Nikolina Rusenova

Integration of Pharmacokinetic and Pharmacodynamic Indices of Marbofloxacin in Turkeys

ABSTRACT Fluoroquinolones are extensively used in the treatment of systemic bacterial infections in poultry, including systemic Escherichia coli bacillosis, which is a common disease in turkey flocks. Marbofloxacin has been licensed for use in various mammalian species, but not as yet for turkeys, although its kinetic properties distinguish it from other fluoroquinolones. For example, the longer half-life of marbofloxacin in many animal species has been appreciated in veterinary practice. It is generally accepted that, for fluoroquinolones, the optimal dose should be estimated on the basis of the pharmacokinetic (PK) and pharmacodynamic (PD) characteristics of the drug under consideration. Knowledge of these specific data for the target animal species allows the establishment of an integrated PK-PD model that is of high predictive value. In the present study, the antibacterial efficacy (PD indices) against a field isolate of Escherichia coli O78/K80 was investigated ex vivo following oral and intravenous administration of marbofloxacin to turkeys (breed BUT 9; six animals per group) at a dose of 2 mg/kg of body weight (BW). At the same time, the serum concentrations of marbofloxacin were measured at different time intervals by a standardized high-performance liquid chromatography method, allowing the calculation of the most relevant kinetic parameters (PK parameters). The in vitro serum inhibitory activity of marbofloxacin against the selected E. coli strain, O78/K80, was 0.5 μg/ml in the blood serum of turkeys, and the ratio of the maximum concentration of the drug in serum to the serum inhibitory activity was 1.34. The lowest ratio of the measured serum concentration multiplied by the incubation period of 24 h to the serum inhibitory activity required for bacterial elimination was lower than the ratio of the area under the serum concentration-time curve (AUC) to the serum inhibitory activity. These first results suggested that the recommended dose of 2 mg/kg BW of marbofloxacin is sufficient to achieve a therapeutic effect in diseased animals. However, considering the risk of resistance induction, the applied dose should be equal to an AUC/MIC of >125, the generally recommended dose for all fluoroquinolones. According to the PK-PD results presented here, a dose of 3.0 to 12.0 mg/kg BW per day would be needed to meet this criterion. In conclusion, the results of the present study provide the rationale for an optimal dose regimen for marbofloxacin in turkeys and hence should form the basis for dose selection in forthcoming clinical trials.

Pharmacokinetic-pharmacodynamic modelling of danofloxacin in turkeys.

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-1h-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 μg/ml, four times higher than in broth. The lowest effective ex vivo AUC24/MIC ratios required for bacteriostasis, bactericidal activity, and total killing of E. coliO78/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 μ g/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.

Differential Infection Patterns and Recent Evolutionary Origins of Equine Hepaciviruses in Donkeys

ABSTRACT The hepatitis C virus (HCV) is a major human pathogen. Genetically related viruses in animals suggest a zoonotic origin of HCV. The closest relative of HCV is found in horses (termed equine hepacivirus [EqHV]). However, low EqHV genetic diversity implies relatively recent acquisition of EqHV by horses, making a derivation of HCV from EqHV unlikely. To unravel the EqHV evolutionary history within equid sister species, we analyzed 829 donkeys and 53 mules sampled in nine European, Asian, African, and American countries by molecular and serologic tools for EqHV infection. Antibodies were found in 278 animals (31.5%), and viral RNA was found in 3 animals (0.3%), all of which were simultaneously seropositive. A low RNA prevalence in spite of high seroprevalence suggests a predominance of acute infection, a possible difference from the mostly chronic hepacivirus infection pattern seen in horses and humans. Limitation of transmission due to short courses of infection may explain the existence of entirely seronegative groups of animals. Donkey and horse EqHV strains were paraphyletic and 97.5 to 98.2% identical in their translated polyprotein sequences, making virus/host cospeciation unlikely. Evolutionary reconstructions supported host switches of EqHV between horses and donkeys without the involvement of adaptive evolution. Global admixture of donkey and horse hepaciviruses was compatible with anthropogenic alterations of EqHV ecology. In summary, our findings do not support EqHV as the origin of the significantly more diversified HCV. Identification of a host system with predominantly acute hepacivirus infection may enable new insights into the chronic infection pattern associated with HCV. IMPORTANCE The evolutionary origins of the human hepatitis C virus (HCV) are unclear. The closest animal-associated relative of HCV occurs in horses (equine hepacivirus [EqHV]). The low EqHV genetic diversity implies a relatively recent acquisition of EqHV by horses, limiting the time span for potential horse-to-human infections in the past. Horses are genetically related to donkeys, and EqHV may have cospeciated with these host species. Here, we investigated a large panel of donkeys from various countries using serologic and molecular tools. We found EqHV to be globally widespread in donkeys and identify potential differences in EqHV infection patterns, with donkeys potentially showing enhanced EqHV clearance compared to horses. We provide strong evidence against EqHV cospeciation and for its capability to switch hosts among equines. Differential hepacivirus infection patterns in horses and donkeys may enable new insights into the chronic infection pattern associated with HCV.

Effects of fluoroquinolone treatment on MDR1 and MRP2 mRNA expression in Escherichia coli-infected chickens.

Current knowledge about the expression of ABC transport proteins suggests that their expression is regulated by a variety of factors, including pathological conditions, and in particular inflammatory reactions to infection. As ABC transporters are major determinants of absorption, distribution and excretion of many antimicrobials, modulation of their activity may result in increased or decreased tissue levels of drugs, affecting the efficacy of treatment. As fluoroquinolones have been identified as modulators and substrates of a number of drug transporters, we evaluated the effect of danofloxacin mesylate and enrofloxacin treatment on the levels of expression of MDR1 and MRP2 mRNAs in the intestines and livers of broilers with experimentally induced colibacillosis. MDR1 mRNA expression was significantly decreased in infected animals and was partly restored over 5 days of treatment with orally administered danofloxacin mesylate or enrofloxacin. Changes in the level of expression of MRP2 mRNA were less prominent. The study suggests that the treatment of colibacillosis with fluoroquinolones, which resulted in a significant clinical improvement of the animals, also restored the expression of drug transporters. This is of clinical importance as these ABC transporters significantly contribute to the functionality of important biological barriers, protecting the bird and specific tissues from pathogens and bacterial toxins.

Effect of diclofenac on ocular levels of ciprofloxacin and lomefloxacin in rabbits with endophthalmitis.

Abstract The purpose of this study was to analyze ciprofloxacin and lomefloxacin penetration into the anterior eye tissues after topical instillation in healthy rabbits and with experimental Staphylococcus aureus endophthalmitis. Additionally, effect of diclofenac sodium eye drops on the distribution of both fluoroquinolones in the inflamed eye tissues was investigated. An intense protocol with frequent antibiotic administration was chosen. Samples from aqueous humor were obtained 2 and 6 h after the start of the treatment. Samples from cornea and iris were obtained at the end of the experiment, after euthanasia of the animals. Drug concentrations were measured by HPLC method. The median levels of ciprofloxacin and lomefloxacin in aqueous humor of healthy animals, 2 and 6 h after drug administration were 6.39–9.65 and 5.30–6.81 µg/ml, respectively. Ciprofloxacin levels were neither changed from the inflammation nor after instillation of diclofenac. In contrary, lomefloxacin concentrations in aqueous humour of inflamed eye were significantly increased 12.15–15.08 µg/ml, especially after diclofenac administration (17.12–27.76 µg/ml). Levels of both fluoroquinolones in cornea (13.08 µg/g for ciprofloxacin and 12.25 µg/g for lomefloxacin) and in iris (0.84 µg/g for ciprofloxacin and 1.34 µg/g for lomefloxacin) were higher than MIC and MBC values against S. aureus ATCC 25923. Although higher lomefloxacin concentrations were observed in the aqueous humor after instillation of diclofenac, the levels of both fluoroquinolones in iris and in cornea were not significantly changed. Topical administration of lomefloxacin and diclofenac in combination improved penetration of the antibacterial agent in the aqueous humor which can be of clinical importance.

Development of Multiplex PCR for Fast Detection of Paenibacillus Larvae in Putrid Masses and in Isolated Bacterial Colonies

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.

Detection of Staphylococcus Aureus Among Coagulase Positive Staphylococci from Animal Origin Based on Conventional and Molecular Methods

Abstract The present study aimed to detect Staphylococcus aureus (S. aureus) among other coagulase positive staphylococci from animal origin by using conventional methods (biochemical tests and latex agglutination) and a molecular method, based on the nuc gene, as the gold standard and to assess the usefulness of these methods. For this purpose, total of 344 staphylococcal isolates were collected and analysed. A total of 156 isolates suspicious for S. aureus were detected by a conventional biochemical method - 88 from cows, 18 from goats, 7 from pigs, 17 from poultry, 7 from rabbits and 19 from dogs. The majority of S. aureus strains gave typical biochemical reactions with the exception of 30 (19.2%) and 25 (16%) that were VP negative and weak positive in fermenting mannitol, respectively. Twelve strains were found to be non-haemolytic (7.7%) and four strains did not ferment trehalose (2.6%). Other staphylococci were identified as S. pseudintermedius (n = 103), S. hyicus (n = 23) and the rest were coagulase-negative staphylococci. Latex agglutination test resulted in rapid positive reactions with S. aureus with exception of 5 strains (3.2%) from cow mastitis milk. Positive agglutination reactions were also established with S. pseudintermedius, and S. hyicus. PCR confirmed all strains that were preliminary identified as S. aureus by amplification of 270 bp fragment of nuc gene specific for this species. The atypical reactions in certain strains established in this study have shown that the precise detection of S. aureus from animal origin should be done by combination of conventional and molecular methods.