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].

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.

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.

Effects of competition on acute phase proteins and lymphocyte subpopulations - oxidative stress markers in eventing horses.

The aim of the study was to evaluate markers of the acute phase response (APR) in eventing horses by measuring acute phase proteins (APP) (haptoglobin, Hp, and serum amyloid A, SAA), lysozyme, protein adducts such as pentosidine-like adducts (PENT), malondialdehyde adducts (MDA), hydroxynonenal adducts (HNE) and total advanced glycation/glycoxidation end products (AGEs), complete blood count and lymphocyte subpopulations (CD4+, CD8+ and CD21+) both at rest and at the end of an eventing competition. Blood samples were collected from eight Warmblood horses (medium age 10 ± 3) during an official national 2-day event competition at rest (R) and 10 min after the arrival of the cross-country test on the second day. Exercise caused a significant increase in red blood cell number, haemoglobin, packed cell volume, neutrophils, white blood cell and lymphocyte number; however, these values remained within the normal range. The CD4+ and CD8+ cells significantly increased, whereas the CD21+ lymphocytes decreased; a significant increase in serum SAA, lysozyme and protein carbonyl derivates was also observed. Two-day event causes significant changes in APR markers such as lysozyme, protein carbonyl derivates (HNE, AGEs, PENT) and lymphocyte subpopulations. The data support the hypothesis that 2-day event may alter significantly APR markers. Limitations of the study were the relatively small sample size and sampling time conditioned by the official regulations of the event. Therefore, further studies are needed to investigate the time required for recovery to basal values in order to define the possible effects on the immune function of the athlete horse.

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.

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.

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.

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).

Antimicrobial susceptibility and mechanism of resistance to fluoroquinolones in Staphylococcus intermedius and Staphylococcus schleiferi.

The objective of the present study was to determine the antimicrobial susceptibility of 136 canine isolates of Staphylococcus intermedius and 10 canine isolates of S. schleiferi subspecies coagulans to 16 fluoroquinolones (FQs), and to investigate the mechanisms of resistance in the nonsusceptible isolates. Of the 136 of S. intermedius tested 98.5% were susceptible to all 16 FQs whereas only 40% of the 10 isolates of S. schleiferi subspecies coagulans were susceptible. Two isolates of S. intermedius and six isolates of S. schleiferi, were found to be resistant to 13 out of 16 FQs, while they retained their susceptibility to fourth generation FQs such as gatifloxacin, moxifloxacin and trovafloxacin. Sequencing of the quinolone-resistance determining regions of gyrA and grlA genes showed that in S. intermedius, dichotomous resistance to FQs was associated with the occurrence of one alteration in GyrA-84 and one in GrlA-80, while in S. schleiferi the same pattern of resistance was observed in isolates showing these changes only in gyrA. This study is the first to screen FQs of the second, third and fourth generation for antimicrobial resistance in clinical isolates of S. intermedius and S. schleiferi of canine origin, and to describe mutations in gyrA and grlA associated with FQ resistance in these bacterial species.

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.

Pharmacokinetics of clomipramine and desmethylclomipramine after single-dose intravenous and oral administrations in cats.

A cross-over study was performed in six adult spayed cats to determine the pharmacokinetics of clomipramine and its metabolite, desmethylclomipramine (DCMP) after intravenous (0.25 mg/kg) and oral (0.5 mg/kg) single-dose administrations. Plasma clomipramine and DCMP were measured by high-performance liquid chromatography at regular intervals for up to 30 h. Intravenous clomipramine best fit a two-compartmental model yielding an elimination rate constant of 0.037-0.09 h(-1) from which a mean half-life of 12.3 h was calculated. Mean clomipramine AUC(0--infinity) (ngxh/mL), clearance (L/hxkg), V(ss) (L/kg) and MRT (h) values were 652.5, 0.393, 5.0, and 13.5, respectively. Compartmental modeling for clomipramine, after oral administration, and DCMP after both administrations, produced wide parameter estimates and plots of residuals indicated poor goodness of fit. Noncompartmental analysis yielded mean AUC(0--30 h) (ngxh/mL), C(max) (ng/mL) and T(max) (h) of 948.3, 87.5 and 6.2 for clomipramine, and 613.8, 34.8, and 12.8 for DCMP respectively after oral administration. Clomipramine bioavailability was 90%. The present study showed marked pharmacokinetic variability for clomipramine and DCMP through biphasic absorption and potential genetic variability in clomipramine metabolism. It was concluded that population pharmacokinetics would allow better characterization of clomipramine variability that may explain the variability in clinical response noted in cats.

Safety of azamethiphos in eel, seabass and trout.

The safety of azamethiphos (AZA), an organophosphorous insecticide and the active ingredient of Salmosan, was evaluated in the European eel, seabass and rainbow trout. Fish were bathed in 0.1 ppm AZA for a period of 60, 120 or 240 min. After termination of each treatment fish were transferred to clean aquaria and randomly sampled over 21 days. Compared to controls, brain acetylcholinesterase (AChE) was inhibited up to 44, 56 and 62% in eels, seabass and trout, respectively, with the inhibition being significant for up to 4 days in eels and seabass and 7 days in trout. As result of the AChE depression, fish displayed motor hyperactivity and erratic jumping at the onset of treatment. Mortality was observed only in trout following exposure for 240 min. A variable correlation observed among species between the level of exposure, the reduced activity of brain AChE and the signs of toxicity suggest that brain AChE should be considered as an indicator of exposure rather than as an index of toxicity of AZA. The present data indicate that at the therapeutic dosage of 0.1 ppm AZA for 1h can be safely used in eels, seabass and trout. The extended treatment times up to 240 min were equally safe for eels and seabass but not for trout.

A comparative kinetic study of thiamphenicol in pre-ruminant lambs and calves.

Eight healthy Holstein-Friesian calves and 8 Massese lambs of either sex (10-15-days old) were used to evaluate the pharmacokinetics of thiamphenicol after intravenous (i.v.) and oral (p.o.) administration (30 mg/kg). Plasma concentrations of thiamphenicol were determined by high-performance liquid chromatography on blood samples collected over 24h following treatment. Pharmacokinetic variables of the drug were calculated for both species and after both administration routes. After intravenous administration of thiamphenicol, a rapid distribution phase was followed by a slower elimination phase and, when thiamphenicol was administered p.o., the bioavailability was about 60% in both species. The higher volume of distribution and the longer biological elimination half-lives in pre-ruminant compared with adult animals indicate that thiamphenicol distributes widely into the extravascular compartment of pre-ruminants. Interspecies differences were observed in the kinetic behaviour of thiamphenicol with respect to peak plasma concentration (C(max)), time of peak plasma concentration (T(max)), elimination half-life (T(1/2)) and total clearance (Cl(B)). In conclusion intravenous or oral administration of 30 mg/kg of thiamphenicol provides plasma concentrations higher than minimum effective concentrations inhibiting bacterial growth (MICs) against most pathogens in pre-ruminant lambs and calves.