Chemical composition of the essential oils of serbian wild-growing Artemisia absinthium and Artemisia vulgaris

The chemical composition of the aerial and root essential oils, hydrodistilled from Artemisia absinthium L. and Artemisia vulgaris L. (wild-growing populations from Serbia), were studied by gas chromatography, gas chromatography-mass spectrometry, and 13C nuclear magnetic resonance. During the storage of plant material under controlled conditions, a significant decrease of essential oil yields (isolated directly after drying and after 1 year of storage) and significant differences in their chemical compositions were observed. A possible mechanism for the observed oil component interconversion has been discussed. The noticeable differences in the chemical composition of the oils isolated from roots and aerial parts of A. absinthium and A. vulgaris were also correlated with the diverging biosynthetic pathways of volatiles in the respective plant organs. The antimicrobial activities against the common human pathogens of all of the isolated oils were tested according to National Committee on Clinical Laboratory Standards. The oils showed a broad spectrum of antimicrobial activity against the tested strains. Therefore, these oils can be used as flavor and fragrance ingredients.     

Cross-sectional survey on Toxoplasma gondii infection in cattle, sheep and pigs in Serbia: seroprevalence and risk factors

Toxoplasmosis is a globally distributed zoonosis with a clinical impact in the unborn fetus and in the immunosuppressed individual. In Serbia, studies of risk factors for Toxoplasma gondii infection in humans have shown that the relatively high prevalence is associated mainly with consumption of undercooked meat and/or meat products. However, data on T. gondii infection in domestic animals mostly used for human consumption are scarce. We thus conducted a cross-sectional survey on the seroprevalence of T. gondii infection in a representative sample of cattle, sheep and pigs from different regions of Serbia between June 2002 and June 2003, and analyzed the main risk factors associated with the infection. Sera from 611 cattle (yearlings and adults of both sexes), 511 ewes, and 605 pigs (market-weight and sows), were examined for T. gondii antibodies by the modified agglutination test. The seroprevalences determined were 76.3% in cattle, 84.5% in sheep and 28.9% in pigs. The antibody levels ranged from 1:25 to 1:400 in cattle, and up to 1:25,600 in sheep and to 1:12,800 in pigs. Among the seropositive, the proportion of high antibody levels (> or =1:1600), suggestive of acute infection, was 10% in sheep, and 4% in pigs. Possible association of the infection with biologically plausible risk factors including gender, age, herd size/farm type, type of housing, feeding practices and region, was analyzed by univariate analysis, and variables significant at P< or =0.1 were included in multivariate logistic regression models. The results showed that risk factors for cattle were small herd size (odds ratio, OR=2.19, 95% confidence interval, CI=1.28-3.75, P=0.004) and farm location in Western Serbia (OR=2.04, 95% CI=1.10-3.79, P=0.024), while housing in stables with access to outside pens was protective (OR=0.37, 95% CI=0.21-0.67, P=0.001). In sheep, an increased risk of infection was found in ewes from state-owned flocks (OR=4.18, 95% CI=2.18-8.00, P<0.001) vs. private flocks, and, interestingly, also in those from Western Serbia (OR=4.66, 95% CI=1.18-18.32, P=0.028). In pigs, the risk of infection was highly increased in adult animals (OR=3.87, 95% CI=2.6-5.76, P<0.001), as well as in those from finishing type farms (OR=3.96, 95% CI=1.97-7.94, P<0.001). In addition to providing data on the current T. gondii seroprevalence in meat animals in Serbia, the results of this study show the main risk factors associated with infection, thereby pointing to the type of preventive measures to reduce T. gondii infection.     

Integrated standardization concept for Angelica botanicals using quantitative NMR

Despite numerous in vitro/vivo and phytochemical studies, the active constituents of Angelica sinensis (AS) have not been conclusively identified for the standardization to bioactive markers. Phytochemical analyses of AS extracts and fractions that demonstrate activity in a panel of in vitro bioassays, have repeatedly pointed to ligustilide as being (associated with) the active principle(s). Due to the chemical instability of ligustilide and related issues in GC/LC analyses, new methods capable of quantifying ligustilide in mixtures that do not rely on an identical reference standard are in high demand. This study demonstrates how NMR can satisfy the requirement for simultaneous, multi-target quantification and qualitative identification. First, the AS activity was concentrated into a single fraction by RP-solid-phase extraction, as confirmed by an alkaline phosphatase, (anti-)estrogenicity and cytotoxicity assay. Next, a quantitative ¹H NMR (qHNMR) method was established and validated using standard compounds and comparing processing methods. Subsequent 1D/2D NMR and qHNMR analysis led to the identification and quantification of ligustilide and other minor components in the active fraction, and to the development of quality criteria for authentic AS preparations. The absolute and relative quantities of ligustilide, six minor alkyl phthalides, and groups of phenylpropanoids, polyynes, and poly-unsaturated fatty acids were measured by a combination of qHNMR and 2D COSY. The qNMR approach enables multi-target quality control of the bioactive fraction, and enables the integrated biological and chemical standardization of AS botanicals. This methodology can potentially be transferred to other botanicals with active principles that act synergistically, or that contain closely related and/or constituents, which have not been conclusively identified as the active principles.     

The Pollution Load by Nitrogen and Phosphorus in the Cetina River

The objective of the investigations of the Cetina River, located in southern Croatia, was to record specific characteristics and properties of the Cetina waters at nine stations. In addition to measurements undertaken in the Cetina River, the water quality of its most significant springs and tributaries, such as Kosinac, ?ilovka, Studenci and Mala Ruda, Velika Ruda and Grab, has also been measured. The water quality in the Cetina watershed has been evaluated in the following storage reservoirs: Peru?a, Bu?ko Blato and Pran?evi?i. The nitrogen compounds and phosphorus concentrations have been estimated at all these sampling sites over a 3-year period (2005–2008). Concentration levels at the Cetina-Vinali? sampling site for total N (from August 2005 to December 2008) ranged from 0 to 1.759 mg/L, for NH₃–N from 0 to 0.374 mg/L, for NO₃–N from 0.063 to 0.916 mg/L and for PO₄–P from 0 to 0.099 mg/L. The results prove that the Cetina-Vinali? sampling site is not polluted by nitrogen and phosphorus compounds. The river section from Trilj to the Pran?evi?i dam, where the water is used for the water supply of Omi?, Makarska and Dalmatian islands, has been polluted by wastewater because the majority of agricultural area, roads, industry and settlements are located upstream of it. The highest concentration for total N of 1.128 mg/L and of 1,527 total coliforms in 100 mL, expressed as a mean value for a 3-year period of investigations, was found at the sampling site Trilj. The results of concentration changes at the ?ikotina La?a and Cetina Radmanove Mlinice sampling sites show no regularities. The highest concentration for total N of 0.941 mg/L was measured at the Cetina Radmanove Mlinice during 2007. The highest concentration for NO₃–N of 0.916 mg/L was measured at the same sampling site. According to the investigations of the water quality of the Cetina springs and tributaries, the bacteriological most polluted river spring is Kosinac, and the bacteriological most polluted river tributary is Grab. With reference to the water quality in the Cetina storage reservoirs, it may be concluded that the lowest quality standard has been found within the Pran?evi?i storage reservoir regarding nitrogen compounds and phosphorus concentration levels.     

Reproductive disturbance caused by an S-triazine herbicide in pigs

The aim of the study was to assess the effect of subacute treatment with a low dose of atrazine (1,3,5-triazine-2,4-diamine, 6-chloro-N-ethyl-N'-(1-methyl-ethyl), an s-triazine herbicide, on endocrine oestrus regulation in gilts. A group of nine gilts (F1 generation of Swedish Landrace x Large Yorkshire) were treated with 1 mg atrazine/kg body mass daily, mixed to the feed for 19 days before the onset of expected oestrus. Blood samples were obtained by cranial vena cava puncture three times daily at 3-h intervals on five post-treatment days, i.e. before and during oestrus. The serum concentration of oestradiol-17 beta (E2) was determined by the fluoroimmunochemical method. On Day -2 before the onset of expected oestrus, a significantly lower (P < 0.001) E2 concentration was measured in the serum of treated gilts (31.25 +/- 1.95 and 39.32 +/- 1.38 pg/mL) than in the control pigs (51.43 +/- 1.29 and 68.59 +/- 2.99 pg/mL). In contrast, the E2 concentration measured in the serum of treated animals was significantly higher (P < 0.001) on the day of the expected onset of oestrus and on the subsequent two days (35.43 +/- 1.85, 53.92 +/- 1.98 and 60.32 +/- 2.35 pg/mL, respectively) than in the control animals (13.52 +/- 1.79, 21.53 +/- 1.35 and 20.05 +/- 1.46 pg/mL, respectively). Insufficient serum E2 concentration of the treated gilts resulted in a failure of expected oestrus, as indicated also by the state of dioestrus demonstrated by histopathological examination of the uterus.     

Evaluation of ELISA for detection of Trichinella antibodies in muscle juice samples of naturally infected pigs

The performance characteristics of an ELISA test for trichinellosis in pigs applied to muscle juice was assessed using 314 samples collected from pigs located in endemic areas of Croatia. Peptic digestion was used as the reference method. The diagnostic accuracy of the two compared dilutions (1:10 and 1:100) was considered to be high because the area under the curve (AUC) index was 0.922 and 0.920 for each dilution, respectively. In this study the two graph-receiver operating characteristic (TG-ROC) analysis was used as a tool for selecting cut-off points. Sensitivity, specificity, likelihood ratios, efficiency and Youden's index were used as indices of test accuracy. The cut-off values that minimize overall misclassification cost under an assumption of 3% prevalence were calculated. Our results indicate that the ELISA applied to muscle juice is a highly accurate test and can be adapted to process a large number of samples.     

Levamisole mucosal adjuvant activity for a live attenuated Escherichia coli oral vaccine in weaned pigs

The present study tested the hypothesis that levamisole exerts its immunopotentiating activity in weaned pigs vaccinated against colibacillosis by priming the lymphocytes and macrophages in the mesenteric lymph node (MLN). Ten weaned piglets were used and allocated into two equal groups. The experimental group was intramuscularly primed with levamisole at an immunostimulatory dose of 2.5 mg/kg given daily, in three consecutive days, and controls received saline according to the same schedule. Both groups were orally vaccinated with the vaccinal Escherichia coli strain on day 0 and challenged with the virulent E. coli strain 7 days later. All pigs were killed on postchallenge day 6. Upon virulent challenge the health status of the two groups was evaluated by clinical observations, and expression of CD25, SWC7 and SWC9 activation antigens by MLN and spleen T and B cells and macrophages, respectively, was tested using flow cytometry. Priming by levamisole significantly contributed to the effectiveness of a live attenuated oral vaccine against porcine postweaning colibacillosis, as evidenced by a good health status of primed vaccinated vs. un-primed vaccinated pigs. The CD3+, CD25+ and SWC9+ MLN but not spleen T cells and macrophages increased in experimental vs. control pigs, implying that levamisole exerts its potentiating activity in the MLN by augmenting both recruitment and activation of cells that participate in cell-mediated immunity.     

Proportions and phenotypic expression of peripheral blood leucocytes in pigs vaccinated with an attenuated C strain and a subunit E2 vaccine against classical swine fever

The influence of an attenuated classical swine fever virus C strain vaccine and a subunit E2 vaccine against classical swine fever on the peripheral blood leucocyte proportion and phenotypic expression in 12-week-old pigs was studied. The C strain was amplified in minipig kidney cell culture and final product contained 10(4 +/- 0.15) TCID50/ml, while the subunit vaccine contained 32 microg per dose of gp E2. Haematological findings showed that the vaccines did not cause leucopenia or lymphocytopenia and the number of neutrophils and eosinophils during the observation period was within physiological range. The results of the proportion of CD4a+, CD5a+, CD8a+, wCD21+, CD45RA+, CD45RC+ , non-T non-B, SWC3a+ and CD11b+ cells were gained by single-colour flow cytometry. At the end of the trial a significantly increase of percentage of CD4+, CD5a+, CD8+, wCD21+ cells has been found in pigs that received the subunit vaccine and the percentage of CD4+, CD5a+, CD8+, CD45RA+ and CD45RC+ cells was higher in pigs that received the attenuated vaccine. Twenty-eight days after vaccination the percentage of CD4+, CD45RA+ and CD45RC+ was significantly higher in pigs vaccinated with the C strain than in pigs vaccinated with the subunit vaccine. In contrary, the percentage of the wCD21- cells was higher in pigs that received the subunit vaccine. Statistically higher values of SWC3a+ and lower values of CD11b+ cells was observed in pigs that received the attenuated vaccine than in pigs vaccinated with the subunit vaccine. Taken altogether, our results showed that the subunit vaccine produced a better stimulation of B cells and CD11b+ monocytes/macrophages /granulocytes/NK cells, whereas the attenuated vaccine induced a higher response of Th cells, naive/memory cells and macrophages/neutrophils. Thus, both vaccines were able to influence the porcine immune system, by activating different subsets of the immune effector/accessory cells.     

Characterization of Erwinia amylovora Strains from Croatia

Erwinia amylovora is the causative agent of fire blight, a destructive disease of rosaceous plants. The European population can be divided into several subtypes according to differences in restriction fragment length polymorphism of the XbaI genomic DNA digest analysed with pulsed-field gel electrophoresis. This technique was also used to determine the genetic relatedness of six Croatian isolates to the E. amylovora types found in the countries surrounding Croatia. The isolates belong to the Pt2 pattern type that is characteristic of the East Mediterranean basin. All tested isolates gave essentially the same total cell protein pattern in SDS-polyacrylamide gel electrophoresis. The number of short-sequence DNA repeats in plasmid pEA29 of six isolates was determined by PCR assays and ranged from four to seven. The isolates examined showed high pathogenicity in immature pear fruits. Differences were also revealed in microbiological assays such as amylovoran synthesis, levan formation, siderophore production and colour on coliform medium.     

New aphid species for italy caught by suction trap

Six aphid species new for Italy were caught by suction trap in the Friuli-Venezia Giulia region, Italy. They are:Chaetosiphon chaetosiphon (Nevsky),Illinoia morrisoni (Swain),Myzodium modestum (Hottes),Pseudacaudella rubida (Börner),Schizaphis longicaudata Hille Ris Lambers andTrichosiphonaphis (Xenomyzus)polygonifoliae (Shinji). All species are provided here with characteristic morphological information and line drawings.