Veterinary Drug Assay Laboratory
(Balai Besar Pengujian Mutu dan Sertifikasi Obat hewan)
Gunung sindur, Bogor 16340
Key words: Chlamydiaceae, Chlamydophila, multiplex PCR
Kata kunci : Chlamydiaceae, Chlamydophila, PCR multipleks
Chlamydophila causes a wide variety of diseases in animals and humans (Fukushiand Hirai, 1992; Fukushi and Hirai, 1993a; Grayston et al., 1989; Moulder et al., 1984). The genetic diversity of Chlamydophila is well recognized by chromosomal DNA finger printings (Fukushi and Hirai, 1989), restriction fragment length polymorphism (RFLP) of rRNA gene loci (Fukushi and Hirai, 1993b), RFLP of ompA and groEL genes (Fukushi and Hirai, 1994), and random amplification of polymorphic DNA (RAPD) analysis (Pudjiatmoko et at., 1996). Chlamydophila classified into several genetic groups based on the ompA gene sequence (Kaltenboeck et at., 1993). The previous phylogenetic analysis of the genus Chlamydophila based on 16S rRNA gene sequences showed 6 genetic groups (Pudjiatmoko et aI., 1997). In 1999 the genus Chlamydophila was classified into 6 species: Chlamydophila psittaci, C. felis, C. caviae, C. abortus, C. pecorum and C. pneumoniae (Everett et aI., 1999). A practical method for genotyping Chlamydophila is not available yet.
Chlamydophila strains used in this study included 5 strains (Frt-Hu/Cal 10, Hu/Borg, Prk/6BCT, Prk/ Daruma, Prt/GCPI) of C. psittaci, 1 strain (Gp/Ie) of C. caviae, 7 strains (Fe/Pnl, Fe/ 145, Fe/Cello, Fe/B 166, Fe/C 164, Fe/C429, Fe/C454) of C. felis, 5 strains (Bo/ESST, Bo/Maeda, Bo/Shizuoka, Koala (type 2), Ov/IPA) of C. pecorum, 1 strain (TW/83T) of C. pneumoniae and 6 strains (A/Har-l T, B/TW-5/0T, C/ TW-3/0T, DfUW-3/Cx, EfUW-5/Cx, L2/434/Bu) of C. trachomatis (Pudjiatmoko et aI., 1997). To confirm the specificity of PCR, the following agents were tested: Staphylococcus aureus, Streptococcus pneumoniae (Gifu 8766 strain), Bordetella bronchoseptica (Gifu 1127 strain), Mycoplasma pneumoniae, Escherichia coli (C600 strain), Haemophilus influenzae (Gifu 3191 strain), Klebsiella pneumoniae (Gifu 2929 strain), Leginella pneumophila (SL94-1 strain), Leginella pneumophila (SL94-2 strain), Coxiella burnetii (Nine Mile ATCC YR 615 strain), Orientia tsutsugamushi (Gilliam Strain), Orientia tsutsugamushi (Karp strain) and Orientia tsutsugamushi (Kato strain). These strains were kindly provided by Dr. T. Ezaki of Gifu University, Japan and Dr. A. Tamura of Niigata College of Pharmacy, Japan.
Procedures for preparation of Chlamydophila DNA have been described previously (Fukushi and Hirai, 1989). Extraction of chlamydial DNA from throat swabs, sera and fecal samples was performed according to the protocol of Higuchi (Higuchi, 1989). Briefly, all the samples were suspended in 1 ml of PBS in a 1.5 ml Eppendorf micro centrifuge tube, and centrifuged at 13,000 x g for 20 sec. The pellet was resuspended in 1 ml of washing buffer (10 mM TrisHCI (pH 8.3), 50 mM KCI, 1.5 mM MgC12), and centrifuged at 13,000 x g for 20 sec. The pellet was resuspended in 1 ml lysis buffer (0.32 M sucrose, 10 mM tris-HCI (pH 7.5), 1 % Triton X-lOO), and centrifuged at 13,000 x g for 20 sec. DNA was released from the pellet by adding PCR buffer with nonionic detergents (50 mM KCI, 10 mM Tris-HCI (pH 8.3), 2.5 mM MgCI2, 0.1 mg/mi gelatin, 0.45 % NP40 and 0.45 % Tween 20), and proteinase K (120 pg/ml) to the washed pellet. The mixture was incubated for 1 h at 55° C, boiled for 10 min, and then chilled on ice.
PCR amplification of clinical samples
Chlamydopila 16S rRNA genes were amplified by using a pair of 16SA and 16SB primers in the first PCR. The PCR was performed with 5 ul of throat swab, fecal, cell culture samples or chromosomal DNA of Chlamydopila extracts in a total volume of 50 ul. Five ul of the first PCR product was submitted to the nested PCR using a pair of family- and species-specific DNA primer. The final reaction mixture contained 40 pmol of each primer, 10 mM Tris-HCl (pH 8.3), 50 mM KC1, 1.0-2.0 mM MgCI2, 200 pM each of dATP, dCTP, dGTP, and dTTP, 0.01 % gelatin, and 2.5 units of Taq polymerase (Takara Shuzo Co., Ltd., Kyoto, Japan) in 50 ul. Concentration of MgCl2 was 1.0 mM for ChPsA-ChPsB, ChFeA-CbPsB and ChTrA-ChTrB primers, 1.5 mM for 16SA-16SB, ChA-ChB, and ChPnA-ChPnB primers, and 2.0 mM for ChPeAChPeB primers. The components and procedure for the multiplex PCR were described above except 2 sense primers for C. felis (ChFeA) and C. psittaci (ChPsA) and an antisense primer for C. psittaci (ChPsB) was used in the method. The thermal cycle amplification program for the first PCR was as follows: initial melting at 94°C for 3 min and then 35 cycles with denaturation at 94°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 2 min and the last extension for 7 min follow by holding at 4°C. The program for nested PCR was as follows: initial melting at 94°C for 3 min and then 35 cycles with denaturation at 94°C for 30 see, annealing at 55°C for 30 sec and extension at 72°C for 2 min and the last extension for 7 min follow by holding at 4 C. Five ul PCR products were electrophoresed in 2 % agarose gel. DNA was stained with ethidium bromide and visualized by UV light.
Table 2. Specificity of peR test of 5 species-spesific primer sets of Chlamydophila
Specificity of PCR amplification
To simplify PCR reaction, co-amplification was carried out by simultaneous use of three primers (ChFeA, ChPsA and ChPsB) ina single tube. In this reaction, the ChFeA-ChPsB primer pair produced about 410 bp of the fragment of , C. felis strains, while ChPsA-ChPsB primer pair produced about 250 bp fragment of C. psittaci strains (Figure 4). These primer pairs produced two previously identified fragments with C. caviae strain. However, Chlamydophila strains from other genetic groups did not produce any PCR products.
Sensitivity of PCR amplification
Direct detection of Chlamydophila from clinical specimens
After the specificity and sensitivity of the PCR method were determined, the evaluation of direct detection and genotyping of Chlamydophila from clinical samples was done simultaneously. Chlamydophila organisms were detected by multiplex PCR in 1 liver specimen from 10 parakeets, 2 conjunctival swabs from 11 cats, 2 fecal specimens from 33 crows, and 5 throat swabs from 140 humans. PCR results in parakeets and cats were confirmed by isolation. Isolation was not attempted with crow and human specimens. The species o~ these ten positive PCR amplified DNAs were determined to be a C. psittaci from parakeet, two C. psittaci from crows, a C. psittaci from human, a C. felis from cat, a C. felis from parakeet, and four C. felis from humans. The genotype of isolates from parakeets and cats confirmed the PCR typing results.
The PCR using primers targeted to the 16S rRNA gene were developed for direct identification of Chlamydophila spp. in the present study. The specificity of the amplifications was confirmed by sequencing of DNA from PCR amplified products and isolates from clinical samples. Using a primer set for the family Chlamydiaceae in combination with five primer sets for the existing species C. caviae, C. felis, C. psittaci, C. pecorum, C. pneumoniae, and C. trachomatis, each designated chlamydial species was able to be differentiated. The method was applied for detection and direct typing of Chlamydophila for clinical samples from humans and animals. The results of the study demonstrated that this method may be useful for laboratory diagnosis.
The sensitivity of our method that detect 1 to 50 IFU or 5 to 50 fg is equivalent to other method that use the MOMP gene (Campbell et al., 1992; Valassina et al., 1995), l6S rRNA gene (Gaydos et al., 1992) and plasmid (Valassina et al., 1995). The PCR with species-specific primer pairs could identified species of Chlamydophila. However, no amplification was observed with other bacteria in both of the PCR. The results indicated that the PCR methods revealed specific to detect family Chlamydiaceae and identify genetic groups of Chlamydophila.
Currently 3 genetic groups are recognized for C. Caviae, C. felis and C. psittaci (Pudjiatmoko et al., 1997; Everett et aI., 1999). Differentiation ofthese 3 genetic groups requires 6 primers (3 sense and 3 antisense primers) using 3 sets of tubes. In method of this study, PCR was carried out using three primers (two sense primers and an antisense primer) in a single tube for both detect and identify species C. caviae, C. felis, and C. psittaci simultaneously. This multiplex PCR method uses less reagent, time and labor. The multiplex PCR technique appears to be suitable method for a rapid detection and genotyping of Chlamydophila isolates.
No case of feline chlamydiosis in human has been reported in Japan. It is also rare in other countries. A human case of feline pneumonitis was reported in 1969 (Schachter et al., 1969). Our study found 4 out of 140 high school children have feline pneumonitis. This result indicate that transmission of feline pneumonitis from cats to human is not rare and infection is often subclinical.
Thomas, R., H.C. Davison and AI. Wilsmore. 1990.
Valassina, M., M.G. Cusi, D. Corsaro, C. Buffi, G.