Oxytetracycline and Florfenicol Concentrations in Food-Additive Premixes Authorised for Broiler Chickens: Assessing Degree of Agreement with Manufacturers Labelling.

Antimicrobials premixes are the presentation of choice to administer drugs simultaneously to groups of animals in intensive husbandry systems that require treatment for pathologies of bacterial origin. Among the premixes available for use in poultry, florfenicol and oxytetracycline are commonly administered via food or water. However, their actual concentration in premixes must meet on-label statements to ensure plasma concentrations reach effective therapeutic levels. Hence, this work was designed for the purpose of verifying whether the concentration of antimicrobial present in five premixes matched their on-label statement. Three oxytetracycline premixes, and two of florfenicol, were analysed using a Xevo TQ-S micro UPLC-MS/MS, and an ABSciex API4000 HPLC-MS/MS, respectively. Analytical methodologies were implemented and validated, showing an R2 ≥ 0.99 for the calibration curves. Oxytetracycline was detected in these premixes at concentrations exceeding on-label statements by 13.28%, 21.54%, and 29.68%, whereas florfenicol concentrations detected in premixes were 13.06% and 14.75% lower than expected. Consequently, this work shows that the concentration of active ingredients that are present in commercial formulations effectively differ from those stated on premix labels, and it also highlights how unpredictable their range of variability might be. This must be addressed through solid and updated laws that guarantee an effective pharmaceutical product.

Determination of five antimicrobial families in droppings of therapeutically treated broiler chicken by high-performance liquid chromatography-tandem mass spectrometry.

Antimicrobials are currently used in poultry for disease treatment. However, their excretion in bird feces may contaminate the environment. Considering this, the objective of this work was to quantify antimicrobials residues concentrations in therapeutically treated broiler chicken droppings throughout the post-treatment period. For this aim a multiresidue method using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was validated. Forty-eight male broiler chickens were distributed and treated with commercial formulations of 5 different antimicrobials. Results showed that oxytetracycline and 4-epi-oxytetracycline, presented the highest concentrations during all sampling period, detecting concentrations of 1471.41 µg kg-1 at the last sampling point (day 22 post-treatment). Florfenicol, tylosin, enrofloxacin, and ciprofloxacin were eliminated and detected in treated chicken droppings until d 18 post-treatment. Sulfachloropyridazine decrease gradually during post-treatment period until day 30. Results demonstrate that studied antimicrobials in treated chicken droppings were eliminated for prolonged periods, therefore becoming a significant route of residues dissemination into the environment.

Detection of Antimicrobial Residues in Poultry Litter: Monitoring a Risk through a Selective and Sensitive HPLC-MS/MS Method.

Tetracyclines, sulphonamides, and quinolones are families of antimicrobials (AMs) widely used in the poultry industry and can excrete up to 90% of AMs administrated, which accumulate in poultry litter. Worryingly, poultry litter is widely used as an agriculture fertilizer, contributing to the spread AMs residues in the environment. The aim of this research was to develop a method that could simultaneously identify and quantify three AMs families in poultry litter by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Samples of AMs free poultry litter were used to validate the method according to 657/2002/EC and VICH GL49. Results indicate that limit of detection (LOD) ranged from 8.95 to 20.86 µg kg-1, while limits of quantitation (LOQ) values were between 26.85 and 62.58 µg kg-1 of tetracycline, 4-epi-tetracycline, oxytetracycline, 4-epi-oxytetracycline, enrofloxacin, ciprofloxacin, flumequine, sulfachloropyridazine, and sulfadiazine. Recoveries obtained ranged from 93 to 108%. The analysis of field samples obtained from seven commercial poultry flocks confirmed the adequacy of the method since it detected means concentrations ranging from 20 to 10,364 µg kg-1. This provides us an accurate and reliable tool to monitor AMs residues in poultry litter and control its use as agricultural fertilizer.

Evaluation of Antibiotic Dissemination into the Environment and Untreated Animals, by Analysis of Oxytetracycline in Poultry Droppings and Litter.

Oxytetracycline (OTC) is widely used in broiler chickens. During and after treatment a fraction of OTC is excreted in its original form and as its epimer, 4-epi-OTC in droppings. To address the transfer of OTC into the environment, we evaluated the dissemination of OTC and 4-epi-OTC from treated birds to the environment and sentinels, through the simultaneous analysis of broiler droppings and litter. Male broiler chickens were bred in controlled conditions. One group was treated by orogastric tube with 80 mg kg-1 of OTC and two groups received no treatment (sentinels). OTC+4-epi-OTC were analyzed and detected by a HPLC-MS/MS post the end of treatment. The highest concentrations of OTC+4-epi-OTC were detected in the droppings of treated birds 14-days following the end of treatment (2244.66 µg kg-1), and one day following the end of treatment in the litter (22,741.68 µg kg-1). Traces of OTC+4-epi-OTC were detected in the sentinels' droppings and litter (<12.2 µg kg-1). OTC+4-epi-OTC can be transferred from treated birds to the environment and to other untreated birds. The presence and persistence of OTC+4-epi-OTC in litter could contribute to the selection of resistant bacteria in the environment, increasing the potential hazard to public and animal health.

Assessment of Consumer Perceptions on the Use of Antimicrobials in Production Animals in Chile.

Successful treatment of diseases that sicken food-producing animals requires the use of antimicrobials. However, these drugs could result in serious consequences for human, animal, and environmental health. The emergence of antimicrobial resistance requires better communication with consumers to inform them about the risks associated with the use of these drugs, as well as the benefits gained from their rational use. This work studied consumer perceptions about the use of antimicrobials in the production of animal origin foods by questioning 72 consumers in nine focus groups. The participants were all inhabitants of the Metropolitan Region, Chile, and were assigned to focus groups of 5 to 12 individuals each, according to their age: age groups I, II, and III spanned age ranges of 25 to 35, 36 to 50, and older than 50 years, respectively. Consumers regarded the use of antimicrobials in farm animals as a relevant and necessary practice, as long as it was done adequately by veterinarians to ensure productivity and to guarantee food quality and safety. Even though most consumers identified antimicrobial use as a potential risk to their health, only a few recognized that antimicrobial resistance could be transferred from animals to humans via food consumption. Also, consumers complained that they did not have access to enough information about foods of animal origin. Consequently, they stated that food industry and regulatory entities should ensure transparency of production processes and also educate the population on this topic. Consumer perceptions about responsible and judicious use of antimicrobials may become an ally in the effort to reduce and improve the use of these drugs in animal production systems.

Determination of Chlortetracycline Residues, Antimicrobial Activity and Presence of Resistance Genes in Droppings of Experimentally Treated Broiler Chickens.

Tetracyclines are important antimicrobial drugs for poultry farming that are actively excreted via feces and urine. Droppings are one of the main components in broiler bedding, which is commonly used as an organic fertilizer. Therefore, bedding becomes an unintended carrier of antimicrobial residues into the environment and may pose a highly significant threat to public health. For this depletion study, 60 broiler chickens were treated with 20% chlortetracycline (CTC) under therapeutic conditions. Concentrations of CTC and 4-epi-CTC were then determined in their droppings. Additionally, this work also aimed to detect the antimicrobial activity of these droppings and the phenotypic susceptibility to tetracycline in E. coli isolates, as well as the presence of tet(A), tet(B), and tet(G) resistance genes. CTC and 4-epi-CTC concentrations that were found ranged from 179.5 to 665.8 µg/kg. Based on these data, the depletion time for chicken droppings was calculated and set at 69 days. All samples presented antimicrobial activity, and a resistance to tetracyclines was found in bacterial strains that were isolated from these samples. Resistance genes tet(A) and tet(B) were also found in these samples.

Determination of Oxytetracycline and 4-Epi-Oxytetracycline Residues in Feathers and Edible Tissues of Broiler Chickens Using Liquid Chromatography Coupled with Tandem Mass Spectrometry.

Antibiotics have been widely used in poultry production for the treatment of bacterial diseases. However, drug residues can remain in products derived from animals after the cessation of the drug therapies. Feathers, in particular, have shown an affinity for antibiotics such as tetracycline, suggesting the persistence of these drugs in nonedible tissue. After the birds are slaughtered, feathers are ground into feather meals, which are used as organic fertilizer or an ingredient in animal diets, thereby entering into the food chain and becoming a potential risk for public health. To evaluate the depletion of oxytetracycline (OTC) and its metabolite 4-epi-oxytetracycline (4-epi-OTC) in the muscles, liver, and feathers, 64 broiler chickens, bred under controlled conditions, were treated orally with a commercial formulation of 10% OTC for 7 days. The analytes were quantified using liquid chromatography-tandem mass spectrometry. OTC and 4-epi-OTC were found in the feathers for 46 days, whereas they were found in the muscle and liver for only 12 and 6 days, respectively. These results prove that the analytes remain in feathers in higher concentrations than they do in edible tissues after treatment with tetracyclines. Thus, feather meals represent a potential source of antimicrobial residue contamination in the food chain.

Increased urinary glucocorticoid metabolites are associated with metabolic syndrome, hypoadiponectinemia, insulin resistance and ß cell dysfunction.

Metabolic syndrome (MetS) may have increased cortisol (F) production caused by 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) in liver and adipose tissue and/or by HPA axis dysregulation. F is then mainly metabolized by liver reductases into inactive tetrahydrometabolites (THMs). We measured THM levels in patients with or without MetS and evaluate the correlation between THMs and anthropometric and biochemical parameters. We recruited 221 subjects, of whom 130 had MetS by ATP III. We evaluated F, cortisone (E), adipokines, glucose, insulin and lipid profiles as well as urinary (24h) F, E and THM levels. ß Cell function was estimated by the HOMA Calculator. We observed that patients with MetS showed higher levels of THMs, HOMA-IR and leptin and lower levels of adiponectin and HOMA-ß but no differences in F and E in plasma or urine. THM was associated with weight (r = +0.44, p<0.001), waist circumference (r = +0.38, p<0.01), glycemia (r = +0.37, p<0.01), and triglycerides (r = +0.18, p=0.06) and negatively correlated with adiponectin (r = -0.36, p<0.001), HOMA-ß (r = -0.21, p<0.001) and HDL (r = -0.29, p<0.01). In a logistic regression model, THM levels were associated with hypertension, hyperglycemia and dyslipidemia. We conclude that MetS is associated with increased urinary THMs but not with F and E levels in plasma or urine. Increased levels of THM, reflecting the daily cortisol production subsequently metabolized, are correlated with hypoadiponectinemia, hypertension, dyslipidemia, insulin resistance and ß cell dysfunction. A subtle increased in glucocorticoid production may further account for the phenotypic and biochemical similarities observed in central obesity and Cushing's syndrome.