| Enrofloxacin (ENR) is an antibiotics of3rd-generation quinolones, and has beenused for prevention and treatment of bacterial diseases in aquatic animals owing to itsefficient, broad-spectrum antimicrobial effects and free of cross-reactivity to otherantibiotics. Chinese mitten crab is a famous and precious aquaculture species, and oftensuffers from bacterial diseases. As a common drug, ENR is also used to control bacterialdiseases of Chinese mitten crab, and thus poses a potential residual risk. For measuringof ENR residual, the high performance liquid chromatography method (HPLC) isproved to be lower in detection limit, and efficient in performance, but involvescomplicated, time-consuming, and cost-intensive pre-treatment procedures. Therefore,by optimizing preparation technique for anti-ENR polyclonal antibodies, we developedan improved ELISA method that was sensitive, specific, quick, and simple for ENRdetermination, which was used in the study to investigate the effects of drugadministration frequency, physical conditions and water temperatures on thepharmacokinetics and residue elimination of ENR in Chinese mitten crab. The mainresults are summarized as follows:1. Female BALB/C mice were injected with conjugates of ENR and cBSA linkedby1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, from which antiseraagainst ENR were collected and used as primary antibodies. The conjugate of ENR andcOVA linked with N,N’-dicyclohexylcarbodiimide was applied for coating antigen, andthen the optimal working concentrations were determined according to OD490≈3.50bychecker-board ELISA. To characterize the affinity of the antisera as IC50(50%inhibitory concentration) and the specificity to ENR, a CI-ELISA with gradientconcentrations of ENR and its analogs, such as ciprofloxacin, as antagonists to blockbinding of antisera with coating antigen was performed. The results suggested that thetiter of the antisera was1.25×10-5, and a linear detection ranged from0.40to125ng/ml with an IC50of8.68ng/ml, less than0.05%cross-reactivity to ciprofloxacin and otheranalogues. The spiking concentrations of ENR in hemolymph, muscle, andhepatopancreas were10,30, and90ng/g (ng/ml), respectively. Average recoveries were72.23%~85.45%,68.66%~74.12%, and50.17%~56.30%. Relative standard deviationswere7.64%~12.26%,12.41%~22.23%and28.28%~35.40%. The immunoassaymethods improved in this study can be used for the detection of ENR residues in aquaticanimal food products.2. A study of following oral administration (10mg/Kg) of ENR was carried out inChinese mitten crab to investigate the effects of different administration frequency,physical conditions and water temperatures on the pharmacokinetics and residueelimination of ENR in Chinese mitten crab. Hemolymph, muscle and hepatopancreaswere collected at5,10min and0.5,1,2,4,8,12,24,48,96,168, and240hpost-gavage. The levels of ENR in tissues were determined by ELISA and the data wereanalyzed with Practical Pharmacokinetics Program (3P97). The results demonstratedthat:(1) With a single oral administration (10mg/Kg) and oral administration at adosage of10mg/Kg for3days at a water temperature of25°C, the ENR level in thehemolymph and muscle peaked, and then declined with different administrationfrequencies. The main pharmacokinetic parameters of ENR in hemolymph, muscle, andhepatopancreas were, respectively, T1/2ka=0.27h,0.27h and2.18h; T1/2α=3.65h,7.39h and19.86h; T1/2β=15.26h,29.94h and42.92h at a single oral administration, andT1/2ka=0.26h,0.24h and0.07h; T1/2α=3.81h,9.04h and23.05h; T1/2β=18.50h,33.21h and48.41h at a multiple oral administration. The distribution and eliminationof ENR at a single oral administration were faster than those for the multiple oraladministrations. The content of ENR in hepatopancreas, hemolymph, muscle, appearedin descending order. The data were analyzed with3P97and suggested that theconcentration-time course of ENR in the three tissues can be described by atwo-compartment open model at different frequency of administration. It is suggestedthat the withdrawal periods of ENR should not be less than375°C d for prevention ofbacterial diseases at25°C following oral administration (10mg/Kg) according tostandards for veterinary drugs residue limits in pollution-free food.(2) Following a single oral administration (10mg/Kg) in the healthy crab and those infected with Aeromonas hydrophila at a water temperature of25°C, the mainpharmacokinetic parameters of ENR in hemolymph, muscle and hepatopancreas ofhealthy crab were respectively T1/2ka=0.27h,0.27h and2.18h; T1/2α=3.65h,7.39h and19.86h; T1/2β=15.26h,29.94h and42.92h and T1/2ka=0.87h,1.35h and2.56h;T1/2α=1.34h,2.59h and8.61h; T1/2β=14.04h,40.28h and7.96h of those infected withA. hydrophila. The distribution and elimination of ENR in the Chinese mitten crabinfected with A. hydrophila were faster than those in healthy crab, while the absorptionof ENR was slower and the content of ENR was less. The levels of ENR inhepatopancreas, muscle, and hemolymph appeared in descending order. The dataanalyzed with3P97suggested that the concentration-time course of ENR in the threetissues can be described by a two-compartment open model at different health condition.It is suggested that the withdrawal periods of ENR should not be less than300°C d fortreatment of bacterial diseases at25°C following oral administration (10mg/Kg)according to standards for veterinary drugs residue limits in pollution-free food.(3) Following a single oral administration (10mg/Kg) in the Chinese mitten crabinfected with A. hydrophila at water temperatures of20,25and30°C, the ENR levels inthe hemolymph and muscle peaked, and then declined, while those in hepatopancreasappeared two peaks, and the level of ENR in the second peak was much higher than thefirst peak at20,25and30°C. The absorption, distribution and elimination of ENR wereaccelerated with ascending of the water temperature. The data analyzed with3P97indicated that the concentration-time course of ENR in the three tissues can bedescribed by a two-compartment open model at different temperatures. The area underthe concentration-time curve from zero to infinity of ENR in the three tissues was foundto negatively correlate with water temperature, whereas total body clearance (CL)showed a positive correlation with water temperature. |
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