Useimmat borrelioosiin sairastuneet saavat kuulla, että heillä ei voi olla enää borrelioosia sillä he ovat saaneet riittäviksi katsotut antibioottihoidot. Seuraavassa tutkimuksessa borrelioosia sairastaville koirille annettiin samoja antibiootteja joita käytetään myös ihmisille. Antibiootit kykenivät vähentämään bakteerien määrää mutta eivät poistamaan niitä kokonaisuudessaan.
Quantitative Spirochetal DNA in Dog Tissue
Reinard K. Straubinger, D.V.M., Ph.D. Reinard K. Straubinger, D.V.M., Dr. Met vet, Ph.D.
Cornell University, College of Veterinary Medicine James A. Baker Institute for Animal Health Hungerford Hill Road Ithaca, NY 14853
Background: B. burgdorferi is known to establish a persistent infection in the mammalian host. However, little is known about the number and the tissue preference of the spirochete. Quantification of B. burgdorferi organisms by culture or staining methods is either impossible or not practical. Current DNA amplification methods are labor intensive and only a limited number of samples can be processed.
Objective: Determine the absolute number of B. burgdorferi organisms by a new method of DNA amplification in a large number of canine tissue and buffy coat samples collected over a 500-day period.
Design: Sixteen specific-pathogen-free beagle dogs were infected with B. burgdorferi by tick challenge. Starting at day 120 after tick exposure, 12 dogs were treated with antibiotics for 30 consecutive days (4 dogs, azithromycin, 25 mg/kg daily, po; 4 dogs, ceftriaxone, 25 mg/kg, daily, iv; 4 dogs, doxycycline, 10 mg/kg, BID, po). The remaining four dogs received no antibiotic therapy. Over a 500-day period, blood samples were taken at two-week intervals and skin punch biopsy samples at monthly intervals. Twenty-five tissue samples were collected during necropsy. DNA of all samples was recovered by phenol/chloroform extraction. Detection and quantification of B. burgdorferi DNA was carried out in 96-well plates in an ABI7700 Sequence Detection System. Primers and a fluorescent-labeled probe designed to bind specifically to the ospA gene of B. burgdorferi strain N40 were used for DNA amplification and quantification. The intensity of fluorescent signal for test samples was compared to a standard curve, generated with DNA in a ten-fold dilution series of a known number of culture-derived low-passage B. burgdorferi organisms suspended in canine buffy coats.
Results: All 16 dogs became infected after tick challenge. In skin biopsy samples of untreated dogs, spirochete numbers peaked at day 60 post infection (< 4 ¥ 106 organisms per 100 ng extracted DNA) and decreased by 100- to 1000-fold during the following six months. Antibiotic treatment did not eliminate the infection, but reduced the number of spirochetes in skin tissue by a factor of 1000 or more. Buffy coat samples were rarely positive (1.6% of 576 buffy coat samples of all dogs). More than 500 days after infection, B. burgdorferi was detectable at low levels (102-104 organisms per 100 ng extracted DNA) in multiple tissue samples in all untreated dogs and in eight of 12 antibiotic-treated dogs. However, more tissue samples were positive in untreated dogs than in antibiotic-treated dogs.
Conclusions: This DNA detection system facilitates the precise and rapid quantification of B. burgdorferi in a large number of tissue and blood samples. Data generated with this technique provide evidence that during the first two months after infection the number of spirochetes increased in the skin of infected dogs and declined thereafter. Interestingly, peak numbers of organisms coincided with the development of the first clinical signs. Antibiotic treatment did not eliminate the infection but decreased the number of organisms.