Valentina Meucci

High Growth Rate of Girls with Precocious Puberty Exposed to Estrogenic Mycotoxins

OBJECTIVE To test the hypothesis that human puberty timing can be advanced by environmental estrogen exposure. STUDY DESIGN We analyzed serum mycoestrogen contamination via high-performance liquid chromatography (HPLC) in 32 girls affected by central precocious puberty (CPP) and in 31 healthy female control subjects. All 32 patients received triptorelin (TR) for more than 12 months after diagnosis. RESULTS Increased serum levels of zearalenone (ZEA; 933.7 +/- 200.3 pg/mL; 95% CI, 723.5-1143.9) and of its congener alpha-zearalenol (106.5 +/- 1.9 pg/mL; 95% CI, 104.5-108.5) contaminated 6 girls with CPP, who were from a bounded Tuscany area. At diagnosis, ZEA levels correlated with patient height (r = 0.906, P < .05) and weight (r = 0.887, P < .05), but not with bone age. In patients who were mycotoxin-positive, height (F = 4.192; P < .01), weight (F = 3.915; P < .01), and height velocity (F = 2.777, P < .05) were higher than patients who were mycotoxin-negative during 12-months TR treatment. Height correlated with weight both in patients who were mycotoxin-positive (r = 0.986, P < .001) and in patients who were mycotoxin-negative (r = 0.994, P < .001). Body mass index, bone age, and gonadal secretion was not different in patient groups before and during TR treatment (P > .05). CONCLUSIONS Mycoestrogenic zearalenone is suspected to be a triggering factor for CPP development in girls. Because of its chemical resemblance to some anabolic agents used in animal breeding, ZEA may also represent a growth promoter in exposed patients.

An optimized digestion method coupled to electrochemical sensor for the determination of Cd, Cu, Pb and Hg in fish by square wave anodic stripping voltammetry

An optimized digestion method coupled to electrochemical detection to monitor lead, copper, cadmium and mercury in fish tissues was developed. Square wave anodic stripping voltammetry (SWASV) coupled to disposable screen-printed electrodes (SPEs) was employed as fast and sensitive electroanalytical method for heavy metals detection. Different approaches in digestion protocols were assessed. The study was focused on Atlantic hake fillets because of their wide diffusion in the human nutrition. Best results were obtained by digesting fish tissue with hydrogen peroxide/hydrochloric acid mixture coupled to solid phase (SP) purification of the digested material. This combined treatment allowed quantitative extraction from fish tissue (muscle) of the target analytes, with fast execution times, high sensitivity and avoiding organic residues eventually affecting electrochemical measurements. Finally, the method has been validated with reference standard materials such as dogfish muscle (DORM-2) and mussel tissues (NIST 2977).

Mycotoxin detection in infant formula milks in Italy

After birth, infant formulas constitute an important or often sole food source for infants during the first months of life. In this study, a survey on the presence of aflatoxin M1 (AFM1) and ochratoxin A (OTA) in the 14 leading brands of infant formulas marketed in Italy was conducted. Mycotoxins were determined by immunoaffinity column clean-up and high-performance liquid chromatography (HPLC) with fluorescence detection. AFM1 was found in two of 185 samples, but at levels below the European legislation limit of 25 ng l−1. OTA was detected in 133 (72%) samples (range = 35.1–689.5 ng l−1). It has been observed that OTA contamination was 80% in the ready-to-use preparations and 63% in the powdered samples. The Scientific Committee for Food (SCF) reviewed the toxicology on OTA and concluded that it would be prudent to reduce exposure to OTA ensuring that exposure is towards the lower end of the range of tolerable daily intakes of 1.2–14 ng kg−1 body weight day−1. OTA was also evaluated by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) and a provisional tolerable weekly intake (PTWI) of 100 ng kg−1 body weight was established. The OTA levels in pre-term ready-to-use infant formulas were sufficient to cause a higher OTA intake than the suggested TDI. The results point out the need to perform controls for prevention programmes especially when attempting to identify risk markers of the infant feed quality.

Antimicrobial susceptibility of Staphylococcus intermedius and Staphylococcus schleiferi isolated from dogs.

The susceptibility to 23 antimicrobial agents was determined in 114 isolates of Staphylococcus intermedius and eight isolates of Staphylococcus schleiferi of canine origin. Overall, 73% of S. intermedius isolates and 37.5% of S. schleiferi isolates were susceptible to all the 23 antimicrobials tested. The large majority of S. intermedius strains retained susceptibility to antimicrobials currently employed in treatment of pyoderma (cephalosporins, cotrimoxazole and association amoxicillin-clavulanic acid) as well as to those effective against staphylococci (fusidic acid, rifampicin and fluoroquinolones). Resistance in S. intermedius was observed mainly against macrolides, chloramphenicol and lincosamides, while S. schleiferi isolates retained susceptibility to all antimicrobials except three of six fluoroquinolones. Although, our results confirm susceptibility to antimicrobials currently employed in pyoderma treatment, the several different resistance patterns observed for S. intermedius emphasize the importance of antimicrobial susceptibility testing of canine staphylococci to choose the most appropriate treatment of infections and to allow the prudent use of antimicrobial drugs in companion animals.

Mycoestrogen Pollution of Italian Infant Food

OBJECTIVE To determine the concentrations of zearalenone and its metabolites in the leading brands of infant formula milks and meat-based infant foods commonly marketed in Italy, and to assess their repercussion in the provisional tolerable daily intakes of these estrogenic mycotoxins. STUDY DESIGN A total of 185 cows milk-based infant formulas and 44 samples of meat-based infant foods samples were analyzed. The analysis of mycotoxins was performed by immunoaffinity column clean-up and high-pressure liquid chromatography with fluorescence detection. RESULTS Zearalenone was detected in 17 (9%) milk samples (maximum 0.76 μg/L). The α-zearalenol was detected in 49 (26%) milk samples (maximum 12.91 μg/L). The β-zearalenol was detected in 53 (28%) milk samples (maximum 73.24 μg/L). The α-zearalanol and β-zearalanol were not detected in milk samples. Although α-zearalenol was detected in 12 (27%) meat samples (maximum 30.50 μg/kg), only one meat-based sample was contaminated by α-zearalanol (950 μg/kg). Zearalenone, β-zearalenol, and β-zearalanol were not detected in meat samples. CONCLUSIONS This study shows the presence of mycoestrogens in infant (milk-based and meat-based) food, and this is likely to have great implications for subsequent generations, suggesting the need to perform occurrence surveys in this type of food.

An optical immunosensor for rapid vitellogenin detection in plasma from carp (Cyprinus carpio)

Vitellogenin (vtg) has proven to be a sensitive and simple biomarker for assessing exposure of fish to environmental estrogens. The aim of this work was to develop a rapid, in the order of minutes, screening method for the detection of fish vtg. The surface plasmon resonance technique (Biacore Xtrade mark) was coupled with immunodetection for the determination of fish vtg in plasma and mucus from carp (Cyprinus carpio). Monoclonal anti-vtg antibodies were linked on the sensor surface through chemical cross-linking via a capturing antibody. A simple regeneration process allowed the reuse of the sensor surface. Sensor optimisation was carried out using carp vtg. The developed immunosensor was tested with vtg spiked samples and with plasma and mucus from fish exposed to 17beta-estradiol (E2). Vitellogenin could be detected in the ppm range in buffer as well as in plasma and mucus. Good discrimination between control and exposed samples was obtained. The results were compared with ELISA and a correlation coefficient of R(2)=0.85 (n=9) between the two methods indicated that the immunochemical biosensor could be used for the analysis of vtg in fish plasma samples. The assay time was 20min hence allowing for rapid sample screening.

Fluoroquinolone resistance and molecular characterization of gyrA and parC quinolone resistance-determining regions in Escherichia coli isolated from poultry

Escherichia coli are a common inhabitant of the gastrointestinal tract of mammals and birds; nevertheless, they may be associated with a variety of severe and invasive infections. Whereas fluoroquinolones (FQ) have been banned in the United States for use in poultry production, the use of these antimicrobials in poultry husbandry is still possible in the European Union, although with some restrictions. The aim of this study was to investigate the FQ resistance of 235 E. coli isolates recovered from chickens and turkeys. Minimum inhibitory concentrations were determined by a microdilution method, whereas mutations in the quinolone resistance-determining regions of the target genes, gyrA and parC, were detected by a PCR-based method. High resistance rates (>60%) were observed for nalidixic acid, flumequine, and difloxacin, whereas resistance to ciprofloxacin, danofloxacin, enrofloxacin, marbofloxacin, and sarafloxacin was less frequently reported (<40%). Sixty-four isolates (27.2%) showed full susceptibility toward the tested FQ, but 57 isolates (24.2%) were resistant to all tested FQ. The remaining 114 E. coli isolates (48.5%) were grouped in 5 different resistance patterns. Isolates resistant only to flumequine or nalidixic acid or both possessed 1 gyrA mutation, whereas isolates with further resistance to enrofloxacin, difloxacin, danofloxacin, and sarafloxacin had in addition 1 or 2 parC substitutions. Two gyrA mutations coupled with 1 substitution in parC were detected in isolates resistant to all tested FQ. The number of mutations and their correlation with the in vitro activity of FQ reflected the currently accepted model, according to which a single gyrA substitution is associated with resistance or decreased susceptibility to older quinolones, whereas further gyrA or parC substitutions are needed for a higher level of resistance.

Cytotoxic effects of oxytetracycline residues in the bones of broiler chickens following therapeutic oral administration of a water formulation

Tetracyclines, which represent one of the most commonly used antibiotics for poultry, are known to be deposited in bones, where they can remain, despite the observation of appropriate withdrawal times. The aim of the study was to determine the concentration of oxytretracycline (OTC) residues in the bone and muscle of chickens, following the oral administration of a commercially available liquid formulation, and to test their cytotoxic effects on an in vitro cell culture model. Seventy-two 1-day-old broiler chickens were randomly allotted into 2 groups (control and treated animals). OTC (40 mg/kg BW) was administered via drinking water during the 1 to 5 and 20 to 25 days of life periods. At the end of the trial, the birds were slaughtered and the OTC residues in the target tissues were measured by means of liquid chromatography (LC) - tandem mass spectrometry (MS/MS). Cytotoxicity was assessed by evaluating the pro-apoptotic effect of the bone residues on the K562 erythroleukemic line and on the peripheral blood mononuclear cells (PBMC). In all the animals, the OTC residues in the muscle were far below the established MRL of 100 μg/kg. The OTC levels in the bones of the treated animals were instead found in the parts per million (ppm) range. Cell cytotoxicity was assessed by evaluating the pro-apoptotic effect of OTC bone residues on the haematopoietic cell system. This in vitro system has revealed a significant pro-apoptotic effect on both the K562 cell line and PBMC cultures. This result suggests potential human and animal health risks due to the entry of tetracycline residues contained in the bones of treated livestock into the food-chain. This could be of concern, particularly for canine and feline diets, as meat, bone meal, and poultry by-products represent some of the main ingredients of pet foods, especially in the case of dry pet food. Further studies are needed to define the underlying mechanisms of cytotoxicity and to evaluate the in vivo toxicological implications due to the observed in vitro effects.

Redox status evaluation in dogs affected by mast cell tumour.

Oxidative stress status has been evaluated in depth in human medicine and its role in carcinogenesis has been clearly established. The purpose of this prospective study was to evaluate antioxidant concentrations and oxidative stress in dogs with mast cell tumours (MCTs) that had received no previous treatments, and to compare them to healthy controls. In 23 dogs with mast cell tumour and 10 healthy controls, oxidative status was assessed using the Reactive Oxygen Metabolites-derived compounds (d-ROMs) test, antioxidant activity was measured by the Biological Antioxidant Potential (BAP) test, and α-tocopherol levels were evaluated using high-performance liquid chromatography and ultraviolet analysis. At baseline, dogs with MCT had significantly higher d-ROMs (P < 0.00001) and lower BAP (P < 0.0002) compared with healthy controls. However, no significant difference was observed for α-tocopherol (P = 0.95). Results suggest that oxidative stress pattern and oxidative defence barrier are altered in dogs with newly diagnosed MCT compared with control dogs. Future studies are needed in order to assess the prognostic role of oxidative stress and to evaluate the impact of different therapeutic approaches.

Plasma Procalcitonin Concentration in Healthy Horses and Horses Affected by Systemic Inflammatory Response Syndrome

Background The diseases most frequent associated with SIRS in adult horses are those involving the gastrointestinal tract. An early diagnosis should be the goal in the management of horses with SIRS. Objective The objective of this study was to evaluate the plasma procalcitonin (PCT) concentration in healthy and SIRS horses to assess differences between the two groups. Animals Seventy‐eight horses (30 healthy and 48 SIRS). Methods Prospective in vivo multicentric study. Horses were classified as SIRS if at least 2 of the following criteria were met: abnormal leukocyte count or distribution, hyperthermia or hypothermia, tachycardia, tachypnea. Healthy horses showed no clinical or laboratory signs of SIRS. Plasma PCT concentrations were measured with a commercial ELISA assay for equine species. Results were expressed as mean±standard deviation. T‐test for unpaired data was performed between healthy and SIRS group. SIRS group was divided in 4 subgroups and t‐test was performed between healthy versus each subgroup. Results PCT concentrations in healthy and SIRS horses were 18.28 ± 20.32 and 197.0 ± 117.0 pg/mL, respectively. T‐test showed statistical differences between healthy versus SIRS group and between healthy versus all subgroups. Conclusions and Clinical Importance Results showed an increase in PCT concentration in SIRS horses as previously reported in humans and dogs. PCT could be used as a single assay in equine practice for detection of SIRS.