Entourage effect for phenolic compounds on production and metabolism of mammary epithelial cells.

Primary culture of mammary epithelial cells (MEC) was exposed to ethyl-acetate, chloroform and hexane extracts of Pistacia lentiscus (lentisk). The hexane extract contained mainly ethyl gallate whereas the chloroform extract contained mainly ethyl-gallate with smaller amount of gallic acid, and the ethyl-acetate extract contained mainly rutin, gallic acid and myricetin. Ethyl acetate extract increased secretion of protein and fat and improved mitochondrial activity. The enhancing effect on protein production was attributed to myricetin, one of the polyphenols in the ethyl-acetate extract whereas gallic acid did not affect protein production or secretion. Interestingly, exposure to the isolated polyphenols did not improve mitochondrial productivity and activity as effectively as exposure to the complete plant extract. The results indicated that polyphenols improve production of milk constituents by MEC, through different modes of action for different polyphenols suggesting an additive or even synergistic effect on production traits of mammary cells.

Pistacia lentiscus extract enhances mammary epithelial cells' productivity by modulating their oxidative status.

We assessed the potential of phenolic compounds from Pistacia lentiscus (lentisk) to enhance production of milk constituents in bovine mammary epithelial cells (MEC). MEC were exposed to 0 (control), 1 or 10 ppm of polyphenols from lentisk ethanolic extract (PLEE) for 24 h. PLEE were absorbed by the MEC plasma membrane, but also penetrated the cell to accumulate in and around the nucleus. PLEE increased triglyceride content in the cell and its secretion to the medium, and significantly increased intracellular lipid droplet diameter. Compared to control, PLEE increased dose-dependently the lactose synthesis, secretion of whey proteins, and contents of casein. To evaluate mitochondrial activity under pro-oxidant load, MEC were preincubated with PLEE and exposed for 2 h to H2O2. Exposure to H2O2 increased the proportion of cells with impaired mitochondrial membrane potential twofold in controls, but not in PLEE-pre-treated cells. Accordingly, proton leakage was markedly decreased by PLEE, and coupling efficiency between the respiratory chain and ATP production was significantly enhanced. Thus, lentisk polyphenols divert energy to production of milk fat, protein and lactose, with less energy directed to cellular damage control; alternatively, PLEE enables MEC to maintain energy and oxidative status under extreme metabolic rate required for milk production and secretion, and reduces the limitation on energy required to support production.

Initial assessment of the economic burden of major parasitic helminth infections to the ruminant livestock industry in Europe.

We report a European wide assessment of the economic burden of gastrointestinal nematodes, Fasciola hepatica (common liver fluke) and Dictyocaulus viviparus (bovine lungworm) infections to the ruminant livestock industry. The economic impact of these parasitic helminth infections was estimated by a deterministic spreadsheet model as a function of the proportion of the ruminant population exposed to grazing, the infection frequency and intensity, the effect of the infection on animal productivity and mortality and anthelmintic treatment costs. In addition, we estimated the costs of anthelmintic resistant nematode infections and collected information on public research budgets addressing helminth infections in ruminant livestock. The epidemiologic and economic input data were collected from international databases and via expert opinion of the Working Group members of the European Co-operation in Science and Technology (COST) action COMbatting Anthelmintic Resistance in ruminants (COMBAR). In order to reflect the effects of uncertainty in the input data, low and high cost estimates were obtained by varying uncertain input data arbitrarily in both directions by 20 %. The combined annual cost [low estimate-high estimate] of the three helminth infections in 18 participating countries was estimated at € 1.8 billion [€ 1.0-2.7 billion]. Eighty-one percent of this cost was due to lost production and 19 % was attributed to treatment costs. The cost of gastrointestinal nematode infections with resistance against macrocyclic lactones was estimated to be € 38 million [€ 11-87 million] annually. The annual estimated costs of helminth infections per sector were € 941 million [€ 488 - 1442 million] in dairy cattle, € 423 million [€ 205-663 million] in beef cattle, € 151million [€ 90-213 million] in dairy sheep, € 206 million [€ 132-248 million] in meat sheep and € 86 million [€ 67-107 million] in dairy goats. Important data gaps were present in all phases of the calculations which lead to large uncertainties around the estimates. Accessibility of more granular animal population datasets at EU level, deeper knowledge of the effects of infection on production, levels of infection and livestock grazing exposure across Europe would make the largest contribution to improved burden assessments. The known current public investment in research on helminth control was 0.15 % of the estimated annual costs for the considered parasitic diseases. Our data suggest that the costs of enzootic helminth infections which usually occur at high prevalence annually in ruminants, are similar or higher than reported costs of epizootic diseases. Our data can support decision making in research and policy to mitigate the negative impacts of helminth infections and anthelmintic resistance in Europe, and provide a baseline against which to measure future changes.

Polyphenols from Pistacia lentiscus and Phillyrea latifolia impair the exsheathment of gastro-intestinal nematode larvae.

The infection of grazing ruminants with gastro-intestinal nematodes (GINs) is a severe problem in the Middle East. However, goats that graze the south-western slopes of the Carmel Heights in Israel have very low faecal egg counts, despite high grazing density. We hypothesized that polyphenols from Pistacia lentiscus L. and/or Phillyrea latifolia L. - both prevalent woody species of the region that are consumed by goats - have anthelmintic bioactivity. We tested this hypothesis by using the larval exsheathment inhibition assay (LEIA). Extracts were prepared from leaves of either plant species using 70% ethanol (E70), 100% ethanol (E100), or boiling water (W). Larvae were incubated in a phosphate-buffered saline solution with or without plant extract (1200µg/ml) and then exposed to an exsheathment solution expected to elicit 100% exsheathment after one hour. All extraction methods of P. lentiscus were highly effective at inhibiting larval exsheathment, but higher potency was found for the E70 than for E100 extraction method, while W was intermediate. Only the E70 extract of P. latifolia was highly effective relative to the control. The E70 extract of P. lentiscus had more than 7 times the potency of the E70 extract of P. latifolia. Irrespective of solvent and tannin-equivalent used, P. lentiscus contained more than double the quantity of total polyphenols than P. latifolia. The polyphenols of P. lentiscus consisted mainly of galloyl derivatives (63.6%), flavonol glucosides (28.6%), and catechin (7.8%). In P. latifolia, oleuropein and its derivative tyrosol accounted for 49.3 and 23.1% of phenolics, respectively, the remainder being flavones (luteolin and quercetin) and their glucoside derivatives. Results of the LEIA test suggest that extracts of tannin-rich plants interfere with the very early stage of host invasion and that high concentration of galloylated derivatives may explain anthelmintic activity.

Constructed wetlands combined with UV disinfection systems for removal of enteric pathogens and wastewater contaminants.

Water shortage is an ongoing cardinal issue in the Middle East region. Wastewater reuse offers some remediation, but to-date many rural communities in the Palestinian Authority (PA) and in Jordan are not connected to centralized wastewater treatment plants (WWTPs), many of them are disposing of their wastewater using infiltration septic tanks. This highlights the need for a small, local, low cost WWTP that can directly benefit local communities, producing effluents suitable for unrestricted irrigation. Constructed wetlands (CWs) could offer a solution as they are relatively easy and cheap to construct and maintain, and effective in removal of many pollutants. Nevertheless, pathogen removal in CWs is often not adequate, calling for additional disinfection. Here we describe the use of low-cost, consumer level, UV based disinfection systems coupled to CWs for wastewater treatment in three CWs: in Israel, Jordan and in the PA. Once mature, our adapted CWs reduced chemical oxygen demand (COD) load, and, given proper use of the UV systems, inactivated indicator bacteria (faecal and E. coli) to levels suitable for irrigation, even when UV transmission (UVT) levels were low (∼40%). Our results demonstrate the promise in this combined treatment technique for cheap and simple wastewater treatment suitable for the Middle East region.

Consumption of Pistacia lentiscus foliage alleviates coccidiosis in young goats.

Coccidiosis near weaning is a major cause of diarrhea, ill-thrift, and impaired performance in small ruminants. A recent survey showed that in villages of the Samaria Hills, Israel, shepherds treat young, weaned goat kids afflicted with diarrhea by cutting and feeding them the foliage of Pistacia lentiscus L. (lentisk) or by tethering them close to lentisk bushes which they browse. The aim of the present study was to assess whether lentisk leaves do indeed have anti-coccidial value, and, if positive, to ascertain the role of tannins in this effect. We monitored for 24 (Experiment 1) and 30 (Experiment 2) days the effect of lentisk feeding on the development of naturally occurring coccidiosis in weaned kids artificially infected with parasitic nematodes. In Experiment 1, kids were infected with nematodes and fed lentisk foliage (PIS) or cereal hay (HAY). Coccidiosis developed at the early stage of the nematode infection, when dietary treatments were initiated. Kids in the PIS group had a lower (P<0.02) concentration of oocysts per gram feces (opg). In Experiment 2, aimed at verifying if tannins are the active component in lentisk foliage, coccidiosis occurred at the peak of the nematode infection, before experimental diets were initiated. Dietary treatments were: cereal hay (HAY), or lentisk foliage consumed without (PIS) or with (PISPEG) a 20-g daily supplement of polyethylene glycol (PEG; MW 4000), a molecule that impairs tannin-bonding with proteins. Goats fed the PIS diet had lower fecal opg counts than counterparts of the HAY (P<0.001) and PISPEG (P<0.002) treatments. Fecal opg counts for the HAY and PISPEG treatments did not differ, suggesting that the anti-coccidial moiety in lentisk was indeed tannins. Our results strongly suggest that: (i) in agreement with the ethno-veterinary anecdotal evidence, exposure of young, weaned goat kids to lentisk foliage alleviates coccidiosis; and (ii) this positive effect is associated with tannins. As coccidiosis is a major affliction of kids, providing them with tannin-rich browse near weaning could be an environmentally friendly way of improving their welfare and health status, in particular under bio-organic farm management.

Anthelmintic activity of Pistacia lentiscus foliage in two Middle Eastern breeds of goats differing in their propensity to consume tannin-rich browse.

The Damascus and Mamber breeds of goats thrive in Middle Eastern Mediterranean regions where the tannin-rich (20% of polyethylene glycol-binding tannins) brush species Pistacia lentiscus L. (lentisk) is ubiquitous. In light of the increasing recognition of the anthelmintic activity of plant tannins, we examined the effect of offering lentisk foliage for 24 days on fecal egg excretion in 5.5-month-old Damascus and Mamber kid goats (n=28) following infection with 10,000 L3 larvae of mixed gastro-intestinal nematodes (GIN). Lentisk foliage was consumed with or without a daily supplement of 20 g polyethylene glycol (PEG; MW 4000). Lentisk tannins showed a strong protein-depletive effect that was totally reversed by the addition of PEG. At the peak of infection, kids of the two breeds lost weight unless they were fed with lentisk without PEG. Fecal egg counts (FEC) were lowest - and did not differ from 0 - in kids fed lentisk without PEG, highest in the controls fed hay as roughage, and intermediate in kids fed lentisk and PEG (241, 1293, and 705 eggs per gram, respectively, SEM 180; P<0.001); therefore, the anthelmintic activity of lentisk was only partly attributable to tannins. The suppressive effect of lentisk on FEC ceased when feeding was discontinued, suggesting that female parasites were not killed but their fertility was reversibly impaired. Damascus kids showed lower FEC than their Mamber counterparts, inferring that the effect of foraging on tannin-rich species is only additive to genetic differences between goat breeds in their sensitivity to GIN infection. On the basis of our results we would expect yearlong lentisk grazing to result in no or very low GIN infection, and Damascus goats to have some advantage over Mamber goats where chemical control of GIN is unfeasible. There appears to be a trade-off between the benefits of lentisk tannin as drug and its side-effects (protein depletion) when given at high level; how goats balance this trade-off requires further elucidation.

Ethnobotanical knowledge of local Arab practitioners in the Middle Eastern region.

An ethnobotanical survey was conducted among 31 local Arab practitioners living in Galilee, Negev, Golan Heights and West Bank. Seeing that the Arabic traditional medicine is in danger of disappearing, a multilevel program is required, involving the training of local practitioners, an establishment of a regional medicinal plant botanical garden and a field gene bank for plant preservation.

The potential of rhizosphere microbes isolated from a constructed wetland to biomethylate selenium.

The potential of rhizosphere microbes isolated from common reed [Phragmites australis (Cav.) Trin. ex Steud] plants grown in a subsurface-flow constructed wetland to biomethylate selenate or selenite was studied in liquid cultures under controlled conditions. Total mean percentages of volatilized Se from half-strength Hoagland culture solutions (low C content) supplemented with selenate or selenite and inoculated with cultured rhizosphere microbes after 15 d of incubation were 7.9 and 49.1%, respectively. There was a relative best fit (r = 0.87) between total number of rhizosphere and cultured microbes and the percentage of volatilized Se in Hoagland solution after 15 d of incubation. However, when the same microbes were cultured in tryptic soybean broth (TSB) medium (high C content), the percentages of volatilized Se from selenate and selenite were 1.3 and 1.9%, respectively. The volatilization percentages of Se from selenate or selenite in culture solutions inoculated with rhizosphere suspension instead of cultured rhizosphere microbes were very low (1.2-3.0%) in both cultivation media. In all experiments, selenite was volatilized significantly (p < 0.05) in higher amounts by cultured rhizosphere microbes after 15 d of incubation compared with selenate. Dissolved biomethylated dimethylselenide (DMSe) in water samples taken from the subsurface-flow bed was determined by purging with helium. The DMSe in water samples was indirectly detected up to 2.4 microg Se L(-1), which indicates that part of the produced DMSe was dissolved in the matrix before being released into the atmosphere. Our results show that rhizosphere microbes isolated from common reed plants have a high potential of Se biomethylation and volatilization from selenate and selenite.

Ethnopharmacological survey of medicinal herbs in Israel, the Golan Heights and the West Bank region.

An extensive ethnopharmacological survey was conducted among the most well known Arabic indigenous herbal practitioners in Israel, the Golan Heights and the West Bank in order to evaluate the potential of local plants used in treating different diseases and illnesses. Thirty-one indigenous practitioners' of Arabic traditional medicine ranging in age from 40 to 116 years, were interviewed using a previously prepared questionnaire. The current survey revealed that 129 plant species are still in use in Arabic traditional medicine for the treatments of various diseases. Among these plants, there are 40 species used for treating skin diseases, 27 species for treating kidney and urinary system, 26 species for treating diabetes, 23 species for treating digestive system including stomach and intestinal pain and inflammation, 22 species for treating liver diseases, 16 species for treating respiratory system and coughing, 13 species for treating forms of cancer and nine species for treating weight loss and cholesterol reduction. Additional findings and implications of this current survey including preparation methods and route of use are discussed in this report.

Effects of NaCl and CaCl(2) on Water Transport across Root Cells of Maize (Zea mays L.) Seedlings.

The effect of salinity and calcium levels on water flows and on hydraulic parameters of individual cortical cells of excised roots of young maize (Zea mays L. cv Halamish) plants have been measured using the cell pressure probe. Maize seedlings were grown in one-third strength Hoagland solution modified by additions of NaCl and/or extra calcium so that the seedlings received one of four treatments: control; +100 millimolar NaCl; +10 millimolar CaCl(2); +100 millimolar NaCl + 10 millimolar CaCl(2). From the hydrostatic and osmotic relaxations of turgor, the hydraulic conductivity (Lp) and the reflection coefficient (sigma(s)) of cortical cells of different root layers were determined. Mean Lp values in the different layers (first to third, fourth to sixth, seventh to ninth) of the four different treatments ranged from 11.8 to 14.5 (Control), 2.5 to 3.8 (+NaCl), 6.9 to 8.7 (+CaCl(2)), and 6.6 to 7.2 . 10(-7) meter per second per megapascal (+NaCl + CaCl(2)). These results indicate that salinization of the growth media at regular calcium levels (0.5 millimolar) decreased Lp significantly (three to six times). The addition of extra calcium (10 millimolar) to the salinized media produced compensating effects. Mean cell sigma(s) values of NaCl ranged from 1.08 to 1.16, 1.15 to 1.22, 0.94 to 1.00, and 1.32 to 1.46 in different root cell layers of the four different treatments, respectively. Some of these sigma(s) values were probably overestimated due to an underestimation of the elastic modulus of cells, sigma(s) values of close to unity were in line with the fact that root cell membranes were practically not permeable to NaCl. However, the root cylinder exhibited some permeability to NaCl as was demonstrated by the root pressure probe measurements that resulted in sigma(sr) of less than unity. Compared with the controls, salinity and calcium increased the root cell diameter. Salinized seedlings grown at regular calcium levels resulted in shorter cell length compared with control (by a factor of 2). The results demonstrate that NaCl has adverse effects on water transport parameters of root cells. Extra calcium could, in part, compensate for these effects. The data suggest a considerable apoplasmic water flow in the root cortex. However, the cell-to-cell path also contributed to the overall water transport in maize roots and appeared to be responsible for the decrease in root hydraulic conductivity reported earlier (Azaizeh H, Steudle E [1991] Plant Physiol 97: 1136-1145). Accordingly, the effect of high salinity on the cell Lp was much larger than that on root Lp(r).

Effects of Salinity on Water Transport of Excised Maize (Zea mays L.) Roots.

The root pressure probe was used to determine the effects of salinity on the hydraulic properties of primary roots of maize (Zea mays L. cv Halamish). Maize seedlings were grown in nutrient solutions modified by additions of NaCl and/or extra CaCl(2) so that the seedlings received one of four treatments: Control, plus 100 millimolar NaCl, plus 10 millimolar CaCl(2), plus 100 millimolar NaCl plus 10 millimolar CaCl(2). The hydraulic conductivities (Lp(r)) of primary root segments were determined by applying gradients of hydrostatic and osmotic pressure across the root cylinder. Exosmotic hydrostatic Lp(r) for the different treatments were 2.8, 1.7, 2.8, and 3.4.10(-7) meters per second per megapascals and the endosmotic hydrostatic Lp(r) were 2.4, 1.5, 2.7, and 2.3.10(-7) meters per second per megapascals, respectively. Exosmotic Lp(r) of the osmotic experiments were 0.55, 0.38, 0.68, and 0.60.10(-7) meters per second per megapascals and the endosmotic Lp(r) were 0.53, 0.21, 0.56, and 0.54.10(-7) meters per second per megapascals, respectively. The osmotic Lp(r) was significantly smaller (4-5 times) than hydrostatic Lp(r). However, both hydrostatic and osmotic Lp(r) experiments showed that salinization of the growth media at regular (0.5 millimolar) calcium levels decreased the Lp(r) significantly (30-60%). Addition of extra calcium (10 millimolar) to the salinized media caused ameliorative effects on Lp(r). The low Lp(r) values may partially explain the reduction in root growth rates caused by salinity. High calcium levels in the salinized media increased the relative availability of water needed for growth. The mean reflection coefficients of the roots using NaCl were between 0.64 and 0.73 and were not significantly different for the different treatments. The mean values of the root permeability coefficients to NaCl of the different treatments were between 2.2 and 3.5.10(-9) meters per second and were significantly different only in one of four treatments. Cutting the roots successively from the tip and measuring the changes in the hydraulic resistance of the root as well as staining of root cross-sections obtained at various distances from the root tip revealed that salinized roots had mature xylem elements closer to the tip (5-10 millimeters) compared with the controls (30 millimeters). Our results demonstrate that salinity has adverse effects on water transport and that extra calcium can, in part, compensate for these effects.

Effect of peanut tannin extracts on growth of Aspergillus parasiticus and aflatoxin production.

Twenty-three peanut (Arachis hypogaea L.) genotypes were evaluated for kernel resistance to Aspergillus parasiticus Spear. colonization and aflatoxin contamination when incubated under high relative humidity. Also, tannin-containing extracts from kernel coats (testae) and cotyledons of these genotypes were prepared and tested for their effect on A. parasiticus growth and aflatoxin production in vitro. The lowest degree of colonization, less than 30%, was noted in kernels from the genotypes, Toalson x UF 73-4022 (selections TX-798731 and TX-798736), A72118, SN 55-437, PI337409, and Florunner. Genotypes with low levels of colonization also had the lowest aflatoxin contamination. The coefficient of correlation between infection frequency and aflatoxin contamination was 0.66. Higher levels of tannins were detected in the testae (23.9-97.2 mg g tissue) compared to the cotyledons (0.17-0.82 mg g tissue). Some of the methanol-extracted and water-soluble tannin extracts from testae and cotyledons, when incorporated in yeast extract sucrose liquid medium (100 mg l), significantly inhibited A. parasiticus growth and reduced the levels of aflatoxin produced. There was no overall correlation between the peanut genotypes and the influence of tannin extracts on A parasiticus growth and aflatoxin production. However, correlations were higher for specific genotypes. For example, the coefficient of correlation between the ability of tannin extracts from testae of genotypes PI337409 and TX-798736 to inhibit aflatoxin production was 0.93 and 0.85 respectively.

Does salinity reduce growth in maize root epidermal cells by inhibiting their capacity for cell wall acidification?

The reduction in growth of maize (Zea mays L.) seedling primary roots induced by salinization of the nutrient medium with 100 millimolar NaCl was accompanied by reductions in the length of the root tip elongation zone, the length of fully elongated epidermal cells, and the apparent rate of cell production: Each was partially restored when calcium levels in the salinized growth medium were increased from 0.5 to 10.0 millimolar. We investigated the possibility that the inhibition of elongation growth by salinity might be associated with an inhibition of cell wall acidification, such as that which occurs when root growth is inhibited by IAA. A qualitative assay of root surface acidification, using bromocresol purple pH indicator in agar, showed that salinized roots, with and without extra calcium, produced a zone of surface acidification which was similar to that produced by control roots. The zone of acidification began 1 to 2 millimeters behind the tip and coincided with the zone of cell elongation. The remainder of the root alkalinized its surface. Kinetics of surface acidification were assayed quantitatively by placing a flat tipped pH electrode in contact with the elongation zone. The pH at the epidermal surfaces of roots grown either with 100 millimolar NaCl (growth inhibitory), or with 10 millimolar calcium +/- NaCl (little growth inhibition), declined from 6.0 to 5.1 over 30 minutes. We conclude that NaCl did not inhibit growth by reducing the capacity of epidermal cells to acidify their walls.