Bacterial strains and growth conditions
M. fermentans P-140 was isolated from the respiratory tract of asthmatic patient in our laboratory  and M. fermentans PG-18 was a kind gift of Dr. Gail H. Cassell (University of Alabama at Birmingham).
The strains were grown at 37°C in l liter of E broth consisting of 2.1% (wt/vol.) PPLO broth base (Difco), 20% (vol./vol.) horse serum, 0.002% (wt/vol.) phenol red, 0.25% (wt/vol.) glucose and 10% (vol./vol.) yeast extract.
Forty-nine New Zealand-White rabbits weighing 3 to 4 kg were used. The animal protocol complied with all relevant and institutional policies including the Helsinki Declaration. Blood and throat swabs of animals were checked for the genus mycoplasma by culture and PCR in order to determine that rabbits were mycoplasma free.
Arthritogenic ability of M. fermentans
The right knee joints of 14 rabbits were injected with 0.1 ml of a broth culture that contained 1 × 106 colony forming units per milliliter (CFU/ml) of Mycoplasma fermentans P-140. Also 14 rabbits were injected with 0.1 ml of broth cultures that contained 1 × 106 CFU/ml of Mycoplasma fermentans PG-18. The left joint received no treatment and was considered a control. As negative controls seven rabbits were injected with 0.1 ml of E broth (mycoplasma free) in the right knee joint.
An increase in the joint diameter was considered an indicator of joint swelling. The diameters were measured twice every third day during the experiment. Two rabbits of each experimental group and one from the control group were sacrificed on day 3,5,8,13,20,27 and 34 after the injection. Both the left and right synovia received the same treatment; the synovial membrane was divided in two parts. The presence of viable mycoplasmas was determined by inoculating one half of the synovial membrane in l ml of E broth and ten fold serial dilutions were also cultured an inoculated onto agar . Blood cultures were performed. The presence of aerobic flora in synovium was determined by plating sheep blood agar plates. Knee synovia were removed and immediately fixed in 10% formalin pH 7.0. Samples were embedded in paraffin, sectioned and stained with hematoxilin and eosin.
Arthritis induced by M. fermentans after tracheal injection
Fourteen New Zealand white rabbits were intracheally injected with 0.1 ml of a broth culture that contained 1 × 106 CFU/ml of Mycoplasma fermentans P-140. Valuation of joint swelling and sacrifice of the animals were performed in the same way as in the experiment about M. fermentans arthritogenic ability. Five milliliters of blood were extracted from heart and cultured before sacrifice. Animals were sacrificed by the injection of 5 ml of Anesthesal. Brain, trachea, lungs, heart, spleen, kidney and knee joints were extracted, 1 cm3 of tissue or half of the synovia were deposited in 0.9 ml of E broth and ten fold serial dilutions were also done . Tissue and synovial cultures, and histopathological study of the synovia were performed as described in the experiment of arthritogenic ability of M. fermentans.
A Polymerase Chain Reaction (PCR) test was used to confirm the presence of M. fermentans in synovial tissues. The oligonucleotide primers used for PCR detection were RW004 5'(GGACTATTGTCTAAACAATTTCCC) 3' and RW005 5'(GGTTATTCGATTTCTAAATCGCCT) 3' designed by Wang, which amplified a 206-nucleotide specific gene sequence within the insertion sequence-like element that exist in multiple copies only in the M. fermentans genome . Before we used this PCR based test to detect M. fermentans, we confirmed the specificity of these primers.
The reaction mixture contained 50 mM KCl, 1.5 mM MgCl2, 10 mM Tris-HCL (pH 8.3), 0.2 mM of each deoxynucleotide triphosphate, 6 μM of each primer and 1 unit of AmpliTaq® (Perkin Elmer Cetus, Emerville, CA.) in a total volume of 50 μl. The sample to be analyzed (5 μl) was always added last. A diluted lysate of M. fermentans PG-18 corresponding to 100 CCU and sterile water were used as positive and negative controls respectively. The amplification involved 40 cycles, each consisted of denaturation at 95°C for 25 s, primers annealing at 60°C for 60 s and extension at 72°C for 60 s. The amplified products were analyzed by electrophoresis in 2% agarose gels and visualized by UV light after ethidium bromide staining.
Many researchers use at least three animals for each time point for a better statistical and experimental reproduction. Although, animal models offer several advantages, one disadvantage is the cost. We used 49 rabbits in our study and it represented a considerable cost so we were not able to use a greater amount of animals. In previous studies where we used the rabbit model to test the arthritogenic ability of M. pneumoniae, M. pulmonis and M. arthritidis we injected 6 animals with each strain and we had a small standard deviations and small animal-animal variations. These facts gave us the confidence to use only 2 rabbits in each time point. However, the use of only two animals may lower the statistical power of the results. The Mann Whitney U-statistic was used to assess the statistical difference between articular diameters of M. fermentans PG-18 and P-140 and to compare the recovery of mycoplasmas in the knee synovial samples. The Kruskall-Wallis test was used to compare M. fermentans PG-18, P-140 intraarticular injected, P-140 intracheally injected and control.