Emerging resistance to florfenicol in Actinobacillus pleuropneumoniae isolates on two Italian pig farms.

Actinobacillus pleuropneumoniae is responsible for porcine pleuropneumonia, a highly contagious lung infection. The control of this respiratory disease remains heavily reliant on antibiotics, with phenicols being one of the primary classes of antibiotics used in pig farming. In the present study, we describe three isolates (B2278, B2176 and B2177) of A. pleuropneumoniae resistant to florfenicol attributed to the presence of the floR gene, which were obtained from two pig farms in Italy. Florfenicol susceptibility tests indicated that B2176 exhibited an intermediate susceptibility profile, while B2177 and B2278 were resistant. All three isolates belonged to serovar 6 and tested positive for the presence of the floR gene. Whole genome sequencing analysis revealed that isolates B2176, B2177 and B2278 harbored genes encoding the toxins ApxII and ApxIII, characteristic of strains with moderate virulence. Moreover, phylogenetic analysis demonstrated that these isolates were closely related, with single nucleotide polymorphisms (SNPs) ranging from 8 to 19. The floR gene was located on a novel 5588 bp plasmid, designated as pAp-floR. BLASTN analysis showed that the pAp-floR plasmid had high nucleotide identity (99 %) and coverage (60 %) with the pMVSCS1 plasmid (5621 bp) from Mannheimia varigena MVSCS1 of porcine origin. Additionally, at least under laboratory conditions, pAp-floR was stably maintained even in the absence of direct selective pressure, suggesting that it does not impose a fitness cost. Our study underscores the necessity of monitoring the spread of florfenicol-resistant A. pleuropneumoniae isolates in the coming years.

Mechanism of sturgeon intestinal inflammation induced by Yersinia ruckeri and the effect of florfenicol intervention.

The mechanism by which Y. ruckeri infection induces enteritis in Chinese sturgeon remains unclear, and the efficacy of drug prevention and control measures is not only poor but also plagued with numerous issues. We conducted transcriptomic and 16 S rRNA sequencing analyses to examine the differences in the intestinal tract of hybrid sturgeon before and after Y. ruckeri infection and florfenicol intervention. Our findings revealed that Y. ruckeri induced the expression of multiple inflammatory factors, including il1ß, il6, and various chemokines, as well as casp3, casp8, and multiple tumor necrosis factor family members, resulting in pathological injury to the body. Additionally, at the phylum level, the relative abundance of Firmicutes and Bacteroidota increased, while the abundance of Plesiomonas and Cetobacterium decreased at the genus level, altering the composition of the intestinal flora. Following florfenicol intervention, the expression of multiple apoptosis and inflammation-related genes was down-regulated, promoting tissue repair. However, the flora became further dysregulated, increasing the risk of infection. In conclusion, our analysis of the transcriptome and intestinal microbial composition demonstrated that Y. ruckeri induces intestinal pathological damage by triggering apoptosis and altering the composition of the intestinal microbiota. Florfenicol intervention can repair pathological damage, but it also exacerbates flora imbalance, leading to a higher risk of infection. These findings help elucidate the molecular mechanism of Y. ruckeri-induced enteritis in sturgeon and evaluate the therapeutic effect of drugs on intestinal inflammation in sturgeon.

Impacts of florfenicol on immunity, antioxidant activity, and histopathology of Oreochromis niloticus: a potential protective effect of dietary spirulina platensis.

The misuse of antibiotics enhances the development of resistant microorganisms and decreases the efficacy of treatments. Florfenicol (FF) is one of the antibiotics approved for use in aquaculture in Egypt. Because of its extensive usage, potential negative impacts on aquatic creatures are a major concern. This motivates us to search for an appropriate neoadjuvant to work synergistically with FF and reduce adverse effects. Results from this study will contribute towards improving the understanding of the impacts of FF on Oreochromis niloticus and the possible amelioratory effects of Spirulina platensis algae (SP). O. niloticus (n = 240; 40 ± 2.5 g) were fed on two diets supplemented with or without SP for 4 weeks, then divided into four treatments each in three replicates (n = 60/treatment). G1; was fed a control diet, and the other groups were fed diets supplemented with FF (10 mg /kg of BW, G2), SP (2 g/kg of diet, G3), or FF + SP (G4) for 10 days. Among the four groups, the SP group (G3) had the best immunostimulatory effects as observed by a significant (p < 0.05) elevation in phagocytic activity, phagocytic index, IL6, and TNF-α. The treatment with FF had significantly impacted hepatic and renal tissues, as the values of liver enzymes and creatinine demonstrated tissue deterioration and also resulted in oxidative stress, which was expressed by an increase of GPx, CAT, and SOD in (G2). Additionally, the combined FF + SP improved the hematological parameters and decreased the oxidative damage induced by FF (G4). Thus, it was clear that FF has harmful effects on O. niloticus and that SP can modulate such impacts. These data recommend the use of SP as an effective immunostimulant and a probable adjuvant to FF in O. niloticus diets to attain maximum disease resistance.

Allicin affects the pharmacokinetics of sulfadiazine and florfenicol by downregulating the expression of jejunum P-gp and BCRP in broilers.

Allicin, one of the main bioactive compounds in garlic, is an excellent feed additive. It is unknown whether allicin affects the expression of P-gp and BCRP, 2 important ABC efflux transporters related to the pharmacokinetics of antimicrobials in chickens. In this study, by bidirectional transport test and broiler jejunum in situ perfusion, we found that allicin inhibited the efflux transport of P-gp and BCRP for their substrates sulfadiazine and florfenicol, 2 commonly used antimicrobials in broilers. Furthermore, we observed that allicin decreased the mRNA expression of chicken jejunum P-gp and BCRP. Pretreatment with allicin changed the pharmacokinetic behavior of orally administered sulfadiazine, by increasing AUC (41.85 vs. 31.24, P < 0.01) and Cmax values (9.82 vs. 8.40, P < 0.05) and decreasing CLZ (0.45 vs. 0.62, P < 0.01). Similarly, pretreatment with allicin also altered pharmacokinetics of orally administered florfenicol, by increasing AUC (18.38 vs. 13.52, P < 0.01) and decreasing CLZ. The results indicate that allicin could inhibit jejunum P-gp and BCRP expression and efflux function, thereby increasing the plasma concentrations of their substrates in broilers.

Effects of early florfenicol exposure on glutathione signaling pathway and PPAR signaling pathway in chick liver.

Florfenicol (FFC) is a common antibiotic for animals. The nonstandard and excessive use of FFC can cause veterinary drug residues in animals, pollute soil and marine environment, and even threaten human health. Therefore, it is necessary to study the toxicity and side effects of FFC on animals. Our previous studies have proved that FFC can cause liver injury in chicks, but there are few in-depth studies on the mechanism of FFC causing liver injury at the level of signaling pathway in chicks. Therefore, transcriptome and proteome sequencing were performed and combined analysis was performed. Sequencing results showed that 1989 genes and 917 proteins were significantly changed in chick livers after FFC exposure. These genes and proteins are related to redox, glutathione transferase activity and lipid metabolism. There are 9 significantly different genes and 7 significantly different proteins in glutathione signaling pathway. Oxidative stress may occur in the liver of chicks through the change of activation state of glutathione signaling pathway. And there are 13 significantly different genes and 18 significantly different proteins in PPAR signaling pathway. The changes of PPAR signaling pathway may induce lipid metabolism disorder in liver. The verification results of qPCR and PRM were consistent with the sequencing results. We also detected GSH-Px, GSH, GST, TG, TC and ANDP levels in liver. These changes of biochemical indicators directly confirmed oxidative stress and lipid metabolism disorders were occurred in the livers of chicks treated by FFC. In conclusion, FFC could induce liver injury in chicks by regulating the expression levels of significantly different genes and proteins in glutathione signaling pathway and PPAR signaling pathway.

Ultra-sensitive detection of florfenicol by flow injection chemiluminescence immunoassay based on Nickel/Cobalt bimetallic metal-organic framework nanozymes.

The emergence and progress of metal-organic frameworks (MOFs) with high stability, large surface area, and abundant unsaturated active sites, once again promote the development of nanozymes, making nanozymes more advantageous to replace natural enzymes and will increase the applications of chemiluminescence immunoassay. In this study, a flow injection chemiluminescence immunoassay based on Ni/Co metal-organic framework (Ni/Co-MOF) nanozymes was developed, which can quickly and highly sensitively detect florfenicol (FF) in animal-derived food residues. Ni/Co-MOF0.75 nanospheres can not only form stable immune probes with antibodies but also act as nanozymes to efficiently catalyze H2O2 for amplifying the chemiluminescence signal of the luminol-H2O2 system. In addition, due to good biocompatibility and large specific surface area, carboxyl-modified resin beads are used as a suitable material for loading more coating antigens. Based on the principle of competitive immunity, FF standard solution will compete with coating antigen loaded on the carboxyl resin beads for the limited binding sites on the FF antibody. Under the best experimental conditions, the detection range of FF is 0.0001-1000 ng mL-1, and the detection limit (LOD) is 0.033 pg mL-1 (S/N = 3). Furthermore, this method has been successfully applied to the analysis of actual samples with satisfactory results, which will provide a certain reference for the detection of small molecules in food and environmental analysis.

Antimicrobial, immunological and biochemical effects of florfenicol and garlic (Allium sativum) on rabbits infected with Escherichia coli serotype O55: H7.

Florfenicol (FFC) is a synthetic broad-spectrum antibiotic and garlic has a bactericidal action against coliforms. This study was carried out to compare the antimicrobial, immunological and biochemical effects of florfenicol and garlic, for their ability to treat enteropathogenic Escherichia coli serotype O55: H7 infection in rabbits. Four groups (G1-G4) were included. G1 group was the negative control; G2 group was the infected with a field-isolated strain of E. coli and untreated; G3 group was the infected+treated with FFC for 5 days; and G4 group was the infected+treated with garlic tablets for 14 days. The rabbits were observed for clinical signs, growth performance and mortality rates. Garlic-infused disks had a larger clear zone of inhibition than other antibiotic disks. Garlic treatment improved growth performance, biochemical parameters, and immunological response and reduced the fecal shedding and histopathological lesions in E. coli O55: H7 infected rabbits compared to the other groups. Colonization of E. coli more rapidly declined in G3 & G4 than in G2. Hepatic and intestinal gene expressions; tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were significantly elevated in G2 compared to the other groups, and their levels were elevated more in G3 than in G4. Serum interferon-gamma (IFN-γ) and phagocytic activity were significantly elevated in G4 compared to G3. G3 revealed macrocytic hypochromic anaemia that was confirmed histopathologically by moderate haematopoiesis of the bone marrow. In conclusion, garlic powder can reduce rabbit colibacillosis, like FFC, and can enhance the immune status of rabbits.

Proteome and transcriptome analysis revealed florfenicol via affected drug metabolism and lipid metabolism induce liver injury of broilers.

In order to explore the mechanism of liver injury induced by florfenicol (FFC) in broilers. Sixty broilers were randomly divided into 2 groups: control group: normal drinking water and feed were given every d; FFC group: tap water containing FFC (0.15g/L) was given every d and feed was taken freely; each group was given 5 dd of continuous medication and feed was taken freely. The results showed that compared with the control group, FFC could significantly inhibit the weight gain of broilers (P < 0.05), and significantly inhibit the expression of CYP1A1 and CYP2H1 in liver tissue (P < 0.05). It was found that the expression of genes related to the effect of cytochrome P450 on the metabolism of exogenous substances, the peroxisome proliferators-activated receptors signal pathway, peroxisome pathway and glutathione metabolic pathway in the liver of broilers. The results of qPCR of UDP glucuronosyltransferase family 2A1 (UGT2A1), glutathione S-transferase-like 2 (GSTAL2), hematopoietic prostaglandin D synthase (HPGDS), glutathione S-transferase theta 1(GSTT1), isocitrate dehydrogenase (NADP(+)) 1 (IDH1), acyl-CoA oxidase 2 (ACOX2), fatty acid binding protein 1 (FABP1), adenylosuccinate lyase (ADSL), and phosphoribosyl aminoim idazolesuccino carboxamide synthase (PAICS) genes which were randomly selected from the most significant genes were consistent with those of RNA-seq. The results showed that FFC can affect the drug metabolism and lipid synthesis in the liver of broiler, thus impairing the normal function of liver and the growth and development of broiler.

Modelagem farmacocinética/farmacodinâmica do florfenicol para o tratamento da adenite equina por meio da simulação de Monte Carlo / Pharmacokinetic/pharmacodynamic modeling of florfenicol for the treatment of equine adenitis using Monte Carlo simulation

O objetivo deste trabalho foi avaliar a eficácia do florfenicol na dose usualmente empregada em equinos de 22 mg/kg pelas vias intravenosa, intramuscular e oral para o tratamento de adenite equina por Streptococcus equi. subsp. equi, usando a modelagem farmacocinética/farmacodinâmica (PK/PD – Pharmacokinetic/Pharmacodynamic) e a simulação de Monte Carlo. Foi realizada uma simulação de Monte Carlo a partir dos parâmetros PK, logo depois, efetuou-se a modelagem PK/PD para determinar as taxas de eficácia do antimicrobiano para o tratamento dessa infecção bacteriana, de acordo com o valor da concentração inibitória mínima (CIM), em um intervalo de CIM de 0,125 – 4 μg/mL. Pela via intravenosa, a probabilidade de erradicação bacteriana foi de 100% para CIM até 0,5 μg/mL e efeito bacteriostático com probabilidades de 99% e 80% para CIMs de 2 e 4 μg/mL, respectivamente. Já pelas vias intramuscular e oral a probabilidade de se atingir o índice de erradicação bacteriológica foi de 100% para CIM de até 0,5 μg/mL, contudo, atinge valores de 80% e 81%, respectivamente, para CIM de 1 μg/mL considerando o efeito bactericida (p<0,01). Portanto, através desse estudo é evidenciado a eficácia do florfenicol até a CIM de 0,5 μg/mL para as três vias de administração citadas, entretanto, para CIMs superiores a esse valor, é imprescindível o ajuste da dose farmacológica, evitando falhas na terapêutica e possível resistência microbiana.

The objective of this study was to evaluate the efficacy of florfenicol at the dose usually used in horses of 22 mg/kg by intravenous, intramuscular and oral routes for the treatment of equine adenitis caused by Streptococcus equi. subsp. equi, using Pharmacokinetic/Pharmacodynamic (PK/PD) modeling and Monte Carlo simulation. A Monte Carlo simulation was performed from the PK parameters, then PK/PD modeling was performed to determine the antimicrobial efficacy rates for the treatment of this bacterial infection, according to the minimum inhibitory concentration (MIC) value, in a MIC range of 0.125 - 4 μg/mL. Intravenously, the probability of bacterial eradication was 100% for MICs up to 0.5 μg/mL, and the bacteriostatic effect was 99% and 80% for MICs of 2 and 4 μg/mL, respectively. However, for the intramuscular and oral routes, the probability of reaching the bacteriologic eradication index was 100% for MICs of up to 0.5 μg/mL, however, it reaches values of 80% and 81%, respectively, for MICs of 1 μg/mL considering the bactericidal effect (p<0.01). Therefore, through this study the efficacy of florfenicol is evidenced up to the MIC of 0.5 μg/mL for the three routes of administration cited, however, for MICs higher than this value, it is essential to adjust the pharmacological dose, avoiding failures in therapy and possible microbial resistance.

Unveiling the Role of Sulfur in Rapid Defluorination of Florfenicol by Sulfidized Nanoscale Zero-Valent Iron in Water under Ambient Conditions.

Groundwater contamination by halogenated organic compounds, especially fluorinated ones, threatens freshwater sources globally. Sulfidized nanoscale zero-valent iron (SNZVI), which is demonstrably effective for dechlorination of groundwater contaminants, has not been well explored for defluorination. Here, we show that SNZVI nanoparticles synthesized via a modified post-sulfidation method provide rapid dechlorination (∼1100 µmol m-2 day-1) and relatively fast defluorination (∼6 µmol m-2 day-1) of a halogenated emerging contaminant (florfenicol) under ambient conditions, the fastest rates that have ever been reported for Fe0-based technologies. Batch reactivity experiments, material characterizations, and theoretical calculations indicate that coating S onto the metallic Fe surface provides a highly chemically reactive surface and changes the primary dechlorination pathway from atomic H for nanoscale zero-valent iron (NZVI) to electron transfer for SNZVI. S and Fe sites are responsible for the direct electron transfer and atomic H-mediated reaction, respectively, and ß-elimination is the primary defluorination pathway. Notably, the Cl atoms in florfenicol make the surface more chemically reactive for defluorination, either by increasing florfenicol adsorption or by electronic effects. The defluorination rate by SNZVI is ∼132-222 times higher with chlorine attached compared to the absence of chlorine in the molecule. These mechanistic insights could lead to new SNZVI materials for in situ groundwater remediation of fluorinated contaminants.

Florfenicol causes excessive lipid peroxidation and apoptosis induced renal injury in broilers.

In order to study the effects and mechanism of florfenicol (FFC) on the kidney function of broilers, 180 1-day-old broilers were randomly divided into 6 groups, 30 in each group. Except for the control group, different doses of FFC were added to drinking water in the other 5 groups (0.15 g/L, 0.3 g/L, 0.6 g/L, 1.2 g/L and 1.8 g/L). After continuous administration for 5 days, renal histopathological changes, serum renal function indicators, renal peroxidation products and antioxidant factors, and apoptotic factors were detected in broilers aged 21 and 42 days. The results showed that compared with the control group, the kidney tissue structure was disordered, the glomerulus was atrophic, the cystic cavity was enlarged, and the epithelial cells of renal tubules were seriously vacuolated in broilers of treatment groups. And with the growth of broilers, the kidney injury of broilers in the low-dose FFC group was relieved. FFC significantly increased the contents of uric acid (UA), blood urea nitrogen (BUN), creatinine (CRE) in serum and malondialdehyde (MDA) in kidney of broilers, but significantly reduced the levels of glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) in kidney. FFC significantly inhibited the mRNA relative transcriptional levels of nuclear factor-erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and nicotinamide adenine dinucleotide phosphate: quinone oxidoreductase-1 (NQO-1), and increased the mRNA and protein expression levels of p53, Caspase-3 and Caspase-6 in kidney tissue of broilers. It is concluded that FFC has certain nephrotoxicity to broilers, and its effect on kidney is dose-dependent and reversible. FFC causes intense lipid peroxidation in broiler kidney by inhibiting the expression of related factors in the downstream signal pathway of Nrf2. FFC can also up-regulate the expression of pro-apoptotic factors and accelerate the abnormal apoptosis of renal cells, thus seriously affecting the renal function of broilers.

Evaluation of absorption and depletion of florfenicol in European seabass Dicentrarchus labrax.

The pharmacokinetic properties and residue elimination of florfenicol (FLO) and its amine were investigated in European seabass Dicentrarchus labrax at 24°C. The trial mainly included analysis of FLO in plasma after a single dose dietary administration of 10 mg/kg and in muscle plus skin following a multiple dosing (10 mg kg-1  day-1 for 7 days) to estimate pharmacokinetics and residue depletion, respectively. The maximum plasma concentration of FLO was measured to be 1.64 µg/ml, 4 hr post administration. The elimination half-life (t1/2b ) and the area under the concentration-time curve extrapolated to infinity (AUC0-∞ ) were calculated to be 13.0 hr and 34.7 µg h-1  ml-1 , respectively. Withdrawal times of FLO and its amine were calculated to be 46.3 degree-days, indicating a fast removal from the edible tissues of treated European seabass. Overall, FLO can be considered as a potentially efficient antibacterial agent for farmed European seabass provided that additional efforts will be devoted towards its in vitro and clinical efficacy.

Cutaneous mucosal immune-parameters and intestinal immune-relevant genes expression in streptococcal-infected rainbow trout (Oncorhynchus mykiss): A comparative study with the administration of florfenicol and olive leaf extract.

This study evaluated changes in cutaneous mucosal immunity (total protein (TP) and immunoglobulin (TIg), lysozyme, protease, esterase, and alkaline phosphatase (ALP)) and some immune-related genes expression (tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-8, hepcidin-like antimicrobial peptides (HAMP), and immunoglobulin M (IgM)) in the intestine of rainbow trout (Oncorhynchus mykiss) orally-administrated florfenicol (FFC) and/or olive leaf extract (OLE), experimentally infected with Streptococcus iniae. The juvenile fish (55 ± 7.6 g) were divided into different groups according to the use of added OLE (80 g kg-1 food), the presence/absence of FFC (15 mg kg-1 body weight for 10 consecutive days), and the streptococcal infectivity (2.87 × 107 CFU mL-1 as 30% of LD50-96h). The extract's chemical composition was analyzed using the high-performance liquid chromatography (HPLC) system. The skin mucus and intestine of fish were sampled after a 10-day therapeutic period for all groups, and their noted indices were measured. Our results signified that the oleuropein, quercetin, and trans-ferulic acid were the most obvious active components of OLE which were found by HPLC analysis. The combined use of OLE and FFC could lowered some skin mucus immunological indices (e.g., TP, TIg, and ALP), and the gene expression of inflammatory cytokines (e.g., TNF-α and IL-1ß) of rainbow trout. Moreover, lysozyme and protease activities respectively were invigorated by the FFC and OLE treatment. Also, the use of OLE as a potential medicine induced the gene expression of HAMP. As the prevention approach, it would be recommended to find the best dose of OLE alone or in combination with the drug through therapeutics period before the farm involved in the streptococcal infection.

Growth and cardiovascular development are repressed by florfenicol exposure in early chicken embryos.

Florfenicol (FLO) is one of the most popular antibacterial drugs used in veterinary clinics and aquaculture. The drug was found to decrease the hatchability of eggs laid by treated hens in veterinary clinics and research work. However, the pathological changes in developing embryos and their cardiovascular system and the mechanism underlying FLO-induced embryonic death remain unclear. In the present study, fertilized eggs laid by hens treated with a therapeutic dose of FLO were collected and incubated. Results showed that FLO exposure repressed embryonic development and induced early embryonic death. As a result, FLO decreased the hatchability and increased the proportion of weak chicks. Moreover, FLO exposure led to embryonic lethality and inhibited the development of chick embryos as characterized by decreased weights, lagging distribution of Hamburger-Hamilton stages, and dysplastic eyes. Pathological examination indicated that FLO exposure affected the normal development of the heart in 4.5-day-old chick embryos, as characterized by shorter transverse cardiac diameter, disordered arrangement of trabecular muscles in ventricles, and reduced thickness of ventricular walls. Furthermore, FLO decreased blood vascular densities and downregulated the expression levels of key angiogenesis-related genes, including the vascular endothelial growth factor and fibroblast growth factor, in the yolk sac membrane. These findings indicated that FLO exposure restricted vascular development during early embryonic development. In summary, our data suggest that the restricted growth and abnormal cardiovascular development may be responsible for FLO-induced early embryonic death. Thus, these findings can be useful for guiding the proper use of FLO and in laying a foundation for further studies on the mechanism of FLO-induced embryonic toxicity.

Determination of amphenicol antibiotics and their glucuronide metabolites in urine samples using liquid chromatography with quadrupole time-of-flight mass spectrometry.

A rapid procedure for the determination of amphenicol antibiotics in human urine by liquid chromatography with quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) is proposed. The presence of thiamphenicol (TAP), florfenicol (FF) and chloramphenicol (CAP) in the human body can be attributed to their administration to treat certain diseases or by eating food of animal origin. The TAP, FF and CAP excreted in urine is mainly in the form of glucuronide conjugates, although their free forms may also be excreted to a lesser extent. In the procedure described, the enzymatic hydrolysis of amphenicol glucuronide forms in urine was carried out using ß-glucuronidase and sulfatase at pH 5 (37 °C, overnight) in order to discriminate the free and conjugated forms. Then, amphenicol antibiotics were submitted to dispersive liquid-liquid microextraction (DLLME) for preconcentration. All the parameters affecting DLLME efficiency were optimized, and the following conditions were selected: 0.9 g NaCl in 10 mL of urine, to which 1.2 mL methanol (as dispersant solvent) and 1 mL of 4-methyl-2-pentanone (as extractant solvent) were added. The absence of a matrix effect allowed quantification of the samples against aqueous standards. Detection limits were 29, 6 and 3 pg mL-1 for TAP, FF and CAP, respectively. Relative standard deviations were calculated to evaluate the intra- and inter-day precision and values lower than 10% were obtained in all cases. The trueness of the method was tested through recovery studies, obtaining satisfactory values (83-104%). Ten urine samples obtained from volunteers were analysed and all of them were free of the studied antibiotics.

Determination of Chloramphenicol, Thiamphenicol and Florfenicol in Chinese Gelatin Medicines using Dispersive Solid-Phase Extraction Coupled with Ultra High-Performance Liquid Chromatography-Mass Spectrometry.

This study established a rapid and reliable method to determine chloramphenicol (CAP), thiamphenicol (TAP) and florfenicol (FF) residues in Chinese gelatin medicines. CAP, TAP and FF were extracted from medicine samples using 2% (v/v) ammonium hydroxide in acetonitrile. Trypsin was used to eliminate the matrix effect caused by protein components in gelatin medicines, whereas anhydrous sodium sulfate, C18-N and NH2-PSA adsorbents were applied to reduce matrix effect induced by other components. The analytical method of these drugs was optimized on ultra high-performance liquid chromatography-mass spectrometer (UHPLC-MS/MS) through the analysis of their standard linearity and regression. The optimized extraction and analytical method were validated in one Chinese gelatin medicine sample (Colla corii asini, E Jiao) with three fortification levels (2, 5 and 10 µg/kg), and the recoveries of these drug residues ranged of 87.6-102.7%. The limit of detection and quantification of CAP, TAP and FF in the sample were 0.2 and 0.5 µg/kg, 0.4 and 1.5 µg/kg, and 0.5 and 1.5 µg/kg, respectively. A total of 30 Chinese gelatin medicine samples were analyzed using the established method. No drug residues were found in these samples except for one Testudinis Carapacis et Plastri (1.67 µg/kg FF) and one turtle shell glue (2.55 µg/kg FF).

Increased bioaffinity and anti-inflammatory activity of florfenicol nanocrystals by wet grinding method.

Context: The main objective of the current study is to improve the water solubility of florfenicol (FF) and evaluate changes in its pharmacokinetics and anti-inflammatory activity.Materials and methods: Florfenicol nanocrystals (FF-NC) were prepared by wet grinding combined with spray drying. The characterisations, pharmacokinetics, and anti-inflammatory activity of FF-NC were evaluated.Results: The particle size, polydispersity index (PDI), and zeta potential of FF-NC were 276.4 ± 19.4 nm, 0.166 ± 0.011, and -18.66 ± 5.25 mV, respectively. Compared with FF, FF-NC showed a better dissolution rate in media at different pH. Pharmacokinetic experiments showed the area under the curve (AUC0-t), maximum concentration (Cmax), and mean residence time (MRT) of FF-NC were about 4.62-fold, 2.86-fold, and 1.68-fold higher compared with FF, respectively. In vitro anti-inflammatory experiments showed that FF inhibited the secretion of tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), and synthesis of NO in a dose-dependent manner, while FF-NC showed a stronger anti-inflammatory effect than FF under the same dose.Conclusion: FF-NC are an effective way to improve the bioaffinity and anti-inflammatory effects of FF.

Effects of anemoside B4 on pharmacokinetics of florfenicol and mRNA expression of CXR, MDR1, CYP3A37 and UGT1E in broilers.

Pulsatillae radix, a traditional Chinese medicine (TCM), is often used in combination with florfenicol for treatment of intestinal infection in Chinese veterinary clinics. Anemoside B4 (AB4) is the major effective saponin in Pulsatillae radix. This study aimed to investigate whether the pharmacokinetics of florfenicol in broilers was affected by the combination of AB4. In this study, broilers were given AB4 (50 mg/kg BW), or 0.9% sodium chloride solution by oral administration for 7 days. They were then fed florfenicol orally (30 mg/kg BW) on the eighth day. The results showed that the AUC(0-∞), MRT(0-∞), t1/2z and Cmax of florfenicol were significantly decreased, and the Vz/F and CLz/F were significantly increased by AB4; the mRNA expression levels of CXR, CYP3A37 and MDR1 (except CXR and CYP3A37 in the liver) were up-regulated by AB4. In conclusion, AB4 altered the pharmacokinetics of florfenicol, resulting in lower plasma concentrations of florfenicol, this was probably related to the mRNA expression of CXR, CYP3A37 and MDR1 in the jejunum and liver (except CXR and CYP3A37) increased by AB4. The implications of these findings on the effect of traditional Chinese medicine containing AB4 on the effectiveness of florfenicol in veterinary practice deserve study.

Florfenicol alleviated lipopolysaccharide (LPS)-induced inflammatory responses in Ctenopharyngodon idella through inhibiting toll / NF-κB signaling pathways.

The present study was conducted to evaluate the anti-inflammatory activity of florfenicol (FFC) against lipopolysaccharide (LPS)-induced inflammatory responses in Ctenopharyngodon idella in vivo and in vitro. Head-kidney (HK) macrophages were pre-treated with 10 µg/mL LPS and then exposed to different concentrations of FFC to determine its in vitro anti-inflammatory activity. Inhibitory effect of FFC on inflammatory mediators TNF-α, IL-6 and IL-1ß, as well as LPS-induced nitric oxide (NO) and prostaglandin E 2 (PGE 2) production were assayed by ELISA. The expression level of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were investigated by RT-PCR. Expression level of TLR-related genes (TLR1, TLR2, TLR4, TLR7, TLR8) expression, tumor necrosis factor receptor-associated factor 6 (TRAF6), transforming growth factor-b-activated kinase 1 (TAK1), Myeloid differentiation factor 88 (MyD88), nucleus p65, NF-κBα (IκBα) were measured by RT-PCR after grass carp were treated with 50, 100 and 200 mg FFC/kg body weight for 3 days. Results from in vitro tests demonstrated that FFC dose-dependently inhibited LPS-induced inflammatory cytokines TNF-α, IL-6 and IL-1ß, inflammatory factors NO and PGE 2 production in macrophages. In addition, iNOS and COX-2 expression levels decreased significantly as compared with LPS treated group. In vivo test demonstrated that treatment with FFC prevented the LPS-induced upregulation of TNF-α, IL-6, IL-1ß, NO and PGE 2. The expression level of iNOS, and COX-2 in FFC-treated grass carp were also downregulated as compared with LPS treated fish. Besides, FFC blocked the expression of Toll-like receptor 2 (TLR2) and then suppressed the phosphorylation of nuclear transcription factor-kappa B (NF-κB) p65 and degradation inhibitor of IκBα. Furthermore, administration of FFC inhibited the up-regulation of IRAK4, TRAF6 and TAK1 induced by LPS. These results suggest that the anti-inflammatory properties of FFC might be the results from the inhibition of iNOS, COX-2, IL-6, IL-1ß, and TNF-α expressions through the down-regulation of Toll/NF-κB signaling pathways.

Capped cadmium sulfide quantum dots with a new ionic liquid as a fluorescent probe for sensitive detection of florfenicol in meat samples.

The new ionic liquid capped CdS quantum dots (IL-CdS QDs) as a fluorescent probe was successfully synthesized by a hydrothermal method in a one step process and used for the facile and sensitive determination of florfenicol (FLF) in aqueous media. The new ionic liquid 3-(2-[(5-amino-1,3,4-thiadiazol-2-yl)thio]ethyl)-1-methyl-1H-imidazol-3-ium chloride (IL) was synthesized by introducing 5-amino-1,3,4-thiadiazole-2-thiol as a ligand onto the alkyl chain of the 1-chloroethyl-3-methylimidazolium chloride ILs. This task specific ionic liquid reagent was used for the capping of CdS QDs which played the role of recognition element of FLF. The IL-CdS QDs were characterized by Ultra Violet-Visible absorption -spectroscopy (UV-Vis absorption spectroscopy), Fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction (XRD), and transmission electron microscopy (TEM). Quenching of fluorescence intensity of the IL-CdS QDs was in proportion to the addition of FLF concentration. Under the optimum conditions, the fluorescence intensity ratio of IL-CdS QDs in the presence and absence of FLF versus FLF concentrations gave a linear response according to the Stern-Volmer equation from 0.1 to 20 µg mL-1 (0.3 to 56 µmol L-1) with a limit of detection 0.035 µg mL-1 (0.098 µmol L-1). The developed method was applied to the determination of FLF in fish and chicken meats with satisfactory results. This method revealed some advantages such as high sensitivity, precision and wide linear range to FLF. The proposed method can be utilized for rapid screening the quality of meat products.