Transformation of Petroleum Saturated Hydrocarbons during Soil Bioremediation Experiments

This paper presents transformations of saturated hydrocarbons of petroleum type pollutants during ex situ bioremediation of soil on the pilot heap (halde), during a period of 6 months, within the grounds of Petroleum Refinery Pan?evo (Serbia). Samples for analysis were taken in time intervals of 2 weeks (P₁–P₁₂ samples). Organic substance was extracted by Soxhlet’s method and quantified. Isoprenoid aliphatics, in particular pristane and phytane, and polycyclic aliphatics of sterane and triterpane types in saturated hydrocarbon fractions were analysed by GC-MS (SIM method). Significant amounts of n-alkanes have not been detected. The MS-chromatogram revealed only marginal amounts of pristane and phytane in sample P₁. Pristane and phytane occurred in sample P₈, and in even higher quantities in the final sample P_12. The proceeding bioremediation process was accompanied by the decrease of the relative amounts of pentacyclic terpanes of hopane type, compared to tri- and tetracyclic terpanes. In the initial sample P₁ the distribution of steranes and hopanes follows a pattern, which is characteristic for crude oils. However, their identification by SIM method was not possible in samples P₈ and P₁₂ because of the reduced concentration. The observed changes in the alkane fractions’ compositions may be considered as atypical, referring to the fact that during oil biodegradation under natural conditions, decomposition of isoprenoids occurs much easier and faster than decomposition of polycyclic alkanes of tri-, tetra- and pentacyclic terpane, sterane and diasterane types, after the decomposition of n-alkanes has been almost completed.     

LAMP assay and rapid sample preparation method for on‐site detection of flavescence dorée phytoplasma in grapevine

In Europe the most devastating phytoplasma associated with grapevine yellows (GY) diseases is a quarantine pest, flavescence dorée (FDp), from the 16SrV taxonomic group. The on‐site detection of FDp with an affordable device would contribute to faster and more efficient decisions on the control measures for FDp. Therefore, a real‐time isothermal LAMP assay for detection of FDp was validated according to the EPPO standards and MIQE guidelines. The LAMP assay was shown to be specific and extremely sensitive, because it detected FDp in all leaf samples that were determined to be FDp infected using quantitative real‐time PCR. The whole procedure of sample preparation and testing was designed and optimized for on‐site detection and can be completed in one hour. The homogenization procedure of the grapevine samples (leaf vein, flower or berry) was optimized to allow direct testing of crude homogenates with the LAMP assay, without the need for DNA extraction, and was shown to be extremely sensitive.     

Molecular tracing of the transmission routes of bois noir in Mediterranean vineyards of Montenegro and experimental evidence for the epidemiological role of Vitex agnus‐castus (Lamiaceae) and associated Hyalesthes obsoletus (Cixiidae)

Epidemiological aspects and transmission routes of bois noir (BN), a grapevine yellows disease induced by ‘Candidatus Phytoplasma solani’, have been exhaustively studied in the affected vineyards of continental Europe but not in the Mediterranean coastal zone. Because ‘Ca. Phytoplasma solani’ and its principal vector Hyalesthes obsoletus presumably originate from the Mediterranean, gaining knowledge of the epidemiological peculiarities of the disease in this area is essential for understanding its global spread and diversification, as well as for designing local management strategies. In this study, molecular epidemiology was applied to trace transmission pathways of ‘Ca. Phytoplasma solani’ in the Mediterranean vineyards of Montenegro, using multilocus sequence typing of tuf, vmp1 and stamp genes of the isolates associated with various hosts. Thus, ‘Ca. Phytoplasma solani’ was tracked from a tentative reservoir plant (inoculum source) through an associated vector population to the infected grapevine. Three pathways of transmission were documented, originating from Urtica dioica, Convolvulus arvensis and Vitex agnus‐castus; however, only the route originating from U. dioica was direct, whereas the latter two were overlapping and could be intermixed. Vitex agnus‐castus is a natural source of ‘Ca. Phytoplasma solani’, representing an important link in disease epidemiology in the Mediterranean and a possible origin of several genotypes occurring in central Europe. Experimental confirmation of the role of Vitex‐associated H. obsoletus in BN transmission in Montenegrin vineyards indicates its tentative role as a vector in the wide area of the Mediterranean, where some of the major wine‐producing regions are located.     

Mycorrhizal colonization and lead distribution in root tissues of Norway spruce seedlings

Non-inoculated spruce seedlings (Picea abies Karst.) and spruce seedlings colonized with Lactarius rufus Fr., Pisolithus tinctorius Coker & Couch or Paxillus involutus Fr. were grown for 35 or 37 weeks in a microcosm system on two types of natural forest humus differing in Pb content. Using X-ray microanalysis, the distribution and content of Pb in the tissues of mycorrhizal and non-mycorrhizal root tips were compared. No significant difference in the Pb contents of root cortex cell walls of non-mycorrhizal and seedlings colonized by Lactarius rufus, Pisolithus tinctorius, or indigenous mycorrhizal fungi (mainly Tylospora sp.) was found. However, in root tips of seedlings colonized by Paxillus involutus, due to a higher binding capacity for cations, the Pb content in cell walls of the root cortex were higher than in non-mycorrhizal roots. Pb contents in cell walls of the cortex of mycorrhizal and non-mycorrhizal roots were 3 times higher in plants growing in humus with a high Pb content than in plants growing in humus with a low Pb content. It is concluded that increasing contents of Pb in the organic matter may lead to an increased loading of the apoplast with Pb. The mycobionts tested in this investigation did not exclude Pb from root tissues.     

Antimicrobial susceptibility of Pseudomonas aeruginosa isolates from dogs with otitis externa

Pseudomonas aeruginosa is a common cause of otitis externa in dogs, and treatment of these infections is becoming problematic because of the increasing number of multiresistant strains. The aim of the present study was to compare the in vitro activities of cefepime, ceftazidime, enrofloxacin, ciprofloxacin, gentamicin and ticarcillin/clavulanic acid against 104 strains of P aeruginosa isolated from dogs with otitis externa. Antimicrobial susceptibility and minimum inhibitory concentrations, in μg/ml, were evaluated by the E test (bioMérieux). The most active compound was ceftazidime, with 100 per cent efficiency. The majority of tested strains were susceptible to ticarcillin/clavulanic acid (89.4 per cent), followed by ciprofloxacin (88.5 per cent) and cefepime (60.6 per cent). The highest resistance was observed to enrofloxacin (51.9 per cent) and gentamicin (43.3 per cent). Large numbers of strains were intermediately susceptible to antibiotics registered for use in veterinary medicine in Croatia--enrofloxacin (47.1 per cent) and gentamicin (41.3 per cent).     

Sublethal effects of a synthetic pyrethroid,deltamethrin, on the glycemia,the lipemia,and the gut alkaline phosphatases of honeybees

A sublethal dose (0.1 pmol per honeybee, i.e., approx. 0.9 pmol/g body wt) of the synthetic pyrethroid, deltamethrin, was injected intrathoracically into the hemocoel of emerging bees and the biochemical effects were determined over a period of 3 hr. The variations in hemolymph concentrations of trehalose, glucose, phospholipid, steroid, and diacylglycerol were limited, but large variations were observed for other lipids. Mono- and triacylglycerols increased dramatically 1.5 hr after injections; then a peak of free fatty acid appeared at the same time (3 hr) as the monoacylglycerols reached their maximum and the triacylglycerols started to decrease. Atypical forms of triacylglycerols accumulated from 1.5 hr to a maximum level 2 hr after injection. Then, after 3 hr, the concentrations of regular fatty acids, monoacylglycerols, and atypical triacylglycerols returned to normal while longer-chain fatty acids and different forms of ceride simultaneously appeared. The gut alkaline phosphatase parameters (V_M, K, and Hill coefficient) also showed large variations 1.5 and 2 hr after injection, indicating an initial inhibition phase followed by superimposed induction and inhibition mechanisms. These data are discussed according to the hemolymph-lipid transport scheme from the biosynthetic to the utilization sites. Hormone release associated to mitochondrial alteration could be responsible for the observed effects.     

Toxoplasma gondii infection in slaughter pigs in Serbia: seroprevalence and demonstration of parasites in blood

ABSTRACT: A seroepizootiological study of Toxoplasma gondii infection involving a total of 488 slaughter pigs (468 market-weight pigs and 20 sows) in the Belgrade area, also included examination of the presence of T. gondii in the blood. Blood sampled at the slaughter line was examined for specific antibodies by modified direct agglutination, and blood clots of those seropositive at titres of 1:50-1:12800 were bioassayed in mice. The overall seroprevalence was 9.2%, significantly higher (p = 0.0063) in sows (30.0%) than in market-weight pigs (8.3%). Amongst the 22 bioassays performed, a total of 16 (72.7%) were positive, by observation of T. gondii cysts (12), seropositivity (7, including 3 in which cysts were not detected), and/or detection of T. gondii DNA by real-time PCR (12, including one otherwise negative). The positive bioassays originated from the blood of 12 market-weight pigs and 4 sows. Despite a general increase in the rate of demonstration of T. gondii with the increase in the specific antibody level, the association was not significant (p = 0.101). The risk of infection was 41-fold increased in sows vs market-weight pigs, and 15-fold in pigs from smallholders' finishing type farms vs those from large farrow-to-finish farms. The presence of viable T. gondii in a proportion of the samples indicates that some of the pigs had an active parasitaemia at the time of slaughter, which, along with the seroprevalence established, points to a potential source of human infection in Serbia. This is the first report on parasitaemia in naturally infected swine.     

Monitoring of libido and semen quality parameters in melatonin‐treated French alpine bucks during the non‐breeding season

The aim of this study was to investigate the effects of exogenous melatonin on libido and semen quality parameters in bucks during the non‐breeding season. Twelve bucks of the French alpine breed from 1.5 to 4 years of age were assigned into melatonin (MG) and control (CG) groups, with 6 bucks in each group. The experimental period was 3 months (March–May), divided into six periods of 15 days each. The bucks in the MG group received four melatonin implants at the end of March. Two semen samples were taken from the bucks by artificial vagina once per week and their libido estimated. Volume and spermatozoa concentration, their mass motility and motility, proportion of live and total abnormal and forms with abnormal head and tail were determined in the obtained ejaculate samples. The total number of spermatozoa and functional spermatozoa fraction in the ejaculate was also calculated. The MG bucks had significantly higher mass motility and motility of spermatozoa in the first half of April, and a higher proportion of live spermatozoa in the first and second half of April (p < .05). Differences in libido intensity were not significant. The results indicated that the application of melatonin significantly improved the qualitative parameters of semen in bucks, as seen in increased mass motility, motility of spermatozoa and proportion of live spermatozoa shortly following melatonin insertion. Therefore, the results of the current study are novel regarding the use of melatonin treatment during the non‐breeding season to improve the qualitative parameters of ejaculates in bucks.