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.

Enrofloxacin against Escherichia coli in turkeys: which treatment scheme is effective?

The efficacy of enrofloxacin (ENRO) was evaluated against multidrug-resistant avian pathogenic Escherichia coli correlating the minimum inhibitory concentrations (MIC) of 235 E. coli field strains with its pharmacokinetics (PK) in 50 healthy turkeys (5 groups) with a PK/pharmacodynamic approach. The treatments were as follows: a) single oral gavage and b) single subcutaneous (SC) treatment at the recommended dose of 10 mg/kg; c) single oral gavage, d) 5 d of 10-h pulsed water medication, and e) 5 d of 24-h continuous water medication at the doubled dose of 20 mg/kg. Blood samples were collected at established times over 24 h. Plasma was analyzed using a liquid chromatography tandem mass spectrometry method that was validated in house. A monocompartmental and a noncompartmental model were applied to the data to obtain the PK results. After gavage administration, the mean maximum concentration Cmax/MIC50 and area under the curve AUC0-24/MIC50 ratios were, respectively, 3.07 ± 0.62 and 7.01 ± 1.03 and 25.48 ± 3.04 and 57.2 ± 3.73 for the 10 and 20 mg/kg doses, respectively. After SC administration of 10 mg/kg, Cmax/MIC50 and AUC0-24/MIC50 ratios were 3.45 ± 0.75 and 33.96 ± 7.46, respectively. After the administration of 10-h pulsed or 24-h continuous medicated water at 20 mg/kg, lower values of Cmax/MIC50 (10-h pulsed: 3.45 ± 0.7; 24-h continuous: 3.05 ± 0.48) and AUC0-24/MIC50 (10-h pulsed: 42.42 ± 6.17; 24-h continuous: 53.32 ± 5.55) were obtained. Based on these results, the European Union-recommended dosage of 10 mg/kg seems ineffective to achieve adequate drug plasma concentrations and even the 20 mg/kg by 10 h pulsed or continuous medicated water administration did not reach completely efficacious concentrations in plasma against colibacillosis. Although the results obtained were not completely encouraging, the medicated water should preferably be provided continuously. To conclude about the efficacy of ENRO treatment against colibacillosis, target tissue concentration should be extensively considered.

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.

Pharmacokinetic/pharmacodynamic evaluation of the efficacy of flumequine in treating colibacillosis in turkeys.

Flumequine (FLU) is used in the treatment of systemic bacterial infections in poultry, including colibacillosis, which is a common disease in turkeys. The pharmacokinetic (PK) behavior of FLU administered to 32 healthy turkeys as an oral bolus via gavage or as 10-h pulsed administration in drinking water were compared, using the authorized dose of 15 mg/kg and the double dose of 30 mg/kg. The minimum inhibitory concentrations (MIC) of 235 Escherichia coli field strains isolated from poultry were determined for pharmacodynamics (PD) to develop a PK/PD model. Blood samples were collected at established times over 24 h, and the obtained plasma was analyzed using a liquid chromatography tandem mass spectrometry method that was validated in-house. A monocompartmental model and a noncompartmental model were applied to the data to obtain the PK results. For both types of administration and both dosages, the ratios of the maximum concentration (Cmax)/MIC50 and the area under the plasma concentration-time curve (AUC)/MIC50 achieved were considerably lower than the fluoroquinolone breakpoints usually adopted for efficacy. The Cmax/MIC50 and AUC0-24/MIC50 ratios were, respectively, 0.67 ± 0.09 and 4.76 ± 0.48 and 1.18 ± 0.35 and 7.05 ± 2.40 for the 15 and 30 mg/kg bolus doses, respectively. After 10-h pulsed administration of 15 mg/kg, values of Cmax/MIC50, 0.19 ± 0.02 on d 1 and 0.30 ± 0.08 on d 5 of therapy were obtained, the AUC/MIC50 ratios were 2.09 ± 0.29 and 3.22 ± 0.93 on d 1 and 5, respectively. Higher values were obtained with the doubled dose of 30 mg/kg: the Cmax/MIC50 ratios were 0.49 ± 0.11 on d 1 and 0.69 ± 0.18 on d 5; the AUC/MIC50 ratios were 5.15 ± 1.15 and 6.57 ± 1.92 on d 1 and 5, respectively. Based on these results, FLU administration should be adopted when specific diagnostic findings indicate its efficacy, and revising the dosage scheme to comply with the prudent and responsible use of antimicrobials in veterinary medicine is advisable.

Will extreme weather events influence the toxic impacts of caffeine in coastal systems? Comparison between two widely used bioindicator species.

In the recent years, marine heatwaves (MHWs) have caused devastating impacts on marine life. The understanding of the combined effects of these extreme events and anthropogenic pollution is a vital challenge. In particular, the combined effect of MHWs on the toxicity of pharmaceuticals to aquatic life remains unclear. To contribute to these issues, the main goal of the present investigation was to evaluate how MHWs may increase caffeine (CAF) toxicity on the clam Ruditapes philippinarum and the mussel Mytilus galloprovincialis. Bioaccumulation levels and changes on oxidative stress, metabolic capacity and neurotoxic status related biomarkers were investigated. The obtained results revealed the absence of CAF accumulation in both species. However, the used contaminant generated in both bivalve species alteration on neurotransmission, detoxification mechanisms induction as well as cellular damage. The increase of antioxidant defence mechanisms was complemented by an increase of metabolic activity and decrease of energy reserves. The obtained results seemed magnified under a simulated MHWs, suggesting to a climate-induced toxicant sensitivities' response. On this perspective, understanding of how toxicological mechanisms interact with climate-induced stressors will provide a solid platform to improve effect assessments for both humans and wildlife.

Detection of canine and equine procalcitonin for sepsis diagnosis in veterinary clinic by the development of novel MIP-based SPR biosensors.

Procalcitonin (PCT) has emerged as a promising biomarker for the rapid identification of sepsis both in human and veterinary medicine. Nevertheless, the only analytical method currently available for the detection of PCT in veterinary species, is represented by immunoassays, useful only for research purposes. In this work, we report the development of two biosensors which utilize molecularly imprinted polymers (MIPs) for the detection of canine and equine PCT. Dopamine (DA) and norepinephrine (NE) were used as monomers for the synthesis of the MIP films on surface plasmon resonance (SPR) gold chips and the imprinting efficiency of canine and equine PCT in terms of binding affinity toward the analyte, selectivity, and sensitivity were compared. After optimization in buffer conditions, PCTs calibration was successfully achieved also in animal plasma, with good specificity and reproducibility. More effective protein binding and imprinting was obtained with polynorepinephrine (PNE) for both PCTs, and the SPR biosensors were able to detect the biomarkers in plasma with a LOD of 15 ng mL-1 and 30 ng mL-1 respectively for equine and canine PCT.

Correction to: Plasma alpha-tocopherol determined by HPLC in dogs at different stages of chronic kidney disease: a retrospective study.

The above article originally published with an error present in the article title, "Plasma alpha-tochopherol determined by HPLC in dogs at different stages of chronic kidney disease: a retrospective study" this should instead have read, "Plasma alpha-tocopherol determined by HPLC in dogs at different stages of chronic kidney disease: a retrospective study" [bold text used to highlight problem area].

Clinical utility of urine kidney injury molecule-1 (KIM-1) and gamma-glutamyl transferase (GGT) in the diagnosis of canine acute kidney injury.

The aim of the present study was to evaluate the sensitivity and specificity of urine KIM-1 and urine GGT for the detection of naturally-occurring AKI, compared to healthy control dogs, dogs with stable chronic kidney disease (CKD), and dogs with lower urinary tract disorders (LUTD). The study included AKI grade 1 (n = 21), AKI grade 2 to 5 (n = 11), stable CKD (n = 11), LUTD (n = 15), and healthy dogs (n = 37). Urine KIM-1 (ng/mg) and GGT (U/l) were normalized to urine creatinine (uCr). Statistically significant difference in KIM/uCr (p = 0.0007) and GGT/uCr (p < 0.0001) was found among the study groups. Area under the curve (AUC) for KIM-1/uCr and GGT/uCr as predictors of AKI was 0.81 and 0.91 respectively. Values of KIM-1/uCr of 0.73 ng/mg and of GGT/uCr of 54.33 showed the best combination of sensitivity and specificity (75% and 75.6%; 85.7% and 89.1% respectively). A significant positive correlation (p < 0.0001) between KIM-1/uCr and GGT/uCr was found. Both urine KIM-1/uCr and GGT/uCr seemed to be potentially good markers for the diagnosis of AKI. Dogs with AKI showed significantly higher levels of urine KIM-1/uCr and urine GGT/uCr, compared with healthy dogs. Caution should be used in the evaluation of elevated urine KIM-1/uCr and GGT/uCr in dogs with pre-existing CKD and/or LUTD. Urine KIM-1/uCr and GGT/uCr might have a significant clinical utility, as complementary test, particularly in diagnosis early, non-azotemic stages of AKI.

Plasma procalcitonin concentration in healthy calves and those with septic systemic inflammatory response syndrome.

The diagnosis of sepsis in calves is challenging. Blood culture and clinical signs combined with a complete blood count have been used for the diagnosis of sepsis. Recent literature in humans and animal species has been focused on sepsis-specific biomarkers, such as procalcitonin (PCT), that may more accurately and efficiently diagnose sepsis. The aim of this study was to evaluate plasma PCT concentrations in healthy and septic calves. Twenty healthy control calves and 58 sick calves with septic systemic inflammatory response syndrome (SIRS) based on SIRS score and clinical findings were included. Calves with septic SIRS were further divided in septic SIRS survivors (SSS) and non-survivors (SSNS). Plasma PCT concentrations were measured with a commercial ELISA assay for cattle. A receiver operating characteristic curve was used to determine cut-off values and corresponding sensitivity and specificity for the diagnosis of sepsis. Differences in plasma PCT concentration between groups (control vs. SSS vs. SSNS) were evaluated. Plasma PCT concentrations in healthy calves and those with septic SIRS were 33.3pg/mL (0-44.3pg/mL) and 166.5pg/mL (85.9-233.0pg/mL), respectively (P<0.001). The optimal cut-off value to predict septic SIRS was 67.39pg/mL (81.0% sensitivity, 95.0% specificity). Plasma PCT concentrations were 127.4pg/mL (72.2-216.0pg/mL) and 234.3pg/mL (204.5-309.4pg/mL) in the SSS and SSNS subgroups, respectively. Statistically significant differences were found among groups (control vs. SSS and SSNS, P<0.0001; SSS vs. SSNS, P>0.05). These results confirmed an increase in plasma PCT concentrations in calves with septic SIRS, as previously reported in humans and other species.

Plasma alpha-tochopherol determined by HPLC in dogs at different stages of chronic kidney disease: a retrospective study.

The aim of the present study was to investigate retrospectively the plasma concentration of alpha-tocopherol in dogs with naturally acquired chronic kidney disease (CKD), at different stages of severity. Forty dogs (CKD group) with different stages of CKD (IRIS 1 n=12, IRIS 2 n=8, IRIS 3 n=11, IRIS 4 n=9) and 20 clinically healthy dogs were considered. Plasma alpha-tocopherol was assessed in both groups through high performance liquid chromatography (HPLC). Dogs of CKD group showed significantly lower (p=0.0002) levels of plasma alpha-tocopherol compared with clinically healthy dogs. A significant difference (p<0.04) in the number of patients with plasma alpha-tocopherol > or ≤ 21.5 ppm was found in CKD patients at different stages of severity. No significant correlation between plasma levels of alpha-tocopherol and plasma creatinine was found. In the present study, dogs affected by spontaneous CKD showed significantly lower plasma concentrations of alpha-tocopherol compared with clinically healthy dogs. Plasma alpha-tocopherol deficiency seems to be more severe in IRIS stage 1 and 4, compared with IRIS stage 2 and 3. The finding of marked alpha-tocopherol deficiency in patients in IRIS stage 1 should encourage further studies on the early use of prescription renal diet and antioxidant in this group of patients.

How swimming affects plasma insulin and glucose concentration in Thoroughbreds: A pilot study.

Low intensity exercise increases insulin-stimulated glucose uptake in skeletal muscle and decreases its plasma concentration. In this study, plasma insulin and glucose concentrations were evaluated 5min before and 5, 15, 25, 35, 45 and 60min after an IV bolus of glucose in 12 Thoroughbreds before and after 1 month of submaximal aquatraining exercise, monitored using heart rate and blood lactate. Plasma glucose concentrations were evaluated using a colorimetric enzymatic method, and plasma insulin concentrations with a solid-phase radioimmunoassay method. Pre-training plasma glucose concentrations at 15, 25 and 35min, area under the glucose curve and peak glucose concentration were significantly higher than post-training values (P<0.05). Baseline pre-training plasma insulin concentrations were significantly lower than in the post-training period, and plasma insulin was significantly higher at 45 and 60min in the pre-training period than the post-training period. These results indicate that aquatraining could improve insulin-glucose metabolism in horses.

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.

Cyclosporine monitoring by fluorescence polarization immunoassay.

The monitoring of cyclosporine levels in whole blood and serum has become a routine procedure for the post-transplant management of immunosuppression. We have developed fluorescence polarization immunoassays for cyclosporine in whole blood and in serum using a monoclonal antibody. These assays are fast (20 determinations in less than 22 min), sensitive (25 micrograms/L for whole blood samples and 10 micrograms/L for serum samples), and precise (CV less than 7% in both assays). Cross-reactivities with AM1 (Metabolite 17) and AM4N (Metabolite 21) are less than 8.5 and 2.5%, respectively, for both assays.

A new HPLC method to determine glomerular filtration rate and effective renal plasma flow in conscious dogs by single intravenous administration of iohexol and p-aminohippuric acid.

A high-performance liquid chromatography method to determine iohexol (IOX) and p-aminohippuric acid (PAH) in the plasma of dogs is evaluated according to recovery, reproducibility, and linearity utilizing a gradient pump. The mobile phase consists of 50mM sodium dihydrogen phosphate with 0.5mM tetrabutylammonium chloride, the pH is adjusted to 4.1, methanol is added to the final ratio of 90:10 (v/v), the flow rate is set at 1 mL/min, and separation is achieved with an ODS2 Luna column. The UV detector is set at 254 nm. IOX and PAH are used for evaluation of the effective renal plasma flow (ERPF) and glomerular filtration rate (GFR). The present method tested in three dogs demonstrates the accuracy in the evaluation of ERPF and GFR. Because of its precision and simplicity and low cost, it can be considered a good tool for ERPF and GFR in small animal practice.

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.

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.

Evaluation of methicillin resistance in Staphylococcus intermedius isolated from dogs.

Methicillin and multi-drug resistance were investigated in 136 Staphylococcus intermedius strains of canine origin. The large majority of isolates were found to be mecA-negative by polymerase chain reaction, whereas only four strains were positive for the mecA gene. All mecA-positive strains were confirmed as methicillin-resistant by complementary tests, except for oxacillin disk diffusion, which yielded one false-negative result. A significantly higher resistance to fusidic acid, lincosamides, and cotrimoxazole was observed in methicillin-resistant S. intermedius (MRSI) compared with methicillin-susceptible strains. Although the prevalence of MRSI in dogs appeared to be low, methicillin resistance was confirmed to be associated with multi-drug resistance, suggesting the importance of antimicrobial susceptibility testing of canine S. intermedius strains.