have the highest disease frequency (31.98%). A total of 78 isolates were identified by R. solanacearum-specific primer pairs (PS-1/PS-2 and OLI-1/Y-2), as well as the following tests: restriction fragment length polymorphism analysis, biovar determination, immunofluorescence, biochemical analysis, and pathogenicity. The genetic composition of 36 selected isolates assessed using multilocus sequence analysis with seven genes (adk, gapA, gdhA, gyrB, ppsA, hrpB, and fliC) showed that all isolates originating from Serbian potato were homogeneous. By using the TCS algorithm of concatenated sequences to get insight into the phylogeography of isolates and other R. solanacearum strains deposited in the NCBI database, we showed that their origin is undetermined. Peroxidase (POD) activity was measured in brown rotted potato tubers. A positive correlation was found between POD activity and disease severity rated on the analysed tubers. In general, POD activity increased by 2–22 times in vascular necrotic tissues compared to non-necrotic ones, and depended on disease severity but not on cultivar. Native polyacrylamide gel electrophoresis analysis of POD profiles resulted in a total of 10 distinct POD isoforms, of which PODs 3–5 were highly intensified in response to R. solanacearum.     

Variations in Pectoral Girdle Muscles in Dogs

Muscle variations take the form of additional muscle bundle or belly, unusual muscle origin or termination, as well as complete muscle absence. Knowledge of such variations not only has clinical importance for guiding surgery, but also can help reveal phylogenetic relationships. To improve our understanding of muscle variations in dogs, 57 medium‐sized, cross‐breed male and female adult animals were dissected as part of a gross anatomy course between 2005 and 2011. Variations in pectoral girdle muscles were observed in 7 (12.3%) dogs and took the form of an additional muscle bundle in the brachiocephalicus muscle (n = 2), in the omotransversarius muscle (n = 2), in the cervical part of the rhomboideus muscle (n = 2) and in the cervical part of the serratus ventralis muscle (n = 1). Muscle variation was bilateral in only one dog; it involved the omotransversarius muscle. The variations did not seem to be sex dependent. Such variations can appear regularly in dogs and should be taken into consideration during anatomical dissection.     

Isolation and molecular biological investigations of avian poxviruses from chickens, a turkey, and a pigeon in Croatia

In the last 3 yr, several outbreaks of avian poxviruses (APVs) have been observed in different parts of Croatia. Four strains of APVs, from chickens, a pigeon, and a turkey, were isolated from cutaneous lesions by inoculation onto the chorioallantoic membranes (CAM) of 12-day-old specific-pathogen-free chicken embryos. The resulting proliferative CAM lesions contained eosinophilic cytoplasmic inclusion bodies. The characteristic viral particles of poxvirus were detected in the infected CAM and also in the infected tissues by transmission electron microscopy. Further identification and differentiation of the four various APVs were carried out by the use of a polymerase chain reaction (PCR) combined with restriction enzyme analysis. Using one primer set, which framed a region within the APV 4b core protein gene, it was possible to detect APV-specific DNA from all four tested isolates. PCR results revealed no recognizable differences in size of amplified fragments between the different APVs from chickens, turkey, and pigeon. Restriction enzyme analysis of PCR products using NlaIII showed the same cleavage pattern for turkey and chicken isolates and a different one for the pigeon isolate. Multiplex PCR for direct detection of APV and reticuloendotheliosis virus (REV) was carried out to determine the possible integration of REV in the genome of isolated APVs. The obtained results revealed that REV was present in chicken and turkey strains of poxviruses, whereas the pigeon isolate was negative. It is not known whether the avipoxvirus vaccine strain used in Croatia is contaminated with REV or if the REV is naturally contaminating Croatian field strains of fowl poxvirus. The latter is indicated by the negative REV finding in the pigeon, which was not vaccinated. The results of the present study indicate the reemergence of fowlpox in Croatia, where infections have not been recorded since 1963 and never confirmed etiologically.     

Antimicrobial and free radical scavenging activities of Teucrium montanum

The antimicrobial and free radical scavenging activities of petroleum ether, chloroform, ethyl acetate, n-butanol and water extracts of Teucrium montanum were investigated. Ethyl acetate, chloroform and n-butanol extracts expressed a wide range of inhibiting activity against both Gram (+) and Gram (-) bacteria. n-Butanol extract possessed potent DPPH free radical scavenging activity.     

Immune complex-based vaccine for pig protection against parvovirus

The insoluble immune complexes (ICs) were prepared under the conditions of double immunodiffusion in gel, using the suspension of the ultrasound treated PK-15 cell-line infected with porcine parvovirus (PPV) containing both viral particles and viral proteins, as well as pig or rabbit anti-PPV polyclonal immune sera. The immunodiffusion performed in an agarose gel allows only viral subunits with a molecular mass equal to or less than 1000 kDa, rather than the viral particles, to diffuse through the gel and reach the point where the immunoprecipitate is to be formed. The immunoprecipitation under the conditions of the diffusion ensures the optimal, i.e. equimolar ratio of both immunoprecipitating components, antibody/antigen in the IC. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the Western blot analyses showed the ICs were composed of two proteins, a protein in which molecular mass corresponded to the VP2 of the PPV and a protein with a molecular mass of the IgG. This suggests that the ICs are mainly composed of the VP2 antigen and IgG class antibodies. The potency of the IC-vaccines prepared in the form of a water-in-oil-in-water emulsion was compared with that of a commercially available, inactivated oil vaccine. The vaccination of gilts, 6 weeks before mating, with the IC containing allogeneic pig antibodies, resulted in the development of high and long-lasting anti-PPV antibody titres, similar to those generated by the licenced vaccine (P > 0.01). The content of the virus material administered by the IC was twice lower than that in the licenced vaccine. Neither systemic nor local reactions were observed in the gilts during the period of the trial with the IC vaccine. The number of viable piglets per litter varied between 9 and 12 and no signs of the PPV infection were detected. Rabbits were used as one of the alternative laboratory animal models accepted for the testing of the vaccine against the PPV. The rabbit humoral immune response generated by the IC containing the allogeneic antibodies were higher than that generated by the ICs containing the xenogeneic pig antibodies. It was similar to that generated by two-times higher content of the virus material administered by a commercially available vaccine. The IC-based vaccines belong to non-replicating, subunit vaccines, which are both ecologically convenient and the safest vaccines of all.     

Associations between defensin polymorphism and somatic cell count in milk and milk utility traits in Jersey dairy cows

The aim of the study was to investigate associations between combined defensin genotypes (CDGs) and somatic cell count (SCC) in Jersey cows. The study included a herd of 184 dairy Jersey cows from Wielkopolska region in Poland. Polymerase chain reaction-restriction fragment length polymorphism analysis with TaqI restrictase established the existence of 12 CDGs with a frequency of over 1%. The most frequent were A1A2B1B2C1C2 and A1A2B1B2C1- genotypes with a frequency of 56.9783% and 12.5% respectively. The study also confirmed the existence of a statistically significant association between SCC and the year of study, season, lactation stage and cow. The highest SCC (transformed into a logarithmic scale) was found in the milk of cows with A1-B1-C1C2 genotype, whereas the lowest one in cows with A2-B1B2C1C2 genotype. Another aim of the project was to study the association between CDG and milk production traits, such as daily milk yield and fat and protein content. CDG was found to be a significant factor affecting daily milk yield and non-significant for fat and protein content. The highest daily milk yield was observed in cows with CDGs A1A2B1B2C2- and -A2-B1B2C1-, whereas the lowest one was characteristic of -A2-B1-C1C2 and A1A2B1-C1- animals. Fat content was found to be related to CDG genotype in the opposite way; the highest values were recorded in animals with -A2-B1B2C1- genotype, the lowest - in animals with -A2-B1-C1C2 genotype. Similar results were observed in protein content in milk -A2-B1B2C1-, the highest content and -A2-B1-C1C2, the lowest content. The results confirm the hypothesis of using CDG as an SCC marker. However, further studies should be conducted to confirm these results before CDG can be used as a marker in a mass selection of dairy cows.     

Gas-phase ligation of Fe+ and Cu+ ions with some flavonoids

It was assumed that gas-phase ligation of metal monocations by flavonoids might provide some insight on the intrinsic antioxidant activity of the latter. Thus, the ligation of Fe+ and Cu+ ions by apigenin (1), luteolin (2), kaempferol (3), quercetin (4), myricetin (5), and naringenin (6) was investigated in the gas phase in a Fourier transform mass spectrometer (FTMS). Both of the metal ions, which were produced by laser desorption ionization (LDI), bind consecutively to two neutral flavonoid molecules either with or without the simultaneous loss of some part (H, CO, H2O) of the latter. The flavonoids are present in the instrument at steady concentrations. The formation of flavonoid positive ions by charge exchange is also a common observation but is accompanied, in some cases, by a loss of H, CO, or H2O fragments. The reaction paths and observed fragmentations are presented. The results are supported by DFT B3LYP calculations that indicate a preference for metal ion attack at C-ring and not at the B-ring site considered to be mainly responsible for flavonoid antioxidant activity.     

Essential and toxic elements in seafood available in poland from different geographical regions

The concentrations of 15 elements (Cu, Fe, Mn, Zn, Co, Ni, Cr, Se, Cd, Pb, Hg, Ca, Na, K, and Mg) were determined in the edible parts of shellfish on sale in the local market in Gdańsk. The samples consisted of three groups--crustaceans, molluscs, and surimi--that are processed to different degrees. For the purposes of this analysis, they were dried, homogenized, and digested in an automatic microwave system. The samples were analyzed quantitatively for Cu, Zn, Fe, Mn, Co, Ni, Cr, Mg, Na, K, and Ca (F-AAS), Cd and Pb (GF-AAS), Se (HG-AAS), and Hg (CV-AAS). The elemental levels detected in shellfish were compared to those in cod, herring, pork, beef, chicken, and eggs. The recommended dietary allowance (RDA) of essential elements and the provisional tolerable weekly intake (PTWI) of toxic elements were estimated. With factor analysis of the data, taxonomically different groups of raw and processed shellfish could be distinguished.     

Antioxidant and antiradical activities in extracts of hazelnut kernel (Corylus avellana L.) and hazelnut green leafy cover

Phenolic compounds in the aqueous systems were extracted, from hazelnut kernel (HK) and hazelnut green leafy cover (HGLC), with 80% (v/v) ethanol (HKe and HGLCe) or 80% (v/v) acetone (HKa and HGLCa). The extracts were examined for their phenolic and condensed tannin contents and phenolic acid profiles (free and esterified fractions) as well as antioxidant and antiradical activities by total antioxidant activity (TAA), antioxidant activity in a beta-carotene-linoleate model system, scavenging of DPPH (2,2-diphenyl-1-picrylhydrazyl) radical, and reducing power. Significant differences (p < 0.05) in the contents of total phenolics, condensed tannins, and TAA existed among the extracts that were examined. HGLCa extract had the highest content of total phenolics (201 mg of catechin equivalents/g of extract), condensed tannins (542 mg of catechin equivalents/g of extract), and TAA (1.29 mmol of Trolox equivalents/g of extract) followed by HGLCe, HKa, and HKe extracts, respectively. Five phenolic acids (gallic acid, caffeic acid, p-coumaric acid, ferulic acid, and sinapic acid) were tentatively identified and quantified, among which gallic acid was the most abundant in both free and esterified forms. The order of antioxidant activity in a beta-carotene-linoleate model system, the scavenging effect on DPPH radical, and the reducing power in all extracts were in the following order: HGLCa > HGLCe > HKa > HKe. These results suggest that both 80% ethanol and acetone are capable of extracting phenolics, but 80% acetone was a more effective solvent for the extraction process. HGLC exhibited stronger antioxidant and antiradical activities than HK itself in both extracts and could potentially be considered as an inexpensive source of natural antioxidants.     

Mechanisms of toxicity of 3-alkylpyridinium polymers from marine sponge Reniera sarai

Polymeric 3-alkylpyridinium salts (poly-APS) present in the marine sponge Reniera sarai show a broad spectrum of biological activities. They are lytic to erythrocytes and various other mammalian cells, enabling the transfection of the latter with alien DNA. Furthermore, they show inhibitory effects to marine bacteria and can inhibit fouling of micro- and macroorganisms to submerged surfaces. Finally, poly-APS act as potent cholinesterase inhibitors. The kinetics of acetylcholinesterase inhibition by poly-APS in vitro is complex and comprises several successive phases ending in irreversible inhibition of the enzyme. The latter is accounted for by aggregation and precipitation of the enzyme-inhibitor complexes. Poly-APS are lethal to rats in concentrations above 2.7 mg/kg. Monitoring of the basic vital functions and histopathological analysis showed that the effects directly ascribable to acetylcholinesterase inhibition are only observed after application of lower concentrations of poly-APS. At higher concentrations, such effects were masked by other, more pronounced and faster developing lethal effects of the toxin, such as haemolysis and platelet aggregation.