Boosting plant food polyphenol concentration by saline eustress as supplement strategies for the prevention of metabolic syndrome: an example of randomized interventional trial in the adult population.

Introduction: Phenolic compounds in lettuce can increase by the application of positive stress (eustress) such as moderate saline stress. Phenolic compounds possess antioxidant capacity that is a key factor in the detoxification of excess reactive oxygen species. A double-blinded randomized interventional and placebo- controlled study design was carried out to compare the effect of daily dietary eustress lettuce ingestion in hepatic, lipid, bone, glucose, and iron metabolism. Methods: Forty-two healthy volunteers, 19 female and 23 male participants, were divided into two groups. Participants were randomized into a polyphenol-enriched treatment (PET) arm or control arm. Each arm consumed 100 g/day of control or eustress (polyphenols enriched treatment = PET) lettuce for 12 days. Primary study outcomes were serological analysis for assessing hepatic, lipid, bone, iron, and glucose markers at baseline and after 12 days. Secondary outcomes assessed body composition. Results: Salinity stress reduced plant yield but increased caffeic acid (+467%), chlorogenic acid (+320%), quercetin (+538%), and rutin (+1,095%) concentrations. The intake of PET lettuce reduced PTH, low-density lipoprotein (LDL), cholesterol, alanine transaminase (ALT), and aspartate transaminase (AST) enzyme levels and increased vitamin D and phosphate levels, while iron and glucose metabolism were unaffected. Discussion: Supplementation with eustress lettuce by increasing polyphenols concentration ameliorates hepatic, lipid, and bone homeostasis. Body composition was not affected. Clinical trial registration: https://classic.clinicaltrials.gov/ct2/show/NCT06002672, identifier: NCT06002672.

A New Potential Dietary Approach to Supply Micronutrients to Physically Active People through Consumption of Biofortified Vegetables.

Micronutrients are required in many reactions involved in physical activity and exercise. Most physically active people do not meet the body's needs in terms of micronutrients through diet. The novelty of the present manuscript is the use of an innovative dietary approach to supply micronutrients to physically active people through biofortified food. Therefore, the key point of this study was to verify whether supplementation with biofortified vegetables-and specifically molybdenum (Mo)-enriched lettuce-in healthy volunteers affects essential regulators of body homeostasis and, specifically, hematological parameters, iron and lipid metabolism, and hepatic function. Twenty-four healthy volunteers were allocated in a double-blinded manner to either a control group that consumed lettuce, or the intervention group, which consumed Mo-enriched lettuce, for 12 days. Blood samples were collected at baseline (T0) and after 12 days (T1). We found that supplementation with Mo-enriched lettuce did not affect hematological parameters, liver function, or lipid metabolism, but significantly improved iron homeostasis by increasing non-binding hemoglobin iron by about 37% and transferrin saturation by about 42%, while proteins of iron metabolism (e.g., transferrin, ferritin, ceruloplasmin) were not affected. The serum molybdenum concentration increased by about 42%. In conclusion, this study shows that consumption of Mo-biofortified lettuce ameliorates iron homeostasis in healthy subjects, and suggests that it could be used as a new nutritional supplementation strategy to avoid iron deficiency in physically active people.

Analysis of Body Perception, Preworkout Meal Habits and Bone Resorption in Child Gymnasts.

The beneficial effects of physical activity on body image perception and bone are debated among artistic gymnasts. Gymnasts seem to be at greater risk of developing body dissatisfaction, eating disorders and osteoporosis due to inadequate nutrition and attention to the appearance of the body. The objective of this work was to investigate the association between the artistic gymnast and a more favorable body image compared to their sedentary peers and if a preworkout high-carbohydrate meal (HCM; 300 kcal, 88% carbohydrates, 9% protein, 3% fat) or high-protein meal (HPM; 300 kcal, 55% carbohydrates, 31% protein, 13% fat) is able to attenuate bone resorption in young rhythmic gymnasts. Twenty-eight preadolescent female gymnasts were examined. Self-esteem tests were used to analyze body image perception. Preworkout eating habits were examined by short food frequency questions (FFQ) validated for children. The biomarker of the bone resorption C-terminal telopeptide region of collagen type 1 (CTX) was measured in the urine (fasting, postmeal and postworkout). Gymnasts reported higher satisfaction with their body appearance compared to sedentary peers. Of the gymnasts, 30% did not have a preworkout meal regularly, and the timing of the consumption was variable. Bone resorption was decreased by the HCM, consumed 90 min before the training, with respect to the HPM. The study suggests that playing artistic gymnastics is associated with a positive body self-perception in a child. The variability in preworkout meal frequency and timing need attention to prevent inadequate eating habits in light of the ability of the HCM to reduce acute bone resorption.

Analysis of corticosteroids in samples of animal origin using QuEChERS and ultrahigh-performance liquid chromatography coupled to high-resolution mass spectrometry.

A rapid and sensitive method for the confirmatory analysis of eight synthetic corticosteroids (betamethasone, dexamethasone, prednisolone, 6-methylprednisolone, triamcinolone, flumethasone, beclomethasone, fluocinolone acetonide) is proposed. The method is useful for detecting illegal treatments in different animal species. It consists of an extraction and cleanup using the quick, easy, cheap, effective, rugged, and safe (QuEChERS) strategy. Quantitative determination is achieved by ultrahigh-performance liquid chromatography coupled to high-resolution mass spectrometry with heated electrospray ionization in negative mode. Quantification is performed using surrogate matrix-matched standard calibration curve with dexamethasone-D4 as the internal standard. The method was validated for analyzing liver samples according to the criteria established by Decision 2002/657/EC. Linearity was assessed in the 1-10 µg kg-1 range and linear correlation coefficients were over 0.99 for all the analytes. CCα ranged from 0.04 to 0.16 µg kg-1 for substances without maximum residue limit. The method allows confident quantification and confirmation of corticosteroids in liver samples, and its simplicity makes it suitable for analyzing large numbers of samples.

Analysis of ß2-agonists in cattle hair samples using a rapid UHPLC-ESI-MS/MS method.

A simple and efficient method was developed for simultaneous analysis of five illegal residual ß2-agonists in cattle hair. ß2-Agonists were quantified by ultra high performance liquid chromatography coupled with electrospray ionisation tandem mass spectrometry operating in positive multiple-reaction monitoring mode. The method was validated as quantitative confirmatory method according to the EU Decision 2002/657/EC: instrumental linearity, specificity, precision, recovery, decision limit (CCα) and detection capability (CCß) were evaluated. The recovery were greater than 90% and the method appeared suitable for the control of these ß2-agonists in cattle hair samples with LOQ values between 4.9 and 5.5 µg/kg. This method could represent a simple and cheap approach to confirm ß2-agonists contamination of cattle for feeding in a not invasive way and before slaughter operations.

Fatty acids determination in Bronte pistachios by gas chromatographic method.

A gas chromatographic with flame ionization detector (GC-MS FID) method for the identification and quantification of fatty acids based on the extraction of lipids and derivatisation of free acids to form methyl esters was developed and validated. The proposed method was evaluated to a number of standard FAs, and Bronte pistachios samples were used for that purpose and to demonstrate the applicability of the proposed method. In this regard, repeatability, mean and standard deviation of the analytical procedure were calculated. The results obtained have demonstrated oleic acid as the main component of Bronte pistachios (72.2%) followed by linoleic acid (13.4%) and showed some differences in composition with respect to Tunisian, Turkish and Iranian pistachios.

Validation of the TrichinEasy® digestion system for the detection of Anisakidae larvae in fish products.

Anisakis and other parasites belonging to the Anisakidae family are organisms of interest for human health, because of their high zoonotic potential. Parasites belonging to this family can cause Anisakiasis, a parasitological disease caused by the ingestion of raw, infested fish products. Furthermore, evidence from the EFSA (European Food Safety Authority; EFSA 2010) has highlighted the allergological potential of nematodes belonging to the Anisakis genre. The detection and identification of Anisakidae larvae in fish products requires an initial visual inspection of the fish sample, as well as other techniques such as candling, UV illumination and artificial digestion. The digestion method consists of the simulation of digestive mechanics, which is made possible by the utilization of HCl and pepsin, according to EC Regulation 2075/2005. In this study, a new Anisakidae larvae detection method using a mechanical digestion system called Trichineasy® was developed. A total of 142 fish samples, belonging to 14 different species, were examined to validate the method. A reaction mixture with 100 g of sample, 10 g of pepsin (1:10000 NF) and 50 ml of 10% HCl at 36 ± 1°C for 20 minutes was evaluated to be the best condition for the digestion of fish samples. These parameters have also allowed the detection of viable larvae after digestion. The results confirm this instrumentation as a valuable and safe tool for the detection of Anisakidae larvae in fishery products.

Anisakidae in Fishing Products Sold in Sicily.

One of the parasite diseases associated with the consumption of raw fish that occurs with some frequency is the anisakiasis, a human disease caused by the accidental ingestion of larval nematodes of the genus Anisakis, family Anisakidae. At the National Reference Centre for Anisakiasis (C.Re.N.A.) from October 2012 to February 2013, a number of 231 bony fish (Trichiuridae, Engraulidae, Scombridae and Clupeidae) were received from different fishing sites in Sicily. Anisakis pegreffii is the main species detected in fish, as identified by molecular analysis based on polymerase chain reaction-restriction fragment length polymorphism, while Anisakis simplex sensu stricto was found only in Scomber scombrus caught in the Mediterranean Sea (Fishing Areas 37), in the Spanish coast (Fishing Areas 37) and in the Atlantic Ocean (Fishing Areas 34). Larvae of the genus Pseudoterranova were found only in fish caught in the Norwegian Sea.

Development and validation of two PCR tests for the detection of and differentiation between Anaplasma ovis and Anaplasma marginale.

Anaplasma ovis and Anaplasma marginale are tick-transmitted bacteria that cause anaplasmosis in domestic and wild animals. Recent results show that some domestic and wild animals and ticks are susceptible to both A. ovis and A. marginale, thus supporting the need to differentiate between these species in hosts and ticks diagnosed with Anaplasma infection. However, although anaplasmosis is one of the most common diseases of grazing animals worldwide, rapid and effective tests are not available for the detection of and discrimination between these 2 Anaplasma species. The objective of this research was to develop an easy and reliable method to identify and discriminate between the closely related pathogens A. ovis and A. marginale. A. ovis and A. marginale major surface protein 4 (msp4) gene sequences were retrieved from different geographic strains and aligned to design 2 sets of primers in a region with significant differences between the 2 species, but completely conserved among strains. PCR reactions using these primers were 100% species-specific and detected all strains from each pathogen previously identified with other methods. The 2 sets of primers designed for the specific PCR amplification of A. ovis and A. marginale allow easy-to-detect and discriminate between the 2 pathogens, thus avoiding the time-consuming sequencing or multi-gene amplification procedures. This PCR provides a tool for the detection of A. ovis and A. marginale in ticks and in wildlife and domestic hosts.

Molecular identification of tick-borne pathogens in Nigerian ticks.

A molecular epidemiology investigation was undertaken in two Nigerian states (Plateau and Nassarawa) to determine the prevalence of pathogens of veterinary and public health importance associated with ticks collected from cattle and dogs using PCR, cloning and sequencing or reverse line blot techniques. A total of 218 tick samples, Amblyomma variegatum (N=153), Rhipicephalus (Boophilus) decoloratus (N=45), and Rhipicephalus sanguineus (N=20) were sampled. Pathogens identified in ticks included piroplasmids (Babesia spp., Babesia bigemina and Babesia divergens), Anaplasma marginale and Rickettsia africae. Piroplasmids were identified in A. variegatum, A. marginale was found in R. decoloratus, while R. africae was detected in all tick species examined. Ehrlichia spp. and Theileria spp. were not identified in any of the ticks examined. Of the 218 ticks examined, 33 (15.1%) contained pathogen DNA, with the presence of B. divergens and R. africae that are zoonotic pathogens of public health and veterinary importance. The variety of tick-borne pathogens identified in this study suggests a risk for the emergence of tick-borne diseases in domestic animals and humans, especially amongst the Fulani pastoralists in Plateau and Nassarawa states of Nigeria.

Characterization of Anaplasma phagocytophilum and A. ovis infection in a naturally infected sheep flock with poor health condition.

Anaplasma species are transmitted by ticks and cause diseases in humans and animals. These pathogens infect sheep, an economically important domestic animal worldwide. The current study was designed to characterize in 200 animals the infection with Anaplasma phagocytophilum and Anaplasma ovis and the genetic diversity of A. ovis strains collected from a naturally infected sheep flock with poor health condition. Sheep had 98% seroprevalence to Anaplasma spp. antibodies. PCR results confirmed the presence of A. phagocytophilum and A. ovis DNA in 11.5% and 37% of the sheep, respectively. Concurrent infections were detected in 6.5% of the sheep. Seventy-one adult ticks were collected from 45 sheep with infestations ranging from one to 15 ticks per animal. The analysis of A. ovis msp4 sequences demonstrated a previously unreported polymorphism for this pathogen with 17 different haplotypes in infected sheep. These results demonstrated that, although A. ovis msp4 haplotypes may be less variable when compared with Anaplasma marginale and A. phagocytophilum strains on a global scale, genetic polymorphisms occur in this locus in strains obtained from an infected sheep flock with poor health condition.

Prevalence and genotypes of Anaplasma species and habitat suitability for ticks in a Mediterranean ecosystem.

Anaplasma species are tick-transmitted pathogens that impact veterinary and human health. Sicily is one of the locations where these pathogens are endemic. Sicily represents a typical Mediterranean ecosystem to study Anaplasma infection and tick habitat suitability. The aims of this study were (i) to characterize by 16S rRNA and species-specific msp4 gene PCR the prevalence and genotypes of A. marginale, A. phagocytophilum, and A. ovis in the most abundant host species in Sicilian provinces and (ii) to correlate differences between hosts and between western and eastern Sicily with the habitat suitability for ticks in these regions. Differences were found in the prevalence of Anaplasma spp. between different hosts and between western and eastern provinces. The differences in Anaplasma prevalence between different hosts may be explained by pathogen host tropism. The differences between western and eastern provinces correlated with the tick habitat suitability in these regions. The analysis of Anaplasma genotypes suggested a higher host and regional specificity for A. phagocytophilum than for A. marginale and A. ovis strains, a finding probably associated with the broader host range of A. phagocytophilum. The presence of identical A. marginale genotypes in the two regions may reflect cattle movement. The results for A. ovis suggested the possibility of some genotypes being host specific. These results provide information potentially useful for the management of tick-borne diseases caused by Anaplasma spp. in Sicily and other Mediterranean regions and may contribute to the development of models to predict the risks for these tick-borne pathogens.

Serologic and molecular characterization of tickborne pathogens in lions (Panthera leo) from the Fasano Safari Park, Italy.

Lions (Panthera leo) are an endangered species threatened by illegal hunting, habitat loss, and infectious diseases. Little is known about the tick-borne pathogens that infect lions and could contribute to population declines. The objective of this study was to characterize Rickettsia spp., Anaplasma phagocytophilum, and Coxiella burnetii infections in 10 lions from the Fasano Safari Park in Italy by serology, polymerase chain reaction, and sequence analysis. Although animals did not show clinical signs of tick-borne diseases, evidence of infection with C. burnetii, spotted fever group Rickettsia sp., and A. phagocytophilum were found in 50%, 20%, and 10% of the lions, respectively. One of the lions tested positive for all three pathogens. This study is the first report of molecular evidence of infection with C. burnetii, Rickettsia sp., and A. phagocytophilum in lions and provides evidence that these felids become infected and serve as hosts for tick-transmitted bacteria.

Molecular characterization of Anaplasma platys strains from dogs in Sicily, Italy.

BACKGROUND: The genetic diversity of Anaplasma platys (Rickettsiales: Anaplasmataceae) strains is currently poorly defined. The present study was designed to characterize A. patys strains in dogs from Palermo, Sicily, Italy, using a combination of PCR and sequence analysis of the 16S rDNA, heat shock operon groESL and citrate synthase (gltA) genes. RESULTS: Blood was collected from 344 dogs (111 pet dogs, 122 pound dogs and 111 hunting dogs) during 2003-2005 in the Province of Palermo, Sicily, Italy. The prevalence of A. platys in dogs in Sicily, as demonstrated by PCR and sequence analysis of the 16S rDNA, groESL and gltA genes, was 4%. None of the samples were positive for A. marginale, A. centrale, A. ovis and A. phagocytophilum DNA. Three different gltA genotypes of A. platys were identified in dogs from Sicily. Two of the gltA sequences of Sicilian A. platys strains were different from sequences reported previously. However, one of the gltA, 16S rDNA and groESL sequences were identical to the sequence of A. platys strains from other regions of the world characterized previously. CONCLUSION: At least three different strains of A. platys were identified in dogs from Sicily by PCR and sequence analyses of the 16S rDNA, groESL and gltA genes. The results reported herein suggested that genetic diversity of A. platys strains may be similar to A. ovis, but lower than the diversity reported for A. marginale and A. phagocytophilum. This lower genetic diversity may have resulted from restricted movement of infected hosts compared to A. marginale-infected cattle and/or the limited host range of A. ovis and A. platys as compared with A. phagocytophilum. These results expand our knowledge about A. platys and encourage further research for analysis of the genetic variation of A. platys strains worldwide.