Prevalence of antibodies against Simkania negevensis in a healthy ...

FEMS Immunology and Medical Microbiology 43 (2005) 21–27 www.fems-microbiology.org

Prevalence of antibodies against Simkania negevensis in a healthy Japanese population determined by the microimmunofluorescence test Tetsuya Yamaguchi a,b,*, Tsutomu Yamazaki b,c,d, Miyuki Inoue c, Chikako Mashida e, Kiyotaka Kawagoe e, Motohiko Ogawa b, Sadashi Shiga b, Yuichi Nakagawa a, Toshio Kishimoto b, Ichiro Kurane b, Kazunobu Ouchi f,g, Takehiko Ohzeki a a

d

Department of Pediatrics, Hamamatsu University School of Medicine, Hamamatsu, Japan b Department of Virology I, National Institute of Infectious Diseases, Tokyo, Japan c Department of Pediatrics, Saitama Medical School, Saitama, Japan Department of Infectious Diseases and Infection Control, Saitama Medical School, Saitama, Japan e Hitachi Chemical Co., Ltd. Hitachi, Japan f Department of Pediatrics, Kawasaki Medical School, Kurashiki, Japan g Department of Pediatrics, Saiseikai Shimonoseki General Hospital, Shimonoseki, Japan Received 29 February 2004; received in revised form 27 June 2004; accepted 30 June 2004 First published online 24 July 2004

Abstract Simkania negevensis has been associated with bronchiolitis in infants and community-acquired pneumonia in adults. Reports of exposure to this microorganism are only available from Israel, North America and Western Europe. Currently, no standard method for diagnosis of S. negevensis infection has been established nor have prevalence rates been shown in Japan. For the first time we demonstrated the ability of the microimmunofluorescence (MIF) test to detect S. negevensis-specific immunoglobulin G and exposure to S. negevensis in Japan. The positive rate in healthy volunteers was 4.3% (25/588), with rates increasing with age. Results indicate the usefulness of the MIF test as a serological method for detecting S. negevensis-specific antibodies. A standard serological test for infection with S. negevensis is needed. 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Simkania negevensis; Serological test; Indirect immunofluorescence antibody test; Antibody specificity

1. Introduction Simkania negevensis, an obligate intracellular gramnegative bacterium [1], now belongs to the new family Simkaniaceae in the order Chlamydiales. This microorganism has been associated with bronchiolitis in infants [2] and community-acquired pneumonia in adults [3]. *

Corresponding author. Tel.: +81-53-435-2312; fax: +81-53-4352311. E-mail address: [email protected] (T. Yamaguchi).

Reports of exposure to this microorganism are available from various parts of the world, such as Israel, North America, and Western Europe [4–6]. However, to date, no report is available from East Asia, including Japan. Evidence of infection with S. negevensis has been supported by culture, polymerase chain reaction (PCR), and in-house enzyme-linked immunosorbent assay (ELISA) [2–5]. But no standard method for diagnosis of S. negevensis infection has been established. Concerning members of the Chlamydiaceae closely related to S. negevensis, a laboratory method for diagnosis of infection

0928-8244/$22.00 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsim.2004.06.023

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T. Yamaguchi et al. / FEMS Immunology and Medical Microbiology 43 (2005) 21–27

has relied mainly on serological analysis [7]. At present, the gold standard of serological testing for chlamydial infection is the microimmunofluorescence (MIF) test [7,8]. But, prior to our study reported here, the MIF test had not been demonstrated to possess the ability to detect S. negevensis antibodies. In the present study, the first objective was to evaluate the ability of the MIF test to detect S. negevensisspecific IgG. The second was to clarify the exposure to S. negevensis in Japan, as has already been shown in Israel, North America and Western Europe, and establish the age distribution of S. negevensis IgG in a healthy Japanese population.

2. Materials and methods 2.1. Samples Sera were sampled from 588 healthy Japanese volunteers who lived in Shimonoseki City, located in the western part of Japan. Of the 588 volunteers, 296 were 19 years old or younger (male 150, female 146), 95 were from 20 to 39 years of age (male 48, female 47), 77 were from 40 to 59 years old (male 37, female 40), 80 were aged 60–79 years (male 40, female 40), and 40 were 80 years of age or older (male 20, female 20). None had a heart or respiratory disease before serum sampling. Samples were kept frozen at 30 C until use. The research protocol was approved by the Human Subjects Committee at Saiseikai Shimonoseki General Hospital. All volunteers gave their consent to participate in the study. 2.2. Control serum of S. negevensis Positive and negative control sera of S. negevensis were kindly provided by Dr. Maureen G. Friedman (Department of Virology, Faculty of Health Sciences, Ben Gurion University of the Negev). 2.3. Antigens Simkania negevensis Z (American Type Culture Collection VR-1471), and Chlamydophila pneumoniae (C. pneumoniae) AR-39 (kindly provided by Prof. C.-C. Kuo, Department of Pathobiology, University of Washington) were used. 2.4. Culture of S. negevensis Culturing of S. negevensis was performed by a modification of the method of Kahane et al. [9]. Vero cells, kindly provided by Dr. S. Saika (Chiba Prefectural Institute of Public Health), were seeded in 24-well tissue culture plates containing round coverslips for confirming growth of S. negevensis and 75 cm2 culture flasks

for preparation of S. negevensis as antigens. RPMI 1640 medium (Gibco Invitrogen, Tokyo, Japan) supplemented with 10% fetal calf serum (FCS) and 0.2% sodium bicarbonate was used. Monolayers of Vero cells grown for 2 days at 37 C with 5% CO2 in a humidified cabinet were examined for confluency. After removal of the culture medium, microorganisms suspended in sucrose–phosphate–glutamate (SPG) medium (sucrose, 75 g; KH2PO4, 0.52 g; NaHPO4, 1.22 g; glutamic acid, 0.72 g; H2O to 1 liter; pH 7.4 to 7.6) were inoculated to monolayers, and cell cultures were incubated for 120 min at 37 C with 5% CO2 in a humidified cabinet. At the halfway point in the inoculation period, cultures were rocked once gently. After removal of inocula, they were grown at 37 C with 5% CO2 in a humidified cabinet for 5 more days with Ôinoculation mediumÕ consisting of RPMI 1640 supplemented with 10% FCS, 100 lg streptomycin ml1, 100 lg vancomycin ml1and 1.0 lg cycloheximide ml1. 2.5. Indirect fluorescent-antibody staining of S. negevensis inclusions After incubation for 5 days, coverslips were fixed for 10 min in methanol. They were then incubated with rabbit polyclonal hyper-immune sera raised against S. negevensis for 1 h at 37 C, followed by washing and staining with mouse anti-rabbit fluorescein isothiocyanate (FITC)-conjugated monoclonal IgG (F 4151; Sigma–Aldrich Japan KK, Tokyo, Japan) for 1 h at 37 C. 2.6. Preparation of S. negevensis antigens When more than 80% of Vero cells were infected with S. negevensis confirmed by inclusion staining, they were harvested and suspended in SPG medium. After mild sonication, the suspension was subjected to one cycle of differential centrifugation (130g for 10 min and 28,000g for 30 min). The precipitate was suspended in phosphate-buffer saline (PBS), pH 7.2, at 10 ml. This suspension was put onto 10 ml of 30% Urografin and centrifuged at 50,000g and 4 C for 30 min. The precipitate was washed with PBS twice and suspended in PBS; one portion was treated with 0.02% formalin for an antigen and another part was used to determine the number of organisms. To avoid inter-slide variations in the MIF test, the density of organisms to be used as test antigens was made to be approximately 6 · 109 organisms per ml. 2.7. Preparation of C. pneumoniae antigens Culturing of C. pneumoniae was done according to the method by Kuo and co-workers [10]. C. pneumoniae antigens were prepared using the method of Wang et al. [11] with slight modifications.


2.8. Microimmunofluorescence test The MIF test was performed by a slight modification of the method of Wang and Grayston [8,12]. Each of the antigens was mixed in an equal volume with 3% normal yolk sac suspension and dotted onto a slide. The slides were dried for 60 min, fixed with acetone for 30 min at room temperature, dried and stored at 80 C until use. Serial twofold dilutions of sera were tested, beginning with a dilution of 1:8. The slides were placed in a humid box for 60 min at 37 C, then washed in PBS and distilled water three times, respectively. After drying, they were again incubated with goat anti-human FITC-conjugated IgG (F 0132; Sigma) diluted 1:20 in PBS, washed, and dried as above. The slides were read by using a fluorescence microscope with high-power (400·) magnification. The endpoint titer was defined by the highest serum dilution with unequivocally positive fluorescence. The MIF test was performed independently four times by the same three investigators, and a positive interpretation was declared when the investigators agreed each time the test was performed. The cutoff for positivity in the MIF test was a dilution of 1:8. 2.9. Adsorption of positive sera To evaluate cross-reacting antigens, positive sera of S. negevensis were adsorbed with either S. negevensis or C. pneumoniae and then tested by MIF. Antigen concentrations were determined by the Bio-Rad Protein Assay (Nippon Bio-Rad Laboratories, Tokyo, Japan), with bovine serum albumin (BSA) as a standard after treated with formalin. Each purified antigen was diluted in PBS to a final concentration of 0.45 mg ml1. After the addition of 60 ll of PBS, 20 ll of serum sample and 80 ll of the antigen dilution were mixed. The mixture was incubated for 48 h at 4 C and centrifuged at 15,000g for 30 min. The supernatant was collected at a final dilution of 1:8.

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with TBS-1% Triton X-100 (five 5-min periods), membranes were incubated with 2.0 lg ml1 horseradish peroxidase labeled goat anti-human IgG (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD) for 1 h at room temperature, and washed with TBS-1% Triton X-100 again (five 5-min periods). Color development was observed upon the addition of 3, 3 0 , 5, 5 0 -tetramethylbenzidine (Kirkegaard and Perry Laboratories) and stopped by rinsing the blots in distilled water.

3. Results 3.1. Microimmunofluorescence test Serum samples from 588 healthy volunteers were tested for S. negevensis-specific IgG. Fig. 1 shows a positive fluorescence reaction of the MIF test for S. negevensis obtained using a serum sample. The immunofluorescence pattern associated with evenly distributed organisms was similar to that of a chlamydial-positive reaction. The pattern was also the same as that of the positive fluorescent reaction that we obtained by the MIF test on the control serum provided by Dr. Friedman; previously, that serum had been shown by another method to be positive.

2.10. Western blot analysis Antigens (1.0 lg per lane) were separated by gradient precast sodium dodecyl sulfate–polyacrylamide gel electrophoresis (4–20%, PAG mini Daiichi 4/20, Daiichi Pure Chemical Co., Ltd., Tokyo, Japan) according to Laemmli [13]. Molecular masses were determined by using the protein standard (Protein Marker Daiichi II, Daiichi Pure Chemical). After separation, proteins were transferred electrophoretically to 0.45-lm-pore-size nitrocellulose membranes (Bio-Rad) for Western blotting according to Towbin et al. [14]. Following transfer, membranes were rocked overnight at 4 C in 5% BSA in Tris-buffered saline (TBS) for blocking. Membranes were then washed with TBS-1% Triton X-100 (twice for 5 min each time) and incubated for 2 h at room temperature with tested sera diluted 1:200 in 0.2% BSA in TBS. After being washed

Fig. 1. Positive immunofluorescence reaction of the MIF test for S. negevensis obtained from a serum sample in this study. Fluorescence associated with evenly distributed organisms of S. negevensis was quite similar to that of a chlamydial-positive reaction. 30%-Urografin purified S. negevensis strain Z was used as the antigen for the MIF test prepared as described in materials and methods. Antigen was incubated with sample serum for 1 h at 37 C, followed by staining with goat anti-human FITC-conjugated IgG (400·).

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25 positive samples had an IgG titer