RESUMO
The present study was performed to develop a fast and sensitive multiplex polymerase chain reaction protocol for routine diagnostics of American foulbrood. A new approach for detection of Paenibacillus larvae in putrid masses was described. Forty five samples of putrid masses obtained from bee combs suspicious for American foulbrood, a reference strain Paenibacillus larvae (NBIMCC 8478), clinical isolates and 4 strains of closely related bacterial species were included in experiments. Bacterial colonies' DNA was isolated by heat and centrifugation method (standard procedure) and with prepGem commercial kit. DNA from putrid masses was isolated by standard and modified procedure. Three pairs of primers specific for 16S rRNA and one pair specific for 35 kDa metalloproteinase genes of Paenibacillus larvae were tested as single pair and in different combinations as multiplex PCR. The sensitivity of the multiplex PCR protocol for putrid masses, developed in study was 100%, versus 45.2% for the standard protocol. The developed multiplex PCR protocol could be successfully used for rapid and specific detection of Paenibacillus larvae in both putrid masses and isolated bacterial colonies.
Assuntos
Proteínas de Bactérias/genética , DNA Bacteriano/genética , DNA Ribossômico/genética , Metaloproteases/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Paenibacillus/genética , Animais , Abelhas/microbiologia , RNA Bacteriano/genética , RNA Ribossômico 16S/genéticaRESUMO
Colibacillosis is a systemic disease responsible for important economic losses in poultry breeding; fluoroquinolones, including danofloxacin, are used to treat diseased animals. The purpose of the present study was to estimate pharmacokinetic-pharmacodynamic (PK-PD) surrogates for bacteriostasis, bactericidal activity and bacterial elimination against Escherichia coli O78/K80, using a PK-PD approach, for danofloxacin in turkeys after oral administration. Eight healthy turkeys, breed BUT 9, were included in a two-way crossover study. The drug was administered intravenously (i.v.) and orally at a dose rate of 6 mg/kg bw. The values of the elimination half-life and the total body clearance after i.v. administration were 8.64 +/- 2.35 h and 586.76 +/- 136.67 ml kg(-1)h(-1), respectively. After oral administration, the values of the absolute bioavailability and the elimination half-life were 78.37+/- 17.35% and 9.74+/- 2.93 h, respectively. The minimum inhibitory concentration against the investigated strain in turkey serum was 0.25 microg/ml, four times higher than in broth. The lowest effective ex vivo AUC(24)/MIC ratios required for bacteriostasis, bactericidal activity, and total killing of E. coli O78/K80 were 0.416 h, 1.9 h and 6.73 h, respectively. The oral dose of 6 mg/kg used in the present study could be interpreted as being sufficient to eliminate E. coli with an MIC 0.25 microg/ml. However, considering the demand that antimicrobial resistance should be avoided by complete bacterial elimination, PK-PD considerations suggest that an even higher dose of 32 mg/kg per day or 0.7 mg/kcal per day should be evaluated in clinical trials.