Assessment of ceftiofur residues in cow milk using commercial screening test kits.

Ceftiofur, a third-generation cephalosporin, is one of the most used antibiotics in dairy industry. Intramuscular injection of 1 mg/kgBW ceftiofur hydrochloride (HCl) generally results in 0 hour withdrawal time for the milk in dairy cows. Nevertheless, farmers and dairy processors occasionally complain about ceftiofur-based products in case of positive result to a commercial rapid screening test for the presence of violative residues of antimicrobials (inhibitors) in the bulk milk tank. Six lactating cows were injected with a 50 mg/ml ceftiofur HCl-based product at the dosage regimen of 1 mg/kg, intramuscularly, once a day, for five consecutive days, as per label. Milk samples were then collected just before the very last injection (T0) and then at 12, 24, 36, 48, 60, 72, 84 and 96 hours after the last injection. Individual milk samples were tested using three commercial screening test kits for inhibitor residues: DelvotestSP NT, SNAP Beta-Lactam ST Plus and ROSA MRL Beta-Lactam Test. Since bulk tank is screened in real operating conditions, samples were also diluted to 1:4, 1:10 and tested again. For the Delvotest SP NT, which lowest detected concentration is close the MRL of the ceftiofur (100 µg/kg), all results were negative. For the ROSA MRL Beta-Lactam Test and the SNAP Beta-Lactam ST Plus, several samples yielded positive and doubtful results at T0 and T12. However, after dilution to 1:10, all results were negative. Consequently, when used as officially instructed, the tested 50 mg/ml ceftiofur HCl-based injectable veterinary products are safe, and milk should be free of violative residues of ceftiofur. With consideration to the low specificity and the low positive predictive value of commercial screening tests, positive reactions of the bulk milk should be interpreted as false positive or another risky usage of ß-lactam-based medicines in the farm must be investigated.

A New Phe-Free Protein Substitute Engineered to Allow a Physiological Absorption of Free Amino Acids for Phenylketonuria

Abstract An innovative technology (Physiomimic Technology) has been applied to amino acids (AAs) formulated for patients with phenylketonuria, with the objective of masking AA taste and odor and prolonging AA release in the gut, allowing a physiological absorption. This technology entails that the AAs are processed with functional additives that are able to modify their release and their organoleptic features. Two prototypes, obtained using sodium alginate + ethylcellulose (engP-1) or sodium alginate + ethylcellulose + glyceryl dibehenate (engP-2), have been tested for AA prolonged release versus the same AAs (n-engP) without the application of the Physiomimic Technology. In vitro tests indicated that the technology is able to prolong the release of the engineered AAs versus the free compounds. A crossover in vivo kinetic study in pigs showed reduced peak concentrations (Cmax) and, as expected, similar areas under the concentration/time curve (up to 5 hours) for the engineered products versus the free AAs. Significantly lower Cmax values (P < .01) were attained for essential AAs, large neutral AAs, and branched-chain AAs, indicating that the technology is able to reduce the typical absorption peak of free AAs. Taste and odor masking has been obtained as a consequence of the AA coating. The Physiomimic Technology, applied to free AAs, provided AA mixes with improved organoleptic features and with modified AA kinetics sustaining a more physiological AA absorption.

Accumulation and response of willow plants exposed to environmental relevant sulfonamide concentrations.

As a result of manure application to arable lands, agricultural ecosystems are often contaminated by veterinary antibiotics. In this study the aptitude of Salix fragilis L. to accumulate and tolerate sulfadimethoxine (SDM) was evaluated, together with the antibiotic effects on the plant development, with particular attention focused on roots. Results showed an antibiotic presence in root tissues, but not in leaves, after one month of SDM exposure to 0.01, 0.1, 1 and 10 mg l(-1). A hormetic growth of the hypogeal system was observed, however stress symptoms on the root development were only noticed after treatment to the highest dose. Results obtained from a second test, where new cuttings were exposed to 10 mg SDM l(-1) for different periods, suggested that willow tolerance to SDM increased with the exposure duration, probably because of the onset of particular acclimation mechanisms. Therefore, the present work indicates that this woody species could be utilized in the phytoremediation of sulfonamide antibiotics at doses comparable to that found in agricultural ecosystems once obtained appropriate confirmations through future studies at a laboratory and field scale.

A monitoring of chemical contaminants in waters used for field irrigation and livestock watering in the Veneto region (Italy), using bioassays as a screening tool.

In this study, 50 livestock watering sources (ground water) and 50 field irrigation sources (surface water) from various industrialised areas of the Veneto region were monitored for chemical contaminants. From each site, four water samples (one in each season) were collected during the period from summer 2009 through to spring 2010. Surface water samples and ground water samples were first screened for toxicity using the growth inhibition test on Pseudokirchneriella subcapitata and the immobilisation test on Daphnia magna, respectively. Then, based on the results of these toxicity tests, 28 ground water samples and 26 surface water samples were submitted to chemical analysis for various contaminants (insecticides/acaricides, fungicides, herbicides, metals and anions) by means of UPLC-MS(n) HPLC-MS(n), AAS and IEC. With the exception of one surface water sample where the total pesticides concentration was greater than 4 µg L(-1), positive samples (51.9 %) showed only traces (nanograms per liter) of pesticides. Metals were generally under the detection limit. High concentrations of chlorines (up to 692 mg L(-1)) were found in some ground water samples while some surface water samples showed an excess of nitrites (up to 336 mg L(-1)). Detected levels of contamination were generally too low to justify the toxicity recorded in bioassays, especially in the case of surface water samples, and analytical results painted quite a reassuring picture, while tests on P. subcapitata showed a strong growth inhibition activity. It was concluded that, from an ecotoxicological point of view, surface waters used for field irrigation in the Veneto region cannot be considered safe.

Transcriptomic markers meet the real world: finding diagnostic signatures of corticosteroid treatment in commercial beef samples.

BACKGROUND: The use of growth-promoters in beef cattle, despite the EU ban, remains a frequent practice. The use of transcriptomic markers has already proposed to identify indirect evidence of anabolic hormone treatment. So far, such approach has been tested in experimentally treated animals. Here, for the first time commercial samples were analyzed. RESULTS: Quantitative determination of Dexamethasone (DEX) residues in the urine collected at the slaughterhouse was performed by Liquid Chromatography-Mass Spectrometry (LC-MS). DNA-microarray technology was used to obtain transcriptomic profiles of skeletal muscle in commercial samples and negative controls. LC-MS confirmed the presence of low level of DEX residues in the urine of the commercial samples suspect for histological classification. Principal Component Analysis (PCA) on microarray data identified two clusters of samples. One cluster included negative controls and a subset of commercial samples, while a second cluster included part of the specimens collected at the slaughterhouse together with positives for corticosteroid treatment based on thymus histology and LC-MS. Functional analysis of the differentially expressed genes (3961) between the two groups provided further evidence that animals clustering with positive samples might have been treated with corticosteroids. These suspect samples could be reliably classified with a specific classification tool (Prediction Analysis of Microarray) using just two genes. CONCLUSIONS: Despite broad variation observed in gene expression profiles, the present study showed that DNA-microarrays can be used to find transcriptomic signatures of putative anabolic treatments and that gene expression markers could represent a useful screening tool.

Primary hepatocytes as an useful bioassay to characterize metabolism and bioactivity of illicit steroids in cattle.

Cattle hepatocytes have already been used in veterinary in vitro toxicology, but their usefulness as a multi-parametric screening bioassay has never been investigated so far. In this study, cattle hepatocytes were incubated with illicit steroids/prohormones (boldenone, BOLD; its precursor boldione, ADD; dehydroepiandrosterone, DHEA; an association of ADD:BOLD), to characterize their transcriptional effects on drug metabolizing enzymes (DMEs) and related nuclear receptors (NRs), on cytochrome P450 3A (CYP3A) apoprotein and catalytic activity as well as to determine ADD and BOLD metabolite profiling. DHEA-exposed cells showed an up-regulation (higher than 2.5-fold changes) of three out of six NRs, CYP2B22 and CYP2C87; likewise, ADD:BOLD increased CYP4A11 mRNA levels. In contrast, a reduction of CYP1A1 and CYP2E1 mRNAs (lower than 2.5(-1)-fold changes) was noticed in ADD- and DHEA-incubated cells. No effect was noticed on CYP3A gene and protein expression, though an inhibition of 6ß-, 2ß- and 16ß-hydroxylation of testosterone (higher than 60% of control cells) was observed in ADD- and BOLD-exposed cells. Finally, 17α-BOLD was the main metabolite extracted from hepatocyte media incubated with ADD and BOLD, but several mono-hydroxylated BOLD and ADD derivatives were detected, too. Collectively, cattle hepatocytes can represent a complementary screening bioassay, useful to characterize growth promoters metabolite profiling and their effects upon DMEs expression, regulation and function.

Evaluation of the aquatic toxicity of two veterinary sulfonamides using five test organisms.

The aquatic toxicity of sulfaquinoxaline (SQO) and sulfaguanidine (SGD) was evaluated on the following test organisms: Daphnia magna (reproduction test), Pseudokirchneriella subcapitata, Scenedesmus dimorphus, Synecococcus leopoliensis (algal growth inhibition test) and Lemna gibba (duckweed growth inhibition test). Furthermore, the additivity of the two compounds was measured on D. magna (acute immobilisation test) and P. subcapitata (algal growth inhibition test) using the isobologram method. Results show that SQO and SGD are more toxic to green algae and daphnids, respectively, than other veterinary sulfonamides (SAs) and that their mixtures have a less then additive interaction. Taking into account the highest concentrations detected so far in surface waters for SQO (0.112 µg L(-1)) and for SGD (0.145 µg L(-1)) and the lowest NOECs obtained with the five test organisms, divided by an assessment factor of 10, the following PNECs and risk quotients (RQs) were calculated. SQO: PNEC 2 µg L(-1); RQ 0.056. SGD: PNEC 39.5 µg L(-1); RQ 0.004. Consequently, at the concentrations actually detected in the aquatic environment, the two SAs alone should not harm the freshwater organisms. However, it seems advisable, for veterinary mass treatments, the use of other SAs that have a lesser impact on the aquatic environment. Furthermore, considering the high probability of having complex mixtures of different SAs residues in water, each individual contamination should be evaluated by applying to the SAs mixtures the conservative criteria of additivity.

Effects of illicit dexamethasone upon hepatic drug metabolizing enzymes and related transcription factors mRNAs and their potential use as biomarkers in cattle.

In cattle fattening, the illicit use of growth promoters (GPs) represents a major problem. The synthetic corticosteroid dexamethasone (DEX) is the GP mostly used, alone or in combination with other steroids or beta-agonists. Recently, GPs were shown to disrupt some cattle cytochromes P450 (CYPs) at the post-transcriptional level; therefore, the effects of two illicit protocols containing DEX (alone or together with 17beta-estradiol, 17betaE) upon main cattle liver drug metabolizing enzymes (DMEs) mRNAs and related transcription factors were investigated by quantitative real time RT-PCR. Eleven genes, out of the 18 considered, were significantly modulated by GPs. Corticosteroid-responsive genes did not respond univocally, whereas retinoic X receptor alpha (RXRalpha) and estrogen receptor alpha (ERalpha) were upregulated depending on the illicit protocol used. Nowadays, an increasing interest has been noticed toward the detection of biomarkers of response (BMRs) to be used in the screening of GPs misuse in cattle farming. In the present study, CYP2B6-like, CYP2E1, glutathione S-transferase A1- and sulfotransferase A1-like (GSTA1- and SULT1A1-like) mRNAs were significantly modulated regardless of the GP, the illicit protocol, and the animal breed, representing promising BMRs. The usefulness of these BMRs needs to be characterized more in depth.

The toxicity of sulfamethazine to Daphnia magna and its additivity to other veterinary sulfonamides and trimethoprim.

Sulfonamides (SAs), the oldest chemotherapeutic agents used for antimicrobial therapy, still play an important role in veterinary mass treatments. Consequently, traces of these compounds, alone or in combinations, have been repeatedly detected in the environment. Sulfamethazine (SMZ) deserves particular attention not only because it is the most used veterinary SA, but also due to its proven effects on fertility in mice and on thyroid hormone homeostasis in rats. In this study, after evaluating the acute toxicity to Daphnia magna of six veterinary SAs and trimethoprim (TMP), the additivity of SMZ to each other compound was tested using the isobologram method. Two reproduction tests on the same biological model were also performed in order to derive LOEC and NOEC of SMZ. The acute EC(50) was in the range 131-270 mgL(-1) for all the compounds tested with the exception of sulfaguanidine (EC(50)=3.86 mgL(-1)). In acute binary tests SMZ showed a complex interaction with sulfaquinoxaline (superadditivity, additivity or subadditivity) at the three different combination ratios tested, simple additivity to TMP and less than additive interaction when paired to the other SAs. LOEC and NOEC of SMZ obtained from reproduction tests were 3.125 and 1.563 mgL(-1), respectively. In conclusion, SMZ should not harm the crustacean population at environmentally realistic concentrations. Its toxicity is comparable to that of other systemic SAs, and their binary interactions are less than additive. The same can not be entirely said for enteric SAs, and considering that these compounds are administered at high doses and mostly excreted in unmetabolised form, further evaluation of their impact to the aquatic environment seems advisable.