In vitro cell compatibility and antibacterial activity of microencapsulated doxycycline designed for improved localized therapy of septic arthritis

OBJECTIVES: For the treatment of septic arthritis in large animals, the local application of antibiotics as a slow release system may be an appropriate means to reach high local bioactivity and low systemic side effects and drug residues. In this study, doxycycline microspheres were developed and tested in vitro for their drug-release properties, suitability for intra-articular application and antimicrobial activity. METHODS: The development of a slow release system was achieved by microencapsulation of the drug into poly(lactide-co-glycolide) microspheres by a novel ultrasonic atomization method. Drug elution was evaluated from microspheres dispersed in elution medium at pre-defined time points by HPLC. Joint-tissue compatibility was tested on cultured bovine synoviocytes by evaluating the expression of pro-inflammatory cytokine mRNA and the production of nitric oxide (NO). Finally, the antimicrobial activity of the released antibiotic was assessed with gram-negative and gram-positive bacteria exposed to release medium sampled at days 1, 7 and 12 after microsphere suspension. RESULTS: An adequate size of the microspheres, sufficient stabilization of doxycycline in aqueous environment and drug release (25 mg microspheres in 4 mL medium) above MIC for bacteria usually isolated in bovine and equine joints were obtained over 15 days. Although the cytokine mRNA expression reflected the excellent tissue compatibility, the results with NO yielded contradictory results. Antimicrobial tests of the release medium proved to match perfectly the activity of non-encapsulated, free doxycycline as reported in the literature. CONCLUSIONS: The newly developed doxycycline delivery system achieved the target specifications and is ready for in vivo testin

Detection of Borrelia-specific 16S rRNA sequence in total RNA extracted from Ixodes ricinus ticks

A reverse transcriptase - polymerase chain reaction based assay for Borrelia species detection in ticks was developed. The method was based on amplification of 552 nucleotide bases long sequence of 16S rRNA, targeted by Borrelia specific primers. In the present study, total RNA extracted from Ixodes ricinus ticks was used as template. The results showed higher sensitivity for Borrelia detection as compared to standard dark-field microscopy. Method specificity was confirmed by cloning and sequencing of obtained 552 base pairs long amplicons. Phylogenetic analysis of obtained sequences showed that they belong to B. lusitaniae and B. afzelii genospecies. RT-PCR based method presented in this paper could be very useful as a screening test for detecting pathogen presence, especially when in investigations is required extraction of total RNA from ticks

Canine borreliosis: a laboratory diagnostic trial

The aim of this study was to investigate samples from dogs suggestive of active canine borreliosis (group A) by culture and PCR and the detection of antibodies against Borrelia burgdorferi sensu lato in order to confirm a presumptive clinical diagnosis of canine borreliosis by laboratory results. Criteria for such a diagnosis were: history of tick exposure, lameness, neurological signs, nephropathy, lethargy, anorexia, and fever. A total of 302 samples comprising EDTA blood, urine, synovial fluid, cerebrospinal fluid, and tissue (skin, synovial membrane, kidney) from 98 dogs (26 with arthritis, 46 with neurological signs, 21 with nephropathy, 5 with non-specific symptoms) were collected and examined. Moreover, 55 healthy dogs (group B) and 236 dogs with symptoms or injuries unlikely to be associated with borreliosis (group C) were included in this study. Blood serum samples collected from all individuals (n=389) were analysed by ELISA. Twenty-one (21%) out of 98 dogs from group A, 4 (7%) out of 55 from group B and 15 (6%) out of 236 dogs from group C were positive for antibodies against B. burgdorferi sensu lato. The seroprevalences between groups A, B and C differed significantly. None of the corresponding samples investigated by PCR and culture were positive for spirochetal DNA or viable spirochetes. Borrelia afzelii was grown from one EDTA-blood sample but the corresponding blood serum sample remained antibody-negative. Consequently, the etiologic role of B. afzelii in this case is unclear. In approximately 40% of the presumptive canine borreliosis cases, other lesions have been found to be responsible for clinical signs. This study affirms that a definitive diagnosis of canine borreliosis cannot be made by clinical symptoms and serology based on a single consultation. Moreover, this study clearly revealed that the diagnostic sensitivity is enhanced by a thorough consideration and exclusion of other diseases