Study On The Pharmacodynamic Of Danofloxacin To Actinobacillus Pleuropneumoniae In Vitro

Antibiotics have a good effect on the treatment of infectious diseases causing by bacteria and mycoplasma in livestock and poultry.With the increasing use of antibiotics,the resistance caused by irrational and unrestrained abuses of antibiotics is becoming more and more serious.The antimicrobial-resistant mutations of microbes make the therapeutic effect failed and bring losses to the breeding industry.Meanwhile,antimicrobial-resistant genes may also be transmitted by contacting and eating food which will endanger the health of animals and human beings.Currently,the control of antimicrobial resistance is conducting all over the world.The focus is put on the pharmacodynamics(PD)and pharmacokinetics(PK)in vitro and in vivo and then set up the epidemiological cut-off,PK/PD breakpoints and clinical breakpoints.Porcine Contagious Pleuropneumonia is widely distributed all over the world.The disease is characterized by acute onset,short course and high mortality.It has become one of the most important respiratory infectious diseases in modern livestock breeding industry.The pathogen of Porcine Contagious Pleuropneumonia is Actinobacillus pleuropneumoniae which is a polyformus actinobacillus,gram-negative staining,with a capsule and a gasbag and no spore formed.It can move with a flagellum.There are 15 types of serotypes which can be divided into biological type I and biological type II depending on whether they need the oxidant coenzyme I when they grow.Danofloxacin is one of the veterinary fluoroquinolones and has strong effect on gram negative and positive bacteria.It also has high antibacterial activity at low concentrations.Because of its broad antibacterial spectrum,strong antibacterial activity and good dynamics in vivo,it has been widely used in veterinary clinic in China.It is reported that the concentration of danofloxacin in the lungs is about 5~7 times as much as that in the blood through the pharmacokinetic study.So danofloxacin is commonly used to treat the respiratory diseases of livestock and poultry.There is no report on the breakpoint of danofloxacin on Actinobacillus pleuropneumoniae at home and abroad.48 strains of Actinobacillus pleuropneumonia are collected and cultured to conduct the drug susceptibility test in vitro.In order to setup the epidemiological breakpoints,provide a theoretical basis for the establishment of PK/PD breakpoints and guide the clinical prevention and treatment of porcine contagious pleuropneumonia.This paper focuses on the researches in the following aspects:1.Isolation and identification of Actinobacillus pleuropneumoniaPathogens are isolated from clinical samples of suspected pigs with infectious pleuropneumonia in Henan province.20 strains of Actinobacillus pleuropneumoniae are isolated and identified through gram-coloring,microscopic examination,biochemical identification and PCR identification.The purified pathogen is identified by 8 pairs of primers for PCR amplification of different serotypes.There are including 12 strains of serotype 1,1 strain of serotype 3,4 strains of serotype 5 and 3 strains of serotype 7.Compared with the traditional method,the PCR identification method can improve the separation rate,shorten the identification cycle and reduce the economic loss effectively in the treatment of disease.The results show that the serotype 1(60%)is the main type.Porcine Contagious Pleuropneumonia is mainly immune by different serotype vaccines.Serotype identification is an essential part of the prevention and control of the disease in epidemic areas.2.Study the effects of danofloxacin on Actinobacillus pleuropneumoniae in vitro10 strains are selected to do the preliminary drug susceptibility test in vitro.The results showed that the pathogen are resistant to ampicillin,sulfamethoxazole,ceftiofur,spectinomycin and colistin and sensitive to amoxicillin/clavulanic acid,compound sulfamethoxazole/TMP,florfenicol,doxycycline,gentamicin,tetracycline,enrofloxacin,ofloxacin and danofloxacin.Through testing the drug sensitivity of 48 strains of Actinobacillus pleuropneumoniae to danofloxacin,the results of MIC and MBC are mainly centralized at 0.0039 ?g/mL.Selected one strain of Actinobacillus pleuropneumoniae whose MIC and MBC are both 0.0078 ?g/mL to study the growth curve and killing curve.The results show that Actinobacillus pleuropneumoniae is sensitive to danofloxacin.Through the killing curve,danofloxacin shows concentration-dependent antibiotics effect to Actinobacillus pleuropneumoniae.When the concentrations are at MIC and above,the inhibition effect gradually enhances with the increase of concentration.The inhibition of bacteria can be maintained for a period of time when the concentration is lower than MIC.The drug sensitivity of strains currently isolated from piglets decreases compared with strains historically isolated.We should strengthen the management and guidance the use of danofloxacin in veterinary clinic to prolong its lifetime in clinic.The data of pharmacodynamics in vitro is an important guiding significance for the determination of clinical dosage and it is also necessary to make a breakpoint value.3.Establishment of detection method of danofloxacin in pig plasmaThe high performance liquid chromatographic method is used to determine the concentration of danofloxacin in plasma.By optimizing sample processing methods,the standard curve and working curve of danofloxacin in porcine plasma are established.The high performance liquid chromatographic method with UV detector has highly accurate and popularity and its sensitivity can meet the requirement of determination.This method can be used for the pharmacokinetics of danofloxacin in pigs in the future researches.