Extraction, Purification, Quantification, and Stability of Bioactive Spilanthol from Acmella oleracea.

Acmella oleracea is an ethnobotanically significant plant with a relatiwely high content of spilanthol. Due to its broad spectrum of activity, including anti-inflammatory, antioxidant, analgesic, antifungal, and bacteriostatic properties, it is considered a valuable bioactive natural product. In addition, spilanthol as its main bioactive component inhibits facial muscle contractions, making it an attractive ingredient in anti-wrinkle and anti-aging cosmetics. Due to its muscle paralyzing effects, it is called herbal botox. The commercial interest in spilanthol encourages the development of effective methods of isolating it from plant material. The methodology used in this paper allows for the obtaining of extracts from Acmella oleracea with a relatively high content of spilanthol. An effective method of spilanthol extraction from all aerial parts of Acmella oleracea as well as methods of enriching spilanthol concentration in extracts achieved by removing polar and acidic substances from crude extracts was developed. To quantify the concentration of spilanthol, a simple, fast and economically feasible quantification protocol that uses nuclear magnetic resonance (HNMR) was developed. In addition, it has been proven, that oxidation of spilanthol by air gives (2E,7Z)-6,9-endoperoxy-N-(2-methylpropyl)-2,7-decadienamide. The studies on spilanthol solutions stability were carried out and the conditions for the long-time storage of spilanthol solutions have also been developed. Additionally, for confirmation of obtained results a sensitive (LOQ=1 ng/mL), precise (RSD lower than 7%) and accurate (RE lower than 7.5%), new HPLC-MS/MS method was applied.

Herd-level seroprevalence of Fasciola hepatica and Ostertagia ostertagi infection in dairy cattle population in the central and northeastern Poland.

BACKGROUND: Fasciola hepatica and Ostertagia ostertagi infections are widespread in cattle population of Europe, however data on their prevalence in Poland are only fragmentary. Therefore, the cross-sectional study was carried out to determine the herd-level seroprevalence of F. hepatica and O. ostertagi infection in dairy cattle population in the central and north-eastern provinces Poland, and to identify basic local risk factors for these infections. In total, 598 herds were enrolled, 394 (65.9%) in the north-eastern province and 204 (34.1%) in the central province. In each herd the questionnaire survey was conducted and bulk-tank milk (BTM) sample was collected and screened using two indirect immunoenzymatic tests. Optical density ratio (ODR) was regarded as the quantitative proxy of exposure to either of the two parasites. RESULTS: Both Fasciola and Ostertagia ELISA ODR in the north-eastern province was significantly higher than ODR in the central province. At the cut-off value of ODR = 0.27 the true herd-level seroprevalence of F. hepatica was 79.6% (95% CI: 74.0%, 84.3%) in the north-eastern province and 13.0% (95% CI: 5.3%, 21.7%) in the central province. At the cut-off of ODR = 0.50151 of 188 herds (80.3%, 95% CI: 74.1%, 85.4%) were seropositive for O. ostertagi in the north-eastern province and only 70 of 136 herds (51.5%, 95% CI: 43.1%, 59.7%) were seropositive in the central province. Location of a herd in the north-eastern province, longer grazing period practiced in a herd and > 50%-share of grazing grass in roughage were all positively related to the increase in exposure to both parasites. Moreover, the use of hay or haylage as main roughage proved to be positively related to the increase in exposure to F. hepatica. CONCLUSIONS: F. hepatica and O. ostertagi are widespread in cattle population in Poland, however their occurrence at a herd-level varies between different regions of Poland. This diversity can only partly be explained by different herd management, and appears linked to environmental and climate conditions typical for these regions.

Accuracy of a diagnostic model based on serum biochemical parameters in detecting cows at an increased risk of chronic fascioliasis.

In adult cattle Fasciola hepatica infection usually follows a chronic subclinical course, and reduces both the milk yield and milk quality, resulting in considerable financial losses. Effective control of the disease is based on reliable identification of asymptomatically infected individuals, which now requires special parasitological or serological diagnostic tests. It is also known that F. hepatica infection induces alterations in some serum biochemical parameters. Therefore, the study was conducted to develop a model based on serum biochemical parameters allowing to identify cows at higher risk of chronic fascioliasis. Six hundred sixty eight adult dairy cows from 97 herds located in central and northeastern Poland were clinically examined, and blood and fecal samples from them were collected for a routine monitoring of fascioliasis and metabolic profile. Using the combination of fecal sedimentation test and indirect ELISA based on excretory/secretory products 203 cows, apparently healthy in clinical examination, were definitively classed as affected by (47 cows, 23.2%) or free from (156 cows, 76.8%) chronic fascioliasis. Their serum was screened for the activity of 4 enzymes (γ-glutamyl transpeptidase, glutamate dehydrogenase, aspartate aminotransferase, creatine kinase) and concentration of 18 other components (total bilirubin, total protein, albumin, globulin, urea, glucose, non-esterified fatty acids, ß-hydroxybutyrate, sodium, potassium, chloride, calcium, magnesium, copper, zinc, iron, selenium, and haptoglobin). Logistic regression analysis was used to build 4 multivariable models allowing for identification of cows at risk of chronic fascioliasis. Then, the accuracy was compared between the models using the area under the receiver operating characteristic curve (AUROC), and an optimal cut-off value was determined for the most accurate model using Youden J index. The most accurate proved to be the model based on glutamate dehydrogenase activity and globulin, urea (all three positively linked with risk of chronic fascioliasis), and selenium concentration (negatively linked) adjusted by the access to pasture and cow's age. At the optimal cut-off of 0.37 this model had sensitivity of 85.1% (CI 95%:72.3%, 92.6%), specificity of 90.4% (CI 95%:84.7%, 94.1%), positive likelihood ratio of 8.8 (CI 95%:5.4, 14.5), and negative likelihood ratio of 0.16 (CI 95%:0.08, 0.33). This model performed significantly better than model including only γ-glutamyl transpeptidase or model including both γ-glutamyl transpeptidase and aspartate aminotransferase (both also adjusted by the access to pasture and cow's age), and was the only model which performed significantly better than the basic model based solely on the access to pasture and cow's age.

Herd-level seroprevalence of Neospora caninum infection in dairy cattle in central and northeastern Poland.

A serosurvey was carried out to estimate the herd-level seroprevalence of Neospora caninum infection in cattle in central and northeastern Poland. Ninety seven dairy cattle herds from 2 provinces of Poland (Podlaskie, 47 herds and Lodzkie, 50 herds) were randomly enrolled in the study using two-stage cluster method. A simple random selection was applied within each herd to select a sample of adult cows (≥18 month-old). A total number of 734 cows were enrolled in the study. The animals were screened with a commercial competitive ELISA (Bio-X Diagnostics, Belgium). To calculate true herd-level seroprevalence test sensitivity and specificity were adjusted from an individual- to a herd-level using FreeCalc method. The true overall herd-level seroprevalence of N. caninum infection was 56.7% (95% CI: 47.5%, 65.9%). The true herd-level seroprevalence in Podlaskie was 63.3% (95% CI: 43.0%, 83.6%) and 50.5% (95% CI: 32.8%, 68.2%) in Lodzkie province and these figures did not differ significantly between the two provinces (chi2 test p = 0.238). One hundred forty three of 734 cows (19.5%) were seropositive which gave the true overall individual-level seroprevalence of 20.1% (95% CI: 17.4%, 23.2%). Percentage of seropositive cows in each herd varied from 6% to 80%. This study is the first epidemiological investigation of herd-level seroprevalence of N. caninum infection in Polish dairy cattle population. In conclusion, the result of the study confirmed previous data that N. caninum infection is widespread in the Polish cattle population and thus should be considered as a potential cause of spontaneous abortions.

Development of a multiplex fluorescence immunological assay for the simultaneous detection of antibodies against Cooperia oncophora, Dictyocaulus viviparus and Fasciola hepatica in cattle.

BACKGROUND: A major constraint for the effective control and management of helminth parasites is the lack of rapid, high-throughput, routine diagnostic tests to assess the health status of individual animals and herds and to identify the parasite species responsible for these helminthoses. The capability of a multiplex platform for the simultaneous detection of three pasture associated parasite species was evaluated and compared to existing ELISAs. METHODS: The recombinant antigens 14.2 kDa ES protein for Cooperia oncophora, major sperm protein for Dictyocaulus viviparus and Cathepsin L1 for Fasciola hepatica were recombinantly expressed either in Escherichia coli or Pichia pastoris. Antigens were covalently coupled onto magnetic beads. Optimal concentrations for coupling were determined following the examination of serum samples collected from experimentally mono-infected animals, before and after their infection with the target species. Absence of cross-reactivity was further determined with sera from calves mono-infected with Haemonchus contortus, Ostertagia ostertagi and Trichostrongylus colubriformis. Examination of negative serum samples was characterised by low median fluorescence intensity (MFI). RESULTS: Establishment of the optimal serum dilution of 1:200 was achieved for all three bead sets. Receiver Operating Characteristic analyses were performed to obtain cut-off MFI values for each parasite separately. Sensitivity and specificity at the chosen cut-off values were close to, or 100% for all bead sets. Examination of serum samples collected on different days post infection from different animals showed a high reproducibility of the assays. Serum samples were additionally examined with two already established ELISAs, an in-house ELISA using the recombinant MSP as an antigen and a DRG ELISA using Cathepsin L1 for liver fluke. The results between the assays were compared and kappa tests revealed an overall good agreement. CONCLUSIONS: A versatile bead-based assay using fluorescence detection (xMAP technology) was developed to simultaneously detect antibodies against C. oncophora, D. viviparus and F. hepatica in cattle serum samples. This platform provides rapid, high-throughput results and is highly sensitive and specific in comparison to existing serological as well as coproscopical diagnostic techniques.

Modelling the spatial distribution of Fasciola hepatica in dairy cattle in Europe.

A harmonized sampling approach in combination with spatial modelling is required to update current knowledge of fasciolosis in dairy cattle in Europe. Within the scope of the EU project GLOWORM, samples from 3,359 randomly selected farms in 849 municipalities in Belgium, Germany, Ireland, Poland and Sweden were collected and their infection status assessed using an indirect bulk tank milk (BTM) enzyme-linked immunosorbent assay (ELISA). Dairy farms were considered exposed when the optical density ratio (ODR) exceeded the 0.3 cut-off. Two ensemble-modelling techniques, Random Forests (RF) and Boosted Regression Trees (BRT), were used to obtain the spatial distribution of the probability of exposure to Fasciola hepatica using remotely sensed environmental variables (1-km spatial resolution) and interpolated values from meteorological stations as predictors. The median ODRs amounted to 0.31, 0.12, 0.54, 0.25 and 0.44 for Belgium, Germany, Ireland, Poland and southern Sweden, respectively. Using the 0.3 threshold, 571 municipalities were categorized as positive and 429 as negative. RF was seen as capable of predicting the spatial distribution of exposure with an area under the receiver operation characteristic (ROC) curve (AUC) of 0.83 (0.96 for BRT). Both models identified rainfall and temperature as the most important factors for probability of exposure. Areas of high and low exposure were identified by both models, with BRT better at discriminating between low-probability and high-probability exposure; this model may therefore be more useful in practise. Given a harmonized sampling strategy, it should be possible to generate robust spatial models for fasciolosis in dairy cattle in Europe to be used as input for temporal models and for the detection of deviations in baseline probability. Further research is required for model output in areas outside the eco-climatic range investigated.

A study of free radical chemistry: their role and pathophysiological significance.

Oxygen is one of the most important molecules on Earth mainly because of the biochemical symmetry of oxygenic photosynthesis and aerobic respiration that can maintain homeostasis within our planet's biosphere. Oxygen can also produce toxic molecules, reactive oxygen species (ROS). ROS play a dual role in biological systems, since they can be either harmful or beneficial to living systems. They can be considered a double-edged sword because at moderate concentrations, nitric oxide (NO•), superoxide anion, and related reactive oxygen species play an important role as regulatory mediators in signalling processes. Many of the ROS-mediated responses actually protect the cells against oxidative stress and re-establish "redox homeostasis". On the other hand, overproduction of ROS has the potential to cause damage. In the recent decades, ROS has become a focus of interest in most biomedical disciplines and many types of clinical research. Increasing evidence from research on several diseases shows that oxidative stress is associated with the pathogenesis of diabetes mellitus, obesity, cancer, cardiovascular diseases, inflammation, ischaemia/reperfusion injury, obstructive sleep apnea, neurodegenerative disorders, hypertension and ageing.