Fleas frequently infest small mammals and play important vectoring roles in the epidemiology of (re)emerging zoonotic disease. Rodent outbreaks in intensified agro-ecosystems of North-West Spain have been recently linked to periodic zoonotic diseases spillover to local human populations. Obtaining qualitative and quantitative information about the composition and structure of the whole flea and small mammal host coexisting communities is paramount to understand disease transmission cycles and to elucidate the disease-vectoring role of flea species. The aims of this research were to: (i) characterise and quantify the flea community parasiting a small mammal guild in intensive farmlands in North-West Spain; (ii) determine and evaluate patterns of co-infection and the variables that may influence parasitological parameters.Microtus arvalis, Apodemus sylvaticus, Mus spretus and Crocidura russula. We also report on aggregation patterns (variance-to-mean ratio and discrepancy index) and co-infection of hosts by different flea species (Fager index) and used generalized linear mixed models to study flea parameter variation according to season, habitat and host sex.We conducted a large-scale survey stratified by season and habitat of fleas parasitizing the small mammal host guild. We report on the prevalence, mean intensity, and mean abundance of flea species parasitizing Ctenophthalmus apertus gilcolladoi, Leptopsylla taschenbergi and Nosopsyllus fasciatus. Results showed a high aggregation pattern of fleas in all hosts. All host species in the guild shared C. a. gilcolladoi and N. fasciatus, but L. taschenbergi mainly parasitized mice (M. spretus and A. sylvaticus). We found significant male-biased infestation patterns in mice, seasonal variations in flea abundances for all rodent hosts , and relatively lower infestation values for voles inhabiting alfalfas. Simultaneous co-infections occurred in a third of all hosts, and N. fasciatus was the most common flea co-infecting small mammal hosts.Three flea species dominated the system: N. fasciatus and C. a. gilcolladoi dominated the flea community, and a high percentage of co-infections with both species occurred within the small mammal guild. Nosopsyllus fasciatus may show higher competence of inter-specific transmission, and future research should unravel its role in the circulation of rodent-borne zoonoses.The generalist  Yersinia pestis , followed by A. sylvaticus [range 1–29], while M. spretus [range 1–5] and C. russula [range 1–7] had fewer fleas per host. For further analyses of infestation patterns, we focused on the main flea species  and the most frequently captured small mammal hosts   and crop type , but not by host sex. Post-hoc tests (Tukey) indicated that C. a. gilcolladoi infestation was less frequent and severe on voles in July than March, with intermediate values in November. Moreover, voles caught in alfalfa had lower flea prevalence, abundance and intensity than those from fallows. In A. sylvaticus, C. a. gilcolladoi parameters differed between sexes , being greater for male than female hosts. The only exception was mean intensity, which reached higher values in animals trapped during March than in July. In M. spretus, neither variable explained C. a. gilcolladoi prevalence variation. The small sample size for this species did not allows us to analyse intensity or abundance.Variation in N. fasciatus, we found in voles the same pattern as in C. a. gilcolladoi, with differences in the three parameters between crop types  and months . Infestation with N. fasciatus was more frequent and severe during July, and lower levels of flea infestation were found in voles from alfalfas. In A. sylvaticus, N. fasciatus abundance did not differ between sexes, but varied between months , with a higher infestation rate during July and a reduced intensity and abundance during November. The infestation with this flea varied between months in M. spretus although the effect was only marginally significant . The highest prevalence rate was found in July but the mean abundance dropped in March. In M. spretus, N. fasciatus was also more abundant on males than females . The small sample size for this species did not allow modelling intensity variation.Regarding L. taschenbergi, we found that this flea was more abundant on males than on females in M. arvalis . In both mouse species, we found a significant effect of sex  and month . The infestation was more prevalent and severe in July and among males than females in both host species. Furthermore, crop type explained variations in intensity and abundance in A. sylvaticus , with a greater intensity in hosts from cereal fields compared with fallows.Regarding M. arvalis (38.8%) and A. sylvaticus (34.6%) than in M. spretus (13.3%) and C. russula (16.7%). Co-infection patterns with two flea species were diverse among hosts. Associations composed of C. a. gilcolladoi-N. fasciatus were most commonly found in M. arvalis (90.3%), and very few co-infections with L. taschenbergi. In fact, this flea was collected alone in almost 90% of the cases. L. taschenbergi-N. fasciatus associations prevailed in the mouse hosts , although this predominance in A. sylvaticus was similar to the other co-infections . Few hosts (2.3%) harboured three flea species  or in M. spretus , but did in A. sylvaticus , with fewer co-infections in female than male hosts. Considering hosts infested with two or three species, we found no differences between sexes . In terms of co-infection assemblies, male and female wood mice presented similar values of co-infection for all flea pairs . In voles, however, we found a male-biased N. fasciatus-L. taschenbergi co-infection, which occurred more frequently in male than in female hosts .Co-infection rates did not differ between sexes in N. fasciatus, C. a. gilcolladoi and L. taschenbergi and showed species-specificity and marked aggregation patterns. We found strong sex-biased differences in the mouse hosts, lower flea infestation in voles captured from alfalfas, and seasonal variations differing between host and flea species. Interspecific co-infections were frequent, with up to three different flea species in some hosts.The flea community parasitizing the small mammal guild studied here was mainly (99.4%) composed by N. fasciatus) was the most common and most abundant flea species in the studied small mammal community, parasitizing both rodents and insectivores. This flea mainly parasitizes rodents but can facultatively infest a wide range of mammalian hosts [Ctenophthalmus fleas are a generalist group that diverges in their distribution range owing to geographical specificity [Ctenophthalmus species identified here are endemic to the Western Mediterranean area, C. a. gilcolladoi is the typical flea of the open habitats of this region [Leptopsylla taschenbergi was the most abundant flea found on A. sylvaticus and M. spretus, and showed a low infestation rate on common voles, which is consistent with its well-known mouse-specificity [The northern rat flea  were lower in M. arvalis than in mice. Behavioural traits could explain these differences [N. fasciatus and C. a. gilcolladoi despite similar abundances. The host-generalist strategy of N. fasciatus [N. fasciatus, would have access to more hosts, having more resources available and reducing intraspecific competition. Another important aspect to consider is the local asynchrony in abundance fluctuations of the rodent species  would tend to parasitize only the fewer suitable hosts that are available (i.e. A. sylvaticus and M. spretus).Our results show the typical aggregation pattern of fleas on rodents found elsewhere . Poulin ferences , since tasciatus  could exin mice; , 53). ThMicrotus arvalis harboured the highest flea burden and infestation rate, followed by A. sylvaticus. Larger body size and greater complexity of the burrow system can lead to heavier flea infestations [M. spretus and C. russula . Moreover, the fossorial life of M. arvalis could increase the probability of getting infested by naïve fleas because burrows are used by preimaginal stages to develop until their first blood feeding [stations , which c feeding .C. a. gilcolladoi showed higher presence in March/November, with a significant drop in July, and vice versa for N. fasciatus and L. taschenbergi, which is in accordance with previous studies [N. fasciatus has no marked seasonality in Atlantic conditions [N. fasciatus eggs require at least 23 °C during 4 days or a peak of 30 °C for 3 h to hatch [We found seasonal variations in the main flea species whatever the rodent host. In general terms, prevalence models could be easily understood from a flea phenology point of view, since maximum values occurred during the season with the greatest flea activity:  studies , 48, 51.to hatch , 56. TheN. fasciatus also differed between voles and mice during July. Since the intensity and prevalence of voles increased significantly during the summer, the reproductive period of this flea [N. fasciatus reproduction. A possible reason could be that wood mouse nests or their own body are less suitable for this flea compared with voles. This type of differences at the host species level has been described in other small mammal communities [N. fasciatus in mice is reached at minimum mouse population abundance  [A. sylvaticus have a more individualistic behaviour and spend more time inside the burrows during the cold months [Seasonality in flea patterns is common as are variations owing to local climatic conditions and host traits , 22–24. his flea , infestamunities . Therefouitment) , which md months , which cd months .C. a. gilcolladoi on wood mice in March when prevalence patterns differed from those on voles. A noticeable seasonality was found in the latter, following the typical flea phenology with peaks during temperate months [C. a. gilcolladoi may prefer infesting common voles rather than mice in this ecosystem, although no investigation has been done yet to determine the relationship between C. a. gilcolladoi and their hosts in this system. This possible preference was not expected since M. arvalis is a host species that colonized the study region during the 1970s, invading from mountain habitats characterized by Atlantic weather conditions [Ctenophthalmus apertus is endemic to Mediterranean open habitats and absent from Atlantic climatic areas [M. arvalis. A host shift could have possibly occurred if this new host species offered better conditions than other hosts [A similar pattern may be occurring for e months , 51. We nditions . Ctenophic areas ; thus, ier hosts . The coler hosts , 59. FutLeptopsylla taschenbergi infestation in voles also appeared associated with cereal habitats. Wood mice inhabiting cereals had heavier flea burdens compared with other habitats. Some authors have linked animal condition and abundance with the quality and quantity of food supply [We also found differences in flea parameters depending on the habitats used by hosts. Common voles inhabiting alfalfas had lower flea prevalence and abundance than those from other crop types. d supply . The mosd supply . The wood supply , avoidind supply  and withd supply  such as d supply .M. arvalis, except with the less frequent flea species, L. taschenbergi. This male-biased difference is usually linked to sexual size dimorphism, immunosuppression by sexual hormones, or behavioural differences that facilitates flea encounter and horizontal transmission [L. taschenbergi) occurred more often in male than in female voles.Lastly, we found sexual differences in flea infestation in both mice species, with greater prevalence and severity in male than in female hosts. Such patterns were not found in smission . Such pasmission , 66, 67.smission  and the smission  that cousmission  and alsosmission  and thersmission , potentiN. fasciatus and C. a. gilcolladoi) were shared by 40% of the hosts of the small mammal guild. Flea co-occurrence is common in small mammals [N. fasciatus was found in the most abundant co-infections of the four small mammals, with an apparently high tolerance to cohabit with other flea species. In fact, we identified this flea and C. a. gilcolladoi (the two more abundant fleas) in 82.7% of all the co-infections analysed. Notably, previous work [Bartonella spp. prevalences of 65% in N. fasciatus collected from our focal common vole population and of 33% in C. a. gilcolladoi. Additional investigation should therefore be carried out to determine the potential roles of these two fleas in the transmission of bartonelloses. Moreover, N. fasciatus is a typical parasite of Rattus spp. [Mus domesticus in Spain [Rattus and Mus spp. commensal rodents are widespread in rural areas, especially linked to the presence of domestic livestock, and they can be infected by Bartonella sp. [Ctenophthalmus spp. and R. beillardae) were more rarely found. Therefore, their possible role in the circulation cycle of zoonoses among this small mammal host guild would probably be less relevant.In the Mediterranean agricultural landscapes studied here, two dominant flea species  is a recent colonizer and, unlike other small mammal hosts, is characterized by large fluctuations in abundance. The consequences of fluctuating M. arvalis abundances for the transmission cycles of flea-vectored diseases should also be investigated.Six different flea species parasitized the studied small mammal guild inhabiting continental Mediterranean farmland, although Additional file 1: Figure S1. Flea cumulative frequencies in the main small mammal host species. CAA, Ctenophthalmus apertus apertus; CAG, Ctenophthalmus apertus gilcolladoi; CB, Ctenophthalmus baeticus; NF, Nosopsyllus fasciatus; LT, Leptopsylla taschenbergi; RB, Rhadinopsylla beillardae. * Sample size too small (n = 1).


The release of reactive oxygen species (ROS) and oxidative stress is associated with the development of many ailments, including cardiovascular diseases, diabetes and cancer. The causal link between oxidative stress and cancer is well established and antioxidants are suggested as a protective mechanism against cancer development. Recently, an increase in the consumption of antioxidant supplements was observed globally. The main sources of these antioxidants include fruits, vegetables, and beverage. Herbal infusions are highly popular beverages consumed daily for different reasons. Studies showed the potent antioxidant effects of plants used in the preparation of some herbal infusions. Such herbal infusions represent an important source of antioxidants and can be used as a dietary protection against cancer. However, uncontrolled consumption of herbal infusions may cause toxicity and reduced antioxidant activity. In this review, eleven widely consumed herbal infusions were evaluated for their antioxidant capacities, anticancer potential and possible toxicity. These herbal infusions are highly popular and consumed as daily drinks in different countries. Studies discussed in this review will provide a solid ground for researchers to have better understanding of the use of herbal infusions to reduce oxidative stress and as protective supplements against cancer development. The intimate link between nutrition and health is well documented  and peopThe consumption of herbal infusions is very common in the Mediterranean region and globally. In a study conducted on 1260 cancer patients in Palestine, 60.9% were consuming herbs, mostly in the form of decoctions . These dOxidative stress is the unbalance between production and elimination of free radicals and reactive species, like reactive oxygen species (ROS) and reactive nitrogen species (RNS) ,12. OxidIn our body, redox homeostasis balances the oxidative stress via enzymatic and non-enzymatic antioxidants. Several types of antioxidants have critical roles in ROS elimination, including dietary natural antioxidants, like tocopherol, selenium, β-carotene, ascorbic acid, polyphenol metabolites, and synthetic antioxidants . On the contrary, many endogenous antioxidant molecules contribute in this role, such as glutathione, α-lipoic acid, coenzyme Q, ferritin, uric acid, bilirubin, metallothionein, L-carnitine, and melatonin. Endogenous antioxidant enzymes are also involved in balancing the oxidative stress. These enzymes include superoxide dismutase (SOD), glutathione reductase (Gr), thioredoxin reductase (TRX), catalase (CAT), glutathione peroxidases (GPXs) and peroxiredoxins (PRXs) .All cancer phases entailing initiation, promotion and progression are affected by oxidative stress. Oxidative stress has the ability to activate several transcription factors including nuclear factor (NF)-κB, hypoxia inducible factor (HIF)-1α, activator protein (AP)-1, p53, peroxisome proliferator-activated receptor (PPAR)-γ,β-catenin/Wnt, and nuclear factor erythroid 2-related factor 2 (Nrf2). These factors regulate the expression of diverse genes included in immune modulation, inflammatory response, carcinogenesis, metastasis, tissue remodeling and fibrosis ,16. BesiAntioxidants in diet and supplements are widely used to protect cells from the damage induced by ROS. However, some clinical trials have shown conflicting results that do not support this concept. Based on recent studies, antioxidants may increase melanoma metastasis in mice ,22 and aSeveral plants with high antioxidant abilities and total phenolic contents have been screened out to be used as a rich source of natural antioxidants. These plants could be developed into herbal infusions, functional food or pharmaceuticals for the inhibition and treatment of diseases caused by oxidative stress . MedicinCitrus limonum) belongs to the Rutaceae family. GC-MS/MS analysis showed the presence of high concentrations of limonene with percentages in the lemon water extract of 23.271% [2O2. Limonene also defended normal lymphocytes from diseases related to oxidative stress, including cancer [Lemon , suggesting a selectivity of these derivatives towards cancer cells . StudiesZingiber officinale Roscoe) belongs to the Zingiberaceae family. Herbal teas prepared from ginger are used as a folk remedy to treat coughs, colds, and flu. It is also applied as a paste for external applications to treat headaches [Ginger  [ody base . Rosmariody base .The stropigenin) . Wild thpigenin) . By usinpigenin) . Anotherpigenin) . Moreovepigenin) .In osteosarcoma cells, rosmarinic acid showed anticancer effects by suppressing DJ-1 via regulation of the PTEN-PI3K-Akt signaling pathway . PreviouOrganum majorana belongs to the Lamiaceae family. Marjoram is a shrub and a perennial plant native in Asia and the Mediterranean area. It has been used traditionally in the folk medicine as an antifungal, antiviral, and antiparasitic remedy [c remedy . Using tc remedy  and ferrc remedy , methanoc remedy . Recentlc remedy , while gc remedy . The marc remedy . Supportc remedy . The purc remedy . Moreovec remedy .Camellia Sinensis) belongs to the Theaceae family and it is widely consumed in Asian countries [The Palestinian herbal infusion contains green tea, lemon verbena, sage, and citrus lemon. Green tea  belongs to the Verbenaceae family. It is a shrub with scented leaves that grows in both tropical and subtropical regions [Lippia citriodora protected against lipid peroxidation and protein carbonylation. Also, the decoction showed higher antioxidant capacity compared to the infusion [Lemon verbena  belong to the Lamiaceae family. Sage is a perennial shrub, an aromatic and remedial plant endemic in the Mediterranean region [Sage  belongs to the Lauraceaeis family. It is a small evergreen tree with a height of 5–7 m. The plant is charecterized by the aromatic odor and pleasant smell. The biological activities of cinnamon are due to the presence of tannins [E)-cinnamaldehyde (68.95%), as the major component, in addition to benzaldehyde (9.94%), and (E)-cinnamyl acetate (7.44%) [4) that induced lipid peroxidation and hepatic injury in rats. The raised serum AST and ALT enzymatic activities induced by CCl4 were significantly decreased by oral administration of 200 mg/kg of each extract once daily for seven days, as compared to the untreated rats [This infusion contains green tea, lemon verbena, cinnamon, damask rose, chamomile flowers, primrose, and ginger. Cinnamon ( tannins . The bar (7.44%) . Saponin (7.44%) . Cinnamo (7.44%) . Differe (7.44%) . A cinnadehyde 9.%, and , human cancer promyelocytic leukemia (HL-60), human colorectal carcinoma , epidermoid carcinoma (A431), and human cervical carcinoma (SiHa) . The alcosterols . At a coosterols .Rosa damascena Mil.) belongs to the Rosacea family. It is a deciduous shrub and is the most significant aromatic medicinal plant. The damask rose is mainly used in the perfume industry and as a flavoring agent in food products [R. damascena essential oil, a total of 22 compounds were detected by GC-MS analysis. Both citronellol (23.43%) and geraniol (34.91%) were the main scent compounds of the fresh rose flowers [R. damascena flower petals were found to be 12.73% and 32%, respectively. These flavonoids work as potent antioxidant agents [R. damascena displayed anti-free radical activity at a concentration of 50 μg/mL. The leaf cold extraction had the most potent antioxidant activity measured with the FRAP assay at the concentration of 100 μg/mL, compared with the hot methanolic extraction [R. damascena flower extracts showed 74.51% and 75.94% antiradical activities, while the antioxidant activity of fresh flower extract (372.26 mg/g) was higher than that of spent flower extract (351.36 mg/g) [Damask rose (products . In R. d flowers . The entt agents . Moreovet agents . The rost agents . Leaf metraction . Fresh a36 mg/g) . The dam36 mg/g) .Matricaria chamomilla L.) belongs to the Asteraceae family. It is a slow growing aromatic annual plant with branched stems, double feathery shared leaves, and tiny, soft, hollow, lettuce head flowers [Chamomile ( flowers . The for flowers . GC-MS aM. chamomilla was investigated using DPPH assay [p < 0.05) increased by 6.81%, 26.16%, 36.71%, and 45.06%, respectively in chamomile group compared with patients in the control group [The antioxidant activity of PH assay . The antPH assay . The antPH assay . The higPH assay . In a siol group .Primula vulgaris) belongs to the Primulaceae family. The primrose flower is funnel-shaped, with orange spots at the base of its lobes [p-coumaric acid and rutin have been recognized as the main phenolics in primrose water extract [2O2-induced DNA damage in a concentration-dependent manner in fibroblast cells compared to the positive controls (only 20 μM H2O2 treatment) [Primrose (ts lobes . p-couma extract . Primroseatment) . Using Meatment) . Furthereatment) .A recent study on five herbal infusions including lemon and ginger combination, wild thyme, marjoram, Palestinian and Lebanese herbal mix was conducted to evaluate their antcancer activities. The water extract of lemon and ginger combination was found to be the most potent against MDA-MB231, MCF-7, and A549 cell lines. Both lemon and ginger combination and wild thyme separately, showed the highest apoptosis induction and angiogenesis suppression abilities on the MDA-MB231 cell line at concentrations of 3.5 and 4.4 mg/mL, respectively. Furthermore, lemon and ginger combination, wild thyme, marjoram, and the Lebanese herbal drink were the most active extracts in stimulating pinocytosis, respectively while Palestinian herbal drink had a moderate effect .Hibiscus sabdariffa L.) is a well-known species that belongs to the Malvaceae family. The plant is an annual or perennial herb found in tropical and subtropical regions of the world. It is used to produce phloem fibers and as an infusion  [H. sabdariffa [H. sabdariffa contained alkaloids, flavonoids, saponins, tannins, and a high anthocyanin content with the lowest flavonoids and phenolic acid [H. sabdariffa, most of which were fatty acids and ester compounds [H. sabdariffa calyces in two major forms; delphinidine-3-sambubioside and cyanidine-3-sambubioside [H. sabdariffa, which tends to be high when the extraction temperature is in the range of 70–80 °C and the extraction time is from 120 to 150 min. At the same time, it decreases as the solvent to solid ratio is increased [H. sabdariffa extracts and their impact on antioxidant activity have been investigated and have shown that the highest total phenolic content (TPC) resulted in high antioxidant scavenging capacity [H. sabdariffa aqueous extract exhibited a higher ability to scavenge peroxyl radicals in the water environment than in the lipophilic system and exhibited a more potent metal-reducing activity than olive leaf extract [Roselle  ,119. A pbdariffa . Studieslic acid . Gas chrompounds . Ethanimompounds . A previubioside . The relubioside . The antubioside . Extractncreased . Furthercapacity . In a la extract .H. sadbariffa is a promising anticancer plant against different cancer types. The aqueous extract of H. sadbariffa was able to reduce cell viability and induced apoptosis of human adenocarcinoma cell line (MCF-7) [H. sadbariffa calyx was able to inhibit tumor growth, lung metastasis, and tumor angiogenesis. All these effects could be mediated via inhibition of tumor Ras, NF-κB, CD31, and VEGF/VEGF-R-induced angiogenesis [Previous studies have shown that  (MCF-7) . In thisogenesis . In a reogenesis . H. sabdogenesis .Punica granatum L.) is a member of the Punicaceae family and is considered to be of Middle East origin. The plant has also been used in the traditional medicine for ages [P. granatum is a good source of phenolic compounds and anthocyanins, as demonstrated in a previous study [P. granatum peels ethanolic extract has been analyzed using GC-MS chromatography and showed various constituents with antioxidant activity such as decahydro-1-pentadactyl- naphthalene, 5-hydroxymethyl furfural and 1, 3-cyclohexadiene [P. granatum—hydroalcoholic and infusion—exhibited high efficiency in inhibiting DPPH radical and had significant reducing power of the Fe3+/ferricyanide complex. The antioxidant activity of fruit peel extracts has been justified by the presence of a high level of phenolic compounds like ellagic acid and its derivatives, as identified by UPLC-PDA-MS analysis [P. granatum and has shown that the TPC and total flavonoids content (TFC) of arils juices were about 20- and 300-fold inferior to decoctions. Regardless of variety, each decoction revealed better antioxidant and chelating activity compared to the juices [P. granatum peel was assessed using three aqueous extraction techniques; continuous shaking extraction, maceration, and hot water infusion. It was found that infusion method resulted in significant (p < 0.05) level of antioxidant activity compared to other extraction techniques [P. granatum has an impact on ROS, an in vivo study was carried out to test the effect of methanolic extracts of pomegranate seeds and peel on oxidative stress induced by methotrexate. The results have demonstrated a significant reduction in GPX and SOD, and an improvement in MDA values after methotrexate treatment [Pomegranate , triterpenoids , and sterols (β-sitosterol and stigmasterol) [Apiaceae . It is wApiaceae . A recenradicals . Use of . anisum . The resasterol) . Anetholasterol) .A previous study has demonstrated the correlation between total phenolic content and antioxidant activity of both anise and cumin infusions (ground form). It was found that phenolic compounds in these infusions are the main contributors to their free radical-scavenging activity and oxidant reduction potency . AnotherP. anisum extracts and essential oil have shown antiproliferative effect on gastric cancer cells (AGS), and anti-angiogenesis activity in HUVEC cells [P. anisum extract showed significant anticancer activity comparing to the normal cell line [Consumption of anise seed has many medicinal benefits, such as anticancer, hepato-protective and antioxidant abilities. In this regard, a comparative study with cisplatin has conducted to evaluate the cytotoxic effect of (PA) aqueous extract on oral squamous cell carcinoma (KB cell line). It was reported that anise seed extract exhibited anticancer activity by being able to reduce cell viability in dose dependent manner . MoreoveEC cells . Anotherell line .Cuminum cyminum L.) is a small annual herbaceous plant that belongs to the Apiaceae family [C. cyminum has revealed the presence of alkaloids, anthraquinones, coumarins, flavonoids, glycosides, resins, saponins, tannins and steroids [C. cyminum have a higher phenolic content and antioxidant capacity comparing to coriander extracts [Cumin  and neral (26%) [O-glucoside [M. officinalis exhibited strong antioxidant activity, which were 10 times stronger than the antioxidant effects of vitamin C and vitamin B [M. officinalis aqueous extract in reducing Mn-induced brain oxidative stress in mice [M. officinalis has the highest total phenolic content and antioxidant activity compared to the other species [M. officinalis extracts have strong antioxidant capacity and DPPH radical scavenging activities compared to butylated hydroxytoluene (BHT) [ in food ,169. Alt in food . The cheal (26%) ,172,173.al (26%) ,175. Thelucoside . M. offiitamin B . An in v in mice . Further species . Anotherne (BHT) .M. officinalis has shown chemo-preventive effect against hepatocellular carcinoma (HCC) in rats and exhibited antioxidant activity via increasing GSH concentration and inhibiting lipid peroxidation in the liver tissues of HCC rats [M. officinalis have shown cytotoxic activity against three human tumor cell lines: NCI-H460 , MCF-7 (breast adenocarcinoma), and AGS (gastric adenocarcinoma) [The anticancer potency of lemon balm has been previously studied using different types of extracts on various tumor cell lines ,182. AquHCC rats . Lemon bHCC rats . Moreovercinoma) .Rosmarinus officinalis L.) belongs to the mint family Lamiaceae which is widely distributed in the Mediterranean region [O-β-d-glucuronide, 7-O-glucoside, hispidulin, diosmin, genkwanin, hesperidin and isoscutellarein 7-O-glucoside which are found in flowers, leaves, roots and stems of R. officinalis [R. officinalis has anti-inflammatory, hepatoprotective, antidiabetic and antimicrobial activity which depend on the content of the phenolic compounds mainly rosmarinic acid, caffeic acid and carnosic acid [Rosemary  [0 cell population along with suppression of G1-S phase transition and reduction of cyclin D1 expression. As a result, the colonies of 184-B5/HER cells were reduced significantly [In view of the antioxidant and antiproliferative activities of R. officinalis, many in vitro studies confirmed these effects depending on the polyphenolic content. For example, Ðilas et al. investigated the effect of a number of oil-soluble rosemary extracts with varying content of carnosic acid, carnosol and methylcarnosol. Results of this study revealed that the extract with the highest content of carnosic acid had the most powerful scavenging ability to hydroxyl, superoxide and 2,2-diphenyl-1-picrylhydrazyl (DPPH)-free radicals. Tested rosemary extracts also exhibited significant antiproliferative effect indifferent cell lines. In both breast adenocarcinoma (MCF7) and cervix epitheloid carcinoma (HeLa) celll lines, the extracts yielded low IC μgmL−1) . Support μgmL−1) . Pro-apoficantly .50 at 1/1000 and 1/400 dilutions, respectively. Moreover, the extract and its active ingradients enhanced the antiproliferative effect (synergistic effect) of cisplatin in both A2780 and A2780CP70 cells. The effect of rosemary extract was due to the suppression of the expression of Bcl-2, Bcl-x, cIAP-1, HIF-, and HO-1 (anti-apoptosis proteins) and Bax, Fas and FADD (pro-apoptosis molecules). Furthermore, A2780 cells were more sensitive to carnosol, carnosic acid and rosmarinic acid than A2780CP70 cells. Interestingly, rosmary extract upregulated a heme-containing protein, cytochrome C, that was emitted from the mitochondria in response to pro-apoptotic stimuli [50 value was 150 mg/mL after 72 h of exposure in KYSE30 cell lines, however, in AGS cell lines, IC50 value was 1.3 mg/mL after 72 h of exposure [The antiproliferative effect of rosemary extract and its active ingradients carnosol, carnosic acid and rosmarinic acid on human ovarian cancer cells was investigated by Tai et al. Rosmary extract was shown to have significant antiproliferative effect in human ovarian cancer cells (A2780) and cisplatin resistance daughter cell line A2780CP70 with effective IC stimuli . Alterna stimuli . Additioexposure . In prosexposure .In vivo, rosemary polyphenols have antiproliferative effects against colon cancer cells in animal models. For example, when male nude mice were grafted with human colon cancer cells (HT-29) treated with fluid extract of rosemary, the extract exhibited a clear reduction in the tumor size. This outcome was strongly correlated with the role of rosemary extract in sharp increase of intracellular ROS that stimulated necrotic cell death. In addition, Nrf2 gene silencing increased rosemary cytotoxic effects . AnotherA short-term intake of chamomile tea  has beneficial effects on glycemic control and antioxidant status in patients with type 2 diabetes mellitus . ConsumpA randomized controlled trial suggested that aromatherapy  has efficacy in preventing treatment-related salivary gland disorder . InteresMelissa officinalis infusion markedly improved oxidative stress conditions and DNA damage that arises from radiation exposure among radiology staff [Cumin essential oil supplementation enhanced some antioxidative indices, as superoxide dismutase and total antioxidant capacity in patients with metabolic syndrome . Based ogy staff .Altough herbal infusions are widely consumed, some studies have reported a lack of effect or even contraductory responses. For example, one of the common uses of lemon juice was the treatment of high blood pressure. Quercetin, a flavonoid present in citrus lemon, was found to lower lower blood pressure in several rat models of hypertension . InteresIn contrast to a previous study which showed that ginger interferes with iron absorption , a recenH. sabdariffa has been used in the traditional medicine as an anti-hyperlipidemic agent [H. sabdariffa has no significant role to lowering serum lipids when compared with placebo, black tea, or diet [H. sabdariffa on fasting plasma glucose (FPG), total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoproteins (LDL), and triglyceride (TG). It was shown that H. sabdariffa has no significant effect on blood TC, HDL, and TG, however, H. sabdariffa was able to lower both FPG and LDL [ic agent . Randomi or diet . Alterna and LDL .P. granatum seed oil exhibited a hormetic effect when applied to mouse mammary organ culture. This was evident by the high inhibitory effect on on tumor at low tested concentration [Despite the potential activity of phytochemicals as therapeutics for chemoprevention, they have certain limitations that are related to the complex mixture of metabolites, biphasic effects (hormesis), and their bioavailability . Hormesintration . Moreoventration . The maintration .In addition to the abovementioned effects, thymol exhibited a hormetic effect in terms of antioxidant and pro-antioxidant activity, cell viability and DNA genotoxicity . Thyme eHerbal infusions are important sources of antioxidant agents and can be used to reduce oxidative stress and protect against cancer. Diverse phytochemicals are present in these infusions with the ability to activate different mechanisms involved in lowering oxidative stress and enhancing anticancer effects. Extraction temperature and time are important variables for some herbal infusions in order to obtain the highest antioxidant effect. However, uncontrolled consumption of herbal infusions may cause toxicity and reduced antioxidant activity.


Wildlife species carry a remarkable diversity of trypanosomes. The detection of trypanosome infection in native Australian fauna is central to understanding their diversity and host-parasite associations. The implementation of total RNA sequencing (meta-transcriptomics) in trypanosome surveillance and diagnosis provides a powerful methodological approach to better understand the host species distribution of this important group of parasites.Phascolarctos cinereus; southern brown bandicoot, Isoodon obesulus; swamp wallaby, Wallabia bicolor; bare-nosed wombat, Vombatus ursinus), one bird  and one amphibian . Samples corresponded to both clinically healthy and diseased individuals. Sequencing reads were de novo assembled into contigs and annotated. The evolutionary relationships among the trypanosomatid sequences identified were determined through phylogenetic analysis of 18S rRNA sequences.We implemented a meta-transcriptomic approach to detect trypanosomes in a variety of tissues  sampled from native Australian wildlife, comprising four marsupials (koala, We detected trypanosome sequences in all six species of vertebrates sampled, with positive samples in multiple organs and tissues confirmed by PCR. Phylogenetic analysis indicated that the trypanosomes infecting marsupials were related to those previously detected in placental and marsupial mammals, while the trypanosome in the regent honeyeater grouped with avian trypanosomes. In contrast, we provide the first evidence for a trypanosome in the eastern dwarf tree frog that was phylogenetically distinct from those described in other amphibians.Trypanosoma transcripts and from diverse hosts and tissues types, thereby representing an effective means to detect trypanosomes that are divergent in genome sequence.To our knowledge, this is the first meta-transcriptomic analysis of trypanosomes in native Australian wildlife, expanding the known genetic diversity of these important parasites. We demonstrated that RNA sequencing is sufficiently sensitive to detect low numbers of  Trypanosoma species are associated with serious disease [Trypanosoma copemani and T. vegrandis have been associated with population declines of woylies (Bettongia penicillata) in Western Australia (WA) [Trypanosomes are haemoprotozoan parasites that infect a wide range of animal taxa –3. Endem disease , 10, othlia (WA) , 10. It lia (WA) , 11.T. copemani, T. irwini and T. gilletti) [To date, most trypanosome surveillance has been directed toward screening Australian mammals . Marsupials, in particular, have been widely screened, allowing the identification of several trypanosome species (e.g. illetti) , 11–13. illetti) , 14, 15.illetti) , 17. Forilletti) , 19. Othilletti) –26. Howeilletti) , 18.T. irwini, T. gilletti, T. copemani, T. vegrandis and T. noyesi) in koalas [Trypanosoma spp. are present in animals sampled on the east and west coasts of Australia, as well as Tasmania [Multiple trypanosome species have been documented in Australian wildlife. For example, surveillance in marsupials recorded up to five species , and the region encoding the glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH), an enzyme involved in the glycolytic pathway [Diagnosis of trypanosome infection largely relies on microscopy and a variety of molecular techniques . PCR-bas pathway . In rece pathway , 6. In c pathway , and hen pathway –32.Trypanosoma in wildlife, screening different tissues from a variety of native Australian species. From this, we identified trypanosomes in several vertebrate groups from New South Wales (NSW) and Tasmania (TAS), including the identification of a divergent species of Trypanosoma in an amphibian species.Herein, we employed, for the first-time, a meta-transcriptomics approach as a method for the identification and surveillance of Phascolarctos cinereus; southern brown bandicoot, Isoodon obesulus; swamp wallaby, Wallabia bicolor; bare-nosed wombat, Vombatus ursinus), one bird  and one amphibian . The amphibian specimen corresponded to a male diagnosed with severe, multisystemic, chronic trypanosomiasis  during monitoring surveys of wildlife, as well as from road-kill cases in NSW. The bare-nosed wombats were derived from road-kill in southern Tasmania. Following dissection, all tissue samples were stored at -80 °C until molecular analysis Table . In totals Table .Table 1CIn brief, total RNA was extracted from a variety of sample tissues Table  using thde novo into contigs using Trinity v. 2.5.1 [−70 were retained for downstream analyses. Further, contigs corresponding to the stably expressed host mitochondrial marker, cytochrome c oxidase subunit 1 (cox1), were identified based on sequence alignments using DIAMOND. All contigs were aligned to reference sequences using BBMap v.37.98 and cross-validated to DIAMOND results to verify that the matches correspond to the vertebrate host. Abundance was quantified as the sum of relative abundances of contigs for the marker. Sequence contigs were annotated as follows: (i) to find conserved domains and classify protein families, sequences were compared against the Conserved Domain Database (CDD) [http://www.ebi.ac.uk/interpro/); (ii) for gene assignment, all putative trypanosome contigs were aligned against a custom reference sequence database (genome assembly ASM21029v1) using DIAMOND [Sequence reads were trimmed for quality using the Trimmomatic tool  and assev. 2.5.1  with defv. 2.5.1  (Additiose (CDD)  and Inte DIAMOND .Trypanosoma infection via PCR assays using primers targeting 2136-bp (outer) and 320-bp (nested) fragments of the 18S rRNA  was determined by using the Akaike information criterion (AIC) in the ModelFinder program [The trypanosome contigs obtained here were compared with homologous sequences retrieved from GenBank, using  program  implemen program . Phyloge program  availabl program . Nodal s program .n = 1), southern brown bandicoot tail (n = 1), and the eastern dwarf tree frog (n = 1). In total, trypanosomes were detected in 76.47% (13/17) of the individuals screened. With respect to target tissues, we detected trypanosome transcripts across a variety of tissues in infected individuals  of bare-nosed wombats, 71.43% (5 out of 7) of koalas, in both of the swamp wallaby samples, reagent honeyeater  followed by the eastern dwarf tree frog , whereas the lowest number of transcripts was identified in the regent honeyeater   of Trypanosoma.Despite the widespread presence of ts Table . Since s18S rRNA of the SSU. Abundance levels of 18S rRNA contigs ranged from 0.64 to 743.40 TPM. The highest cumulative abundances were identified in the eastern dwarf tree frog (TPM = 46.71) and the swamp wallaby #2 (TPM = 802) , with the highest levels observed in the swamp wallaby #1 sample .To place trypanosome sequences into a phylogenetic context (see below), and hence achieve taxonomic assignment, we identified the contigs targeting the 18S rRNA gene , ear (n = 1), liver (n = 14), lung (n = 1), tail (n = 1), and testes (n = 1). A 320-bp nested fragment corresponding to the 18S rRNA was amplified in all samples containing trypanosomes, as previously identified by meta-transcriptomics . This clade can be further divided into two groups: the first includes trypanosomes from the wallaby and the southern brown bandicoot, while the second group contained trypanosomes from the wallaby and bare-nosed wombat. Strikingly, the trypanosome from the koala fell into a different clade that is related to T. gennarii (nucleotide sequence similarity of 81.30%) and T. freitassi (82.04%) identified in South American marsupials (Monodelphis spp.), T. bennetti (92.56%) in birds  and T. irwini (98.75%) in koalas. Moreover, we identified a trypanosome species in the regent honeyeater that is closely related to the avian trypanosomes T. thomasbancrofti and T. avium that share ~100% and 97% sequence similarity, respectively. Sequence comparisons against avian genotypes 1–4  [T. thomasbancrofti  ) showed netti 92.6% in birIn addition to the trypanosomes related to mammals and birds, we identified a trypanosome species infecting the eastern dwarf tree frog that was divergent from other trypanosomes in amphibians  [We have, to our knowledge for the first time, implemented a meta-transcriptomic approach for detecting uencing) , meta-truencing) . To dateuencing) , 6. UsinT. cruzi, reflecting the change in temperature when the parasite moves from the vector to the mammalian host [Leishmania [Most of the trypanosome transcripts identified in the hosts analyzed were associated with genes encoding ribosomal components, suggesting that ribosome biogenesis and protein synthesis have a central role in the infection process Tables , 3. In tian host , 45. Hspian host , 47. Theishmania , 49. Difishmania , 50.Reoviridae) [Polyomaviridae) in the western barred bandicoot (Perameles bougainville) [Trypanosoma and Myxobolus, confounding the association of disease with any etiological agent. Because our study was limited to vertebrates, it does not provide insights into the potential vector involved in parasite transmission. However, as suggested in previous studies, it is possible that both ticks and dipterans (i.e. flies and mosquitoes) are vectors of these parasites as they can feed on a large variety of hosts including mammals, birds and amphibians [Previous studies have suggested that trypanosomes often have deleterious effects on the health of the infected hosts , 51, 52.viridae) , while tinville) . Similarinville) , 56. In phibians , 57, 58.phibians –61. Cleaphibians , 19.Trypanosoma sp. ABF previously described in the swamp wallaby in NSW [T. cyclops, an exotic trypanosome isolated from the monkey Macaca nemestrina and related to T. theileri-related trypanosomes in ruminants and tabanids. The relatedness among these trypanosome species raises concerns over the potential susceptibility of Australian vectors and vertebrates to infection by exotic trypanosomes and hence the establishment of a zoonotic transmission cycle [Phylogenetic analysis revealed that the trypanosomes identified in native Australian fauna fell into different lineages that were largely concordant with that of the host species from which they were sampled, although we were unable to make taxonomic assignments to the species level. Notably, we identified three distinct clades of marsupial trypanosomes Fig. . The tryy in NSW , and to on cycle , 8. In aon cycle .T. irwini, T. gilletti, T. copemani, T. vegrandis, T. noyesi and Trypanosoma sp. AB-2017 have been described in koalas [Trypanosoma sp. detected in the koala was closely related to T. irwini and the avian exotic trypanosome T. bennetti. Given than the former has been also identified in koalas, the trypanosome detected in the sampled koala likely corresponds to T. irwini. The close relationship between the T. irwini and T. bennetti has been previously documented [Among the trypanosome species infecting marsupials, n koalas , 7, 13. cumented , 13 and cumented , 63. ThiT. thomasbancrofti (genotype 1), and T. thomasbancrofti was originally described in the regent honeyeater [T. avium was identified in the rook (Corvus frugilegus) and associated with serious disease and death in birds, with suggestions that it is transmitted by blackflies (Simulium spp.) [T. thomasbancrofti in the regent honeyeater and highlight the importance of parasitological surveillance in the wild for this species classified as critically endangered (CR) (sensu IUCN).The trypanosome sequence we identified in the regent honeyeater likely belongs to neyeater . This trneyeater . In contum spp.) , 65 and um spp.) . Hence, Trypanosoma with Myxobolus might have detrimental effects on amphibian health. This clearly merits further investigation. To our knowledge, this is the first report of a trypanosome in the eastern dwarf tree frog  database. Relative abundance was calculated for each contig as transcripts per million (TPM).Additional file 3: Table S2. List of PCR primers used in this study for confirmation of trypanosome infection.Additional file 4: Table S3. List of sequences used for phylogenetic analysis.Additional file 5: Table S4. Pairwise sequence identity among 18S rRNA sequences of avian trypanosomes belonging to genotypes 1–4 and the putative T. thomasbancrofti identified in this study. Genotype classification sensu Šlapeta et al. (2016) [. (2016) .Additional file 6: Figure S2. Maximum likelihood tree showing phylogenetic relationships among trypanosomes within the aquatic clade based on the 18S rRNA gene. The trypanosome identified in Litoria fallax is indicated in blue. The hosts of trypanosomes are indicated with colour-coded tips.


The Florida Keys Mosquito Control District has used dry ice-baited light traps to monitor mosquito populations on Vaca Key since 1998. The first site sampled was monitored continuously for almost 20 years until all vegetation was removed.This paper describes a dataset compiled over almost 20 years of continuous trapping along Manor Lane on Vaca Key, Florida. The Florida Keys Mosquito Control District conducted adult mosquito surveillance along Manor Lane in Vaca Key for almost twenty years. Surveillance was accomplished by use of dry ice-baited light traps. Traps were set and retrieved weekly except for interruptions due to unavoidable situations, such as storms and illness. Traps were provisioned with 2 pounds (ca. 1 kg) of dry ice, deployed in the late afternoon and retrieved the following morning. Traps were hung from the same tree limb each time they were set. Mosquitoes were returned to the laboratory, killed by freezing and identified to species. This paper reports data pertaining to 32 mosquito species.These data were collected to document the species composition, seasonal distribution and relative abundance of mosquitoes on Vaca Key, Florida.Monitoring of the mosquito fauna on Vaca Key began in the late summer of 1998 and continued until the winter of 2017. Collections were intended to be made weekly although, due to storms, illness and vacations, this was not always possible.A battery-powered light trap  was baited with approximately two pounds ca. 1 kg) of dry ice and hung from the same tree limb once per week for over 19 years. The trap was deployed in the late afternoon and retreived the following morning. The trap collection was taken to the laboratory, frozen, and all mosquitoes separated, identified, and counted. Data were recorded in spreadsheets. Voucher specimens of all taxa are retained in the synoptic mosquito reference collection maintained by the Florida Keys Mosquito Control District. Data may be found in Suppl. material  kg of dr24°42'78"N, 81°04'56"W.The trap was deployed on Vaca Key, Monroe County, Florida, USA. The coordinates of the trap location trap were: Thirty-two mosquito species were documented during the sampling period.The study site was described by Data collection began on 17 August 1998 and ended on 27 December 2017.Creative Commons Public Domain Waiver (CC-Zero)CSV1Manor Lane BDJData in spreadsheet, csv.38Number of adult female mosquitoes collected per trap per night. NA indicates that the species was not reported and so no numbers are available.9A80E46B-7502-5194-B548-621A6EECD0C210.3897/BDJ.8.e55059.suppl1Supplementary material 1Manor Lane BDJData typeCount data in a csv spreadsheet.Brief descriptionApproximately 19.5 years of near-weekly mosquito collections from Vaca Key, Monroe County, Florida.File: oo_388380.csvhttps://binary.pensoft.net/file/388380Hribar, L.J.


Essential oils are of paramount importance in pharmaceutical, cosmetic, agricultural, and food areas thanks to their crucial properties. However, stability and bioactivity determine the effectiveness of essential oils. Polymeric nanoencapsulation is a well-established approach for the preservation of essential oils. It offers a plethora of benefits, including improved water solubility, effective protection against degradation, prevention of volatile components evaporation and controlled and targeted release. Among the several techniques used for the design of polymeric nanoparticles, nanoprecipitation has attracted great attention. This review focuses on the most outstanding contributions of nanotechnology in essential oils encapsulation via nanoprecipitation method. We emphasize the chemical composition of essential oils, the principle of polymeric nanoparticle preparation, the physicochemical properties of essential oils loaded nanoparticles and their current applications.  For millennia, nature has been considered a valuable source of medicinal agents and an exciting number of modern drugs have been derived from natural sources . RecentlCymbopogon citratus DC. EO when encapsulated by poly (lactide-co-glycolide)-NP as compared to the free oil [Jasminum officinale L. EO was increased after encapsulation in gelatin and arabic gum nanoparticles [Essential oils are generally complex mixtures of volatile organic compounds biosynthesized as secondary metabolites determining the specific aroma, flavor and fragrance of plants ,6. Essenfree oil . Moreovefree oil . In anotarticles .Among the several methods used for developing polymeric NP, the nanoprecipitation method (or solvent displacement) seems to be the most simple and reproducible ,29. In tAccording to the French Agency for Normalization (AFNOR) , an esseZingiber officinale L., Acorus calamus L.), seeds , fruits  and woods  [Vetiveria zizanioides L. [Essential oils could be obtained from flowers , leaves , rhizomes (lbum L.) . Generallbum L.) . Essentioides L. .Essential oils are basically a complex mixture of terpenic hydrocarbons, especially monoterpenes and sesquiterpenes, and oxygenated derivatives like aldehydes , ketones , alcohols , phenols (thymol) and esters  . EssentiSyzygium aromaticum L. [Mentha piperita L. [Origanum vulgare L. [Cinnamomum cassia Persl. [Rosmarinus officinalis L. [Cymbopogon citratus DC. [Thymus vulgaris L. EO [Thymus vulgaris L., Origanum vulgare L., Syzygium aromaticum L., Ocimum basilicum L., Myristica fragrans Houtt. and Petroselinum crispum Mill. show remarkable antioxidant activities due to their phenolic structure which neutralize free radicals and decompose peroxides [Melissa officinalis L. [Saussurea lappa L. [Artemisia herba-alba Asso. [Melaleuca alternifolia Cheel. [Comptonia peregrina L. [Essential oils derived from different parts of aromatic plants have been extensively researched for their nutritional health benefits. Essential oils exhibit excellent antimicrobial properties and the mechanism of action has been studied in detail . The maiticum L. , Mentha erita L. , Origanulgare L. ,38, Cinna Persl. , Rosmariatus DC.  and Thymis L. EO  as largeeroxides . Essentieroxides ,42. Morenalis L. , Saussurlappa L. , Artemisba Asso. , Melaleua Cheel. , and Comgrina L.  show excgrina L. ,49. Furtgrina L. ,20.The use of EO or their derivatives is always confronted by their volatility, chemical un-stability upon exposure to air, light, moisture and heat . HădărugNanoncapsulation has been proposed as a novel approach to overcome the mentioned limitations ,54. SevePolymeric nanoparticles are solid colloidal particles with a diameter ranging from 1 to 1000 nm. They are comprised of nanocapsules and nanospheres. Nanospheres have a monolithic-type structure (matrix) in which active pharmaceutical ingredients (API) are encapsulated within the particles or adsorbed onto their surfaces. While, Nanocapsules are the vesicular system in which the API is confined to a cavity consisting of an inner liquid core surrounded by a polymeric membrane. In this case, the API is usually dissolved in the inner core, and may also be adsorbed to the capsule surface  Figure . With reSeveral preparation methods have been reported in the literature to develop polymeric NP, while the nanoprecipitation is extensively employed ,73,74,75Generally, to produce EO based-nanoparticles by nanoprecipitation method, the polymer and the EO are solubilized in an organic solvent or mixture of solvents. The organic solution is then added to water, with or without a hydrophilic surfactant under moderate magnetic stirring which causes the interfacial deposition of a polymer after displacement of the organic solvent . After tRecently, several EO loaded-NP were developed by a modified nanoprecipitation process developed by Luque-Alcaraz et al. . In thisTo explain the particle formation in the nanoprecipitation method, Joye and McClements pointed that this phenomenon includes four steps: supersaturation, nucleation, growth, and coagulation  Figure . This exWhen the solute concentration is reduced below the critical supersaturation concentration, nuclei growth for condensation or coagulation begins. Condensation is the addition of single molecules to the particles surface. This phase stops when solute concentration is reduced below the equilibrium saturation concentration. Coagulation is another major driving force for particle growth. It consists on the adhesion of particles to each other’s and happens when the attractive forces  dominate the repulsive ones (steric or electrostatic repulsion). By applying mechanical forces like stirring, homogenization, or ultrasound, the particles within the aggregate may be released. On the other hand, the nanoparticles may coalesce via aggregation process leading to the formation of stable particles. The coagulation step depends on collision frequency and efficiency. The collision frequency is the number of collisions per unit time per unit volume, and vary with the variation of particle concentration, particle size, and particle motion. Whereas, the collision efficiency represents the number of collisions which lead to coagulation, and depends on the balance of attractive and repulsive forces between the particles. To stabilize particles against coagulation, stabilizing agents could be added during the process .The nanoprecipitation method involves the use of an organic solvent, which is completely soluble in the external aqueous phase, inducing immediate polymer precipitation. According to the ®RS100 [®L100-55 [®EPO [The polymers used could be biodegradables and biocompatibles; synthetics or naturals. Synthetic polymers like Eudragit®RS100 ,75, Eudr®L100-55 , Eudragi55 [®EPO , poly-ε-55 [®EPO ,66,89, p55 [®EPO ,67 showe55 [®EPO ,73,82. I55 [®EPO .®200 [Hydrophobic surfactants (W/O) could be added to the nanocapsule core to hinder the particle ‘aggregation. Sorbitan esters, like span 20  and phos®200  and leci®200  are comm®F68 [It could consist of mixture of surfactants, used for avoiding particle ‘aggregation; dissolved in the non-solvent. Pluronic®F68 ,65, Twee®F68 ,87 and p®F68 ,87,88,90®F68 ,74,81,82Cymbopogon martini Roxb. EO loaded poly-ε-caprolactone based-nanocapsules with small particle size (282.1 nm), narrow size distribution (poly dispersibility index less than 0.14) and high encapsulation efficiency (99.54%) [Rosmarinus officinalis L. EO was efficiently entrapped in poly-ε-caprolactone based-NP with an average size of 220 nm, zeta potential equal to −19.9 mV and an encapsulation efficiency of about 99% [The solvent displacement technique has been extensively used to encapsulate essential oils in polymeric NP, as illustrated in (99.54%) . Similarbout 99% . Furtherbout 99% . Origanum vulgare L. EO loaded- poly-ε-caprolactone NP prepared by two methods: nanoprecipitation and double emulsion [According to the literature, several works reported the effectiveness of the nanoprecipitation over other methods in term of particle size and oil entrapment efficiency. Shakeri et al. reported that using the nanoprecipitation technique to develop carvacrol loaded-poly (3-hydroxybutyrate) nanocapsules allowed significantly improvement in oil loading (21%) and particles stability  as compared to the dialysis method . Furtheremulsion . For insemulsion . Rosmarinus officinalis L. EO loaded-nanoparticles was studied. Results showed that large particles have been produced when increasing poly-ε-caprolactone quantity. Apparently, high polymer amount results in increasing the thickness of the polymeric shell and thus increasing the particles size instead of their number [Mentha piperita L., Cinnamomum Cassia presl. and Cymbopogon citratus DC., respectively in cellulose acetate based-NP and studied the variation in the particle size. Results revealed that the particle size was in the following order: Cinnamon EO-NP < Peppermint EO-NP < Lemongrass EO-NP [Citrus aurantifolia Christm. on the surface of chitosan-NP decreased the zeta potential from +61.1 to +57.0 mV after EO encapsulation [3+ groups of chitosan following their interaction with lime EO [Rosmarinus officinalis L. EO encapsulation due to its presence in the core of the NP instead of being adsorbed on their surface, as previously reported [Mentha piperita L., Cinnamomum Cassia presl. and Cymbopogon citratus DC., respectively; have in their chemical structure long hydrocarbon tails and heads with functional groups, it is expected that they can act also as a surfactant for the preparation of NP [Currently, several reports have been investigated to determine the effect of raw materials on the physicochemical properties of the NP prepared by solvent displacement method. In this context, the impact of poly-ε-caprolactone amount on the average diameter of r number . With ress EO-NP . This fiss EO-NP . With ress EO-NP . Authorssulation . This fi lime EO . In othereported . By the reported ,82,86,88reported ,71. As ron of NP .Rosmarinus officinalis L. EO loss was investigated [In addition to the factors mentioned above, operating conditions may affect the physicochemical properties of the NP prepared by nanoprecipitation process. In this context, Qiu and coworkers studied the effect of the complexation temperature of starch  on the colloidal properties of methone-loaded starch NP . Resultsstigated . In thisstigated . Similarstigated ,67,71,93stigated ,87,90,94stigated . One mayZanthoxylum rhoifolium L. EO from poly-ε-caprolactone-NP have reached a level of maximal release (81.9%) at 72 h [Zanthoxylum rhoifolium L. EO showed an initial burst effect, followed by a slow release after 12 h. Similar biphasic release profile was already described [Lavandula dentata L. EO release from polyethylene oxide-b-polylactic acid (PEO-b-PLA) copolymers based-NP depends on polylactide molecular weight [Lavandula dentata L. EO from PEO5KDa-b-PLA10KDa nanoparticles was about 40% and this percentage remained the same for 24 h of releasing process. While, for PEO5KDa-b-PLA4.5KDa nanoparticles, only 5% was released. This difference in release amount was related to the nanoparticle average size. In fact, the nanoparticles produced from PEO5KDa-b-PLA10KDa copolymer were significantly smaller than those produced from the PEO5KDa-b-PLA4.5KDa copolymer. Apparently, small nanoparticles favored the diffusion of lavender EO from the inner core to the external medium, leading to increase the amount of EO released [In vitro release of EO from particles prepared by nanoprecipitation technique generally exhibits a controlled manner. In a study carried out by Christofoli and coworkers,  at 72 h . The relescribed ,73,88. Aescribed . SeveralNigella sativa L. EO co-loaded with indomethacin in poly-ε-caprolactone -NP during one month under three different temperatures  [Zanthoxylum rhoifolium L. EO against photodegradation (UV-A and UV-C radiations) was investigated [The great challenge that hinders the feasibility of biological applications of EO in several fields is their instability in the presence of light, heat and humidity . Hence, d 40 °C) . No signstigated . Resultsstigated . Rosmarinus officinalis L. EO loaded-nanocapsules prepared by this technique at small and large scales [With the development of nanocarriers design strategies, scalability and reproducibility remain crucial in the choice of a suitable technique to produce plant oils loaded nanocarriers with predictable properties. With regards to the nanoprecipitation process, Ephrem and coworkers compared the colloidal properties of e scales . Resultse scales . Overall, based on the literature discussed above, the extensive use of nanoprecipitation process for essential oil encapsulation purpose was related to its benefits including (i) rapidity , (ii) siEssential oils have conquered space in several fields due to their pertinent properties. Currently, the biological effects of EO have been widely documented ,98. The Aspergillus flavus and Aspergillus parasiticus are widely involved in food spoilage producing secondary metabolites or mycotoxins that are carcinogenic and cause fatal diseases in both animals and humans. Recently, Luque-Alcaraz et al. investigated the inhibitory effect of Schinus molle L. EO loaded chitosan NP against the filamentous fungi Aspergillus parasiticus involved in spoilage of fruits, vegetables or other substrates rich in carbon sources [Aspergillus parasiticus viability. Similarly, Sotelo-Boyas et al. reported that the inclusion of thyme EO extracted from the stem and leaves of Thymus serpyllum L. in chitosan NP is a feasible alternative to obtain antibacterial nanoparticles, where the activity that each compound presents individually is strengthened [Staphylococcus aureus  for 40 µL of minimum inhibitory volume [Thymus serpyllum L. and Citrus aurantifolia Christm., respectively; to eradicate Pectobacterium carotovorum, plant phytopathogenic bacteria, involved in the decay of fresh fruits and vegetables like potatoes, carrots, radishes, onions, cucumbers, squash, eggplant, peppers, cabbage and tomato [Pectobacterium carotovorum than free chitosan NP and lime EO based-NP. For chitosan free nanoparticles, the number of colonies forming units (CFU) could not be determined (countless colonies). While for chitosan-lime EO- and chitosan-thyme EO- based NP, CFU were found 450 and 240 respectively. The inhibition halo diameter of free chitosan-NP, chitosan-lime EO-NP and chitosan- thyme EO-NP were found 15.0 ± 0.2, 13.0 ± 0.1 and 24.0 ± 0.1 mm, respectively [Zanthoxylum rhoifolium L. EO loaded in poly-ε-caprolactone nanospheres and investigated in vivo activity against Bemisia tabaci populations using tomato as host plant [Rosmarinus officinalis L. EO in poly-ε-caprolactone NP enhances its insecticidal activity against the red flour beetle, Tribolium castaneum [During the last few decades, environmental risks to humans, flora, and fauna and the development of resistance in species of pathogenic microorganisms have increased significantly due to the indiscriminate use of synthetic agrochemicals such as pesticides, herbicides and insecticides. Today, the use of natural compounds especially EO in the agriculture field has brought to the forefront ,99,100.  sources . Apparen sources ,102,103.ngthened . The higy volume . Sotelo-d tomato . Thyme Eectively . Furtherst plant . The biost plant . In anotastaneum . One mayCitrus aurantifolia Christm. EO loaded-NP by nanoprecipitation technique and tested their antibacterial activity against four food-borne bacteria: Staphylococcus aureus, Listeria monocytogenes, Shigella dysenteriae and Escherichia coli [Shigella dysenteriae, with an inhibition halo diameter of 3.5 cm for 40 mL of minimum inhibitory volume [Salmonella typhi, Staphylococcus aureus and Enterococcus coli with minimum inhibitory concentration (MIC) ranging from 182 to 374 mg/mL [The use of synthetic food preservatives is the most common method of postharvest disease control; however, due to the high consumer awareness to the use of synthetic additives, formulations based on low toxicity and more environmentally friendly compounds are more desirable. In light of that, natural extracts like EO or their main components, which are considered as Generally Recognized as Safe (GRAS) are effective alternatives to synthetic products. Currently, several works have evidenced the application of EO as antimicrobial and antioxidant in food packaging . In 2017hia coli . The higy volume . Further74 mg/mL . The enh74 mg/mL . Similar74 mg/mL .Salmonella enterica and the Shiga toxin-producing Escherichia coli (STEC), including Escherichia coli O157:H7. This pathogen can be transmitted to consumers by a variety of food vehicles such as poultry, meat and fresh and dairy products, causing harmful diseases. In 2016, Yegin et al. investigated the inhibition of Salmonella enterica and Escherichia coli O157:H7 in vitro on spinach surfaces by geraniol loaded-pluronic F127 nanoparticles [Salmonella enterica and Escherichia coli O157:H7, a decrease in MIC of geraniol following nano-encapsulation was reported. For Salmonella enterica, the MIC values were 0.25 and 0.70 wt.% with encapsulated and un-encapsulated geraniol, respectively; while for Escherichia coli O157:H7, the MIC values were 0.2 and 0.4 wt.%. The type of NP application onto spinach inoculated with pathogens was investigated and results showed that the immersion was more effective than the spraying technique due to the high contact between pathogen cells attached to spinach surfaces and EO loaded-NP. In this study, more experiments were carried out to investigate the NP absorption and interaction with bacterial membrane lipids. In this context, confocal microscopy was used to characterize the interaction of fluorescent geraniol NP with Escherichia coli O157:H7 cells. Results showed that un-encapsulated Nile Red, used as fluorescent agent, was not absorbed into cell membranes and no fluorescent signal from cells was detected. While, nanoparticles co-encapsulated geraniol and Nile Red were taken up into the internal compartments of Escherichia coli O157:H7 cells as fluorescence was detected from these cells. This finding was attributed to the fact that encapsulation of EO enhances their bio-availability and transport to targeted cells [Other examples of foodborne microbial pathogens which continue to impose significant health burden even in developed countries are articles . For boted cells . Schinus molle L. EO-loaded chitosan nanoparticles to eradicate Colletotrichum gloeosporioides, a phytopathogen fungus responsible for the anthracnose of avocado  [Schinus molle L. EO was shown; and the developed particles demonstrated a high inhibitory effect upon the in vitro viability of Colletotrichum gloeosporioides and low mutagenicity and toxicity. Additionally, authors reported that the inhibitory effect increased proportionally to the used concentration. At a concentration of 0.160 mg/mL, the developed particles exhibited a greater inhibitory effect on radial growth, spore germination and viability of the spores [®RS100) was found to exhibit an antibacterial activity. In 2019, Froiio et al. pointed out a synergetic effect between Eudragit®RS100 and sweet orange EO, extracted from the peels of Citrus sinensis L., to preserve the fresh orange juice against foodborne bacteria: Escherichia coli [In another work study, Chavez-Magdaleno and coworkers developed duction) . In thise spores . Besideshia coli . Chavez-hia coli .Thymus vulagris L. EO loaded-Eudragit L 100-55 nanoparticle [Vitis Vinifera L.) were carried out. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay revealed that the radical scavenging activity was higher for encapsulated EO (73.50 ± 0.76%) as compared to that of un-encapsulated oil (59.62 ± 1.77%). Additionally, the shelf life study demonstrated that grapes treated with EO-loaded-NP maintained their characteristics of color, firmness, titratable acidity, and total soluble solid content for longer time than those without any treatment [Thymus vulgaris L. EO loaded nanosystem [Thymus vulgaris L. EO loaded chitosan-NP at different concentrations for controlling Colletotrichum gloeosporioides in vitro and in situ was investigated [Thymus vulgaris L. EO loaded chitosan-NP did not affect fruit quality since weight loss, dry matter content, total soluble solids and acidity were similar to that of the control fruit [Currently, several reports carried out in situ assays by applying coating formulations containing EO-loaded nanosystems on fresh fruits to investigate their shelf life and to evaluate any damage caused by microorganisms. In this context, Piña-Barrera et al. proposed a promising food packaging system for the preservation of grape against foodborne bacteria basing on particle . The antreatment . Furthernosystem . In anotstigated . Resultsol fruit .Staphylococcus epidermidis [Cymbopogon citratus DC. EO to reduce the ability of Escherichia coli and Candida albicans to develop biofilms in a dose and strain dependent manner as compared to the un-encapsulated EO [Mentha piperita L., Cinnamomum Cassia presl. or Cymbopogon citratus DC. EO and results revealed no significant cytotoxicity on the normal growth and the development of cultured diploid human cells [Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Candida albicans).The ability to form biofilms contributes significantly to the pathogenesis of many microbial diseases ,106 and dermidis . Furtherlated EO . In addilated EO . Afterwaan cells . AdditioCymbopogon Martinii Roxb. EO loaded-NP as an alternative to synthetic antioxidants for cosmetic application [Staphylococcus aureus and Escherichia coli as compared to free EO emulsion [Nigella sativa L. EO to indomethacin in order to boost the anti-inflammatory and analgesic effects of indomethacin [Nigella sativa L. EO [Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus were in the same range as that of the unencapsulated thymol, encapsulated thymol formulation was selected as the optimal formulation since it provided an antibacterial activity for a long period. For instance, cosmetic lotion formulations containing encapsulated thymol provided total suppression of viable bacteria growth over the three months test period, while free thymol showed effective suppression for only 2–4 weeks [Nowadays, EO are remarkably incorporated in cosmetic products due to their ability to enhance the product’ properties and preservation, and to give a marketing image to the final product. Several work studies have been elaborated to incorporate EO in cosmetics and personal care products. Recently, Jummes et al. developed lication . The devemulsion . Furthermethacin . Skin peva L. EO . In anotva L. EO . Althoug–4 weeks . Lavandula dentata L. oil, widely used for its sedative effect, was efficiently entrapped in NP prepared from the diblock copolymer (PEO-b-PLA) for application as antibacterial agent in textiles components used in the footwear industry [Origanum Vulgare L., loaded poly-ε-caprolactone NP to functionalize medical devices for antibacterial activity [During recent decades, the use of EO has emerged to the textile domain and a considerable amount of research has been conducted concerning the benefits of aromatherapic textiles . Lavanduindustry . Variousactivity . In thisactivity .On the whole, the polymeric NP have created a tremendous interest regarding the encapsulation of EO due to their inherent features including, (1) nanometric size which enhances cellular uptake ,86, (2) Currently, growing concern for essential oils with the same efficiency or even more than chemical synthesized drugs has prompted scientists to focalize most of their efforts on developing new approaches to preserve the stability, bioactivity and bioavailability of these bioactive agents. The occurrence of oil nanoencapsulation has been noticed as efficient approach to resolve such restrictions. In this bibliographic paper, we emphasize an overview, recent advances, challenges and applications of essential oils loaded polymeric nanoparticles prepared via nanoprecipitation process. According to the literature discussed above, the nanoprecipitation technique represents an easier, less energy consuming, more reproducible, as well as a widely valid method for the encapsulation of essential oils when compared to other preparation methods. For instance, it provides most suitable nanoparticles in term of size and encapsulation efficiency. Operating conditions management and raw materials selection are key points to obtain formulations bearing suitable characteristics for the in vitro and in vivo applications. Important activities, like antimicrobial, antioxidant, antifungal, anti-inflammatory, pesticidal and insecticidal were enhanced upon the encapsulation of essential oils in polymeric nanoparticles.Although several advances have been reported in the literature, more in vivo studies are needed to provide reliable results. Additionally, toxic effects, accumulation in biological systems and removal mechanisms must be established. Scale-up the nanoprecipitation in industries constitute another important aspect to be taken into consideration due to the fact that using polymeric nanoparticles for the delivery of essential oils is one of the newest approaches in the pharmaceutical technology.


Escherichia Coli (E. coli) pathotypes in Iran is limited. This nation-wide survey aims to provide a comprehensive description of the distribution of five pathogenic E. coli in Iran.Knowledge about the distribution of E. coli-positive cultures underwent PCR testing for the detection of STEC, ETEC, EPEC, EAEC, and EIEC pathotypes. Pathotype frequency by province, age-group, and season was estimated.Stool samples were collected from 1,306 acute diarrhea cases from 15 provinces (2013–2014). E. coli , and 659 (50.5%) were pathogenic E. coli . STEC was the most frequent pathotype (35.4%). ETEC (14.0%) and EPEC (13.1%) were the second and the third most frequent pathotypes, respectively. EAEC (4.3%) and EIEC (0.3%) were not highly prevalent. Fars (88.7%) and Khorasan-e-Razavi (34.8%) provinces had the highest and lowest frequencies, respectively. E. coli pathotypes were more frequent in warmer than cooler seasons, showed the highest frequency among children under five years of age (73%), and had no significant association with participants’ gender.979 diarrhea samples (75.0%) were culture-positive for E. coli may be an important cause of acute diarrhea in adults and children in Iran. STEC and ETEC seem to be widespread in the country with a peak in warmer seasons, impacting the recommended use of seasonal STEC and ETEC vaccines, especially in high-risk groups. Monitoring the incidence of E. coli pathotypes, serotypes, and antibiotic resistance over time is highly recommended for evaluation of interventions.Diarrheagenic  Escherichia coli (E. coli) bacteria, called diarrheagenic E. Coli (DEC), is capable of causing diarrhea in the human host. Different DEC strains (pathotypes) have been determined recently, some of which include Shiga toxin–producing E. coli (STEC), enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), and diffusely adherent E. coli (DAEC) [A subset of i (DAEC) .E. coli pathotypes may vary by geographic region. Some of the STEC serotypes, called O157:H7, are prevalent in the United States and Canada. The non-O157:H7 serotypes, however, are prevalent in Latin America and Europe. ETEC, is a widespread pathotype in the developing world [Current evidence suggests that the incidence of ng world .E. Coli pathotypes over the country [E. Coli pathotypes [E. Coli pathotypes in the epidemiology of diarrheal diseases in different geographical regions and among adults, adolescents, and children of Iran.In Iran, there is a paucity of large-scale studies providing an unbiased estimate of the distribution of major  country . Many of country –9, but othotypes –13. AlsoThis study was approved by the ethics committee of Pasteur Institute of Iran . Participants were informed about the study objectives, assured about the confidentiality of their information, and gave their written informed consent for participation in this study.Cross-sectional.Consisted of all Iranian nationals who were residents of target provinces of Iran and referred to health centers with a chief complaint of acute diarrhea (with/without bleeding). Acute diarrhea was defined as passage of liquid/watery stool for more than 2–3 times a day. Immunologically compromised patients as well as patients with chronic diarrhea were excluded. Also cases in which individuals traveled two weeks prior to the process of recruitment were not included.E. coli-positive samples identified by culture method were sent to the National E. Coli Reference Laboratory (NECRL) in Pasteur Institute of Iran (PII) for molecular analysis and pathotype identification.Of 31 provinces in Iran, 15 were selected in this study (sampling fraction = 50%). The provinces were selected in a way that ensured that all the geographical areas of Iran are included. In each province, three cities were selected, leading to a total number of 45 cities. Stool samples from diarrhea cases were collected at the second half of each month in order to harmonize the time-frame of sampling across all cities. A random sample of collected specimens was then subjected to culture. To ensure data quality and standardize the laboratory procedures across all field centers, we held a series of training workshops where the staff were thoroughly trained about the study procedures. Trainings involved how to collect stool samples, do the cultures, interpret and record culture results, and transfer the samples to NECRL.E. coli characteristics were selected and transferred to SIM medium , Simmons citrate , and MRVP broth  to further check for the presence of E. coli characteristics. Samples that were Indol-positive , MR-positive , VP-negative , and citrate-negative were potentially considered E. coli-positive. Identified E. coli-positive samples were incubated on LB . After O/N incubation, the samples were centrifuged (Eppendorf Germany) and the final products were stored at −70 °C until used for PCR assays.Stool samples were inoculated on MacConkey agar  at 37 °C for 24 hours. After O/N incubation (incubator: Vision Korea), results were checked, and five typical colonies on MacConkey agar (with pink color) were selected and transferred to the triple sugar iron agar media . Colonies with E. coli virulence genes indicative of the five candidate E. coli pathotypes were subjected to PCR assay. The primers used for probe amplification were chosen either from existing literature or designed from available gene sequences  plus seven μl of DDW and forward and reverse primers (1 μl of each) were added to one μl of the sample. For positive and negative controls, 100 kb DNA ladder and ladder mix were used. The processes of denaturation, annealing and extension were performed in the Eppendorf thermo cycler (Germany).For the preparation of samples, ten E. coli pathotypes was estimated in the overall population and in subgroups of age, location (province/city), and time (4 seasons and 12 months). Association of each E. coli pathotype with season and type of diarrhea (with/without bleeding) was assessed using Chi Square test. Statistical tests were considered as significant at 0.05 levels.Data were analyzed in Stata software (version 14). Frequency of E. coli-positive , and were subjected to molecular assays. Pathogenic E. coli were detected in 659 out of 1,305 diarrheal samples . Stx1 (26.1%) and Eae (25.9%) were the most frequent virulence genes. STEC (35.4%) was the most and EIEC (0.3%) was the least frequent pathotype , and lowest in Razavi Khorasan Province, although the prevalence in the latter province was still considerably high  in Iran and was detected in all provinces , Panel BWas the second most frequent pathotype (14.0%) in Iran and was detected in all investigated provinces , Panel CE. coli pathotype in Iran (13.1%). The highest frequency (31.0%) was observed in Khuzestan province. This pathotype was not detected in Esfahan province . Except Kurdistan province with sixteen isolated EAEC pathotypes, other provinces identified less than five EAEC isolates . At the Was a rare pathotype in Iran . No remarkable seasonal and monthly trends could be identified for other pathotypes, probably due to availability of few samples  with STEC  and EIEC  being the most frequent pathotypes. EPEC was highly frequent in children aged 1–5 years, as well as adults aged 30–40 years (16.7% each). ETEC had the highest frequency in children aged 5–10 years (21.4%). This pathotype was also common in adults and geriatrics . This is lower than the previous estimates in different parts of Iran, such as Tehran (20%)  Zanjan Click here for additional data file.S2 File(DOCX)Click here for additional data file.S3 File(DOCX)Click here for additional data file.


Hydatigera taeniaeformis (H. taeniaeformis) is one of the most robust of tapeworm parasites that is widely distributed around the world. Information of proteins of H. taeniaeformis has not previously been reported. Using liquid chromatography tandem-mass spectrometry (LC–MS/MS) analysis, the proteome of H. taeniaeformis metacestode was profiled and a total of 408 proteins were identified. Of these, 26.5% (108/408) were annotated to be associated with metabolic pathways. Consistently, Gene Ontology analysis showed that those identified proteins were mainly classified into metabolic process of the biological processes. A set of metabolic enzymes, including Fructose-1,6-bisphosphate aldolase, enolase, Glucan phosphorylase, and phosphoenolpyruvate carboxykinase, were abundant in H. taeniaeformis metacestodes. In addition, some rare but interesting proteins like antigens (such as tegument protein and Antigen B) were identified. The two recombinant proteins of tegument protein and Antigen B were well-recognized by the sera from the H. taeniaeformis-infected mice. The H. taeniaeformis metacestode proteome might help to find new candidates for the immunodiagnosis and vaccine development and provide valuable information on H. taeniaeformis biology. T. taeniaeformis metacestodes.A total of 408 proteins were identified in Identified proteins were classified mainly to be involved in metabolic process.H. taeniaeformis-infected mice.The two recombinant proteins of tegument protein and Antigen B were well-recognized by the sera from Hydatigera taeniaeformis  is a parasitic helminth belonging to a family of Taenidae. The natural life cycle of H. taeniaeformis alternates between a definitive host (cats or dogs) and an intermediate host (mostly rodents and less frequently lagomorphs and humans)  , 2. Adul humans) , 4. Onco rodents –7. Our rEchinococcus spp. . Identification and characterization of proteins from H. taeniaeformis metacestode might help to find new candidates for the immunodiagnosis and vaccine development and provide valuable information on H. taeniaeformis biology.To date, proteomic studies of several cestodes have been reported, including cus spp. , 9, Taena solium , Taenia datigena , and Taenia ovis . HoweverH. taeniaeformis . In the phylogenetic tree based on cox-1 sequences, both Miaoping (MP) and Wangping (WP) isolates were clustered together with H. taeniaeformis isolated from China , Germany (JQ663994.1), and Japan (AB221484.1) with high probabilities , and then added 6 M urea to the mixture. After centrifugation, the supernatant proteins (approximately 2 mg) were digested with 40 μL trypsin buffer  in 40 μL (25 mM) NH4HCO3) in a 37 °C water bath for 16–18 h. This digest was transferred to clean ultrafiltration tubes fitted with 10 kDa membranes, and centrifuged at 14,000 × g for 10 min. Peptide sequencing was conducted using high performance liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) in Q-Exactive (Thermo Fisher Scientific) as described previously (H. taeniaeformis protein database retrieved from WormBase Parasite1 (http://parasite.wormbase.org/Hydatigera_taeniaeformis_prjeb534/Info/Index/) with following parameters: tryptic-specific peptides, maximum of one missed cleavages, a peptide mass tolerance of 20 ppm and a MS/MS tolerance of 0.5 Da. Label-free quantitation of proteins were calculated by intensity-based absolute quantification (iBAQ). Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for the identified proteins were annotated.eviously . The rawEscherichia coli BL21 (DE3) cells. For the expression and purification of the recombinant proteins, bacteria were induced with 0.5 M isopropyl-b-D-thiogalactoside (IPTG) and the recombinant proteins were purified by Ni Sepharose 6 Fast Flow  and checked by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE gel.The coding sequences of tegument protein and Antigen B genes were amplified with their specific primes. PCR fragments were digested and subcloned into prokaryotic expression plasmid pET-28a. The recombinant plasmids were transformed into H. taeniaeformis . Finally, they were washed and incubated with a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG  for 1 h. Signals were visualized by exposing X-ray films using Pierce ECL Western Blotting Substrate .The recombinant proteins were separated by 12% SDS-PAGE and transferred to a PVDF membranes using a Semi-Dry electrophoretic transfer cell (BioRad) for 30 min. The membranes were incubated with 5% (w/v) non-fat for 1 h and then incubated overnight at 4°C with mouse ant-sera against H. taeniaeformis metacestodes. In total, 408 proteins were identified by at least two unique peptides , and enolase  functional classification, 287 identified proteins were mainly grouped into six categories  . Approxitabolism . Three m enolase . Higher ytoplasm –15. Besiytoplasm . The absscoleces , 16–18, Echinococcus multilocularis , related to posttranslational modification, were very abundant in the H. taeniaeformis metacestodes  is an allergenic molecule of potential clinical importance of the intensity of echinococcosis immune response  by western blot. The recombinant proteins of HtTegP and HtAtB were about 18 and 16 kDa on the SDS-PAGE, respectively (H. taeniaeformis, but not by the negative control serum, indicating the good immunoreactivity of recombinant proteins (E. multilocularis, TegP11 was primarily localized in the surface of protoscoleces and has an immunoregulatory capacity on RAW246.7 macrophages (H. taeniaeformis infection.Three typical nuclear protein homologs grouped into translation group were identified, including an elongation factor EF-2, an adenine nucleotide translocator, and Histones H3/4. The presence of elongation factors has been detected in asiatica . Echinocresponse . Histoneacestode . Severalacestode , tegumenacestode , and putacestode . Early saeformis , 39. A scestodes , 41. It ectively . Westernproteins . Tegumenproteins . A growiproteins , 43. Recproteins . In E. mrophages . Antigenrophages . Severalrophages . FurtherT. taeniaeformis metacestodes, most of which were classified mainly to be involved in metabolic process. The two recombinant proteins of tegument protein and Antigen B were well-recognized by the sera from H. taeniaeformis-infected mice. Identification and characterization of proteins from H. taeniaeformis metacestode might help to find new candidates for the immunodiagnosis and vaccine development.A total of 408 proteins were identified in The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.Animal experiments were approved by Animal Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. All animal experiments in the study were handled in strict accordance with good animal practice of Animal Ethics Procedures and Guidelines of the People's Republic of China.XG performed study design, data analysis, and preparation of the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.