Jpn. J. Infect. Dis., 54, 2001

Laboratory and Epidemiology Communications

Detection of Anti‑Coxiella burnetii Antibodies by ImmunofluorescentTechnique and Enzyme‑Linked Immunosorbent Assay
Yumiko Furuya*, Takashi Katayama, Miyuki Hara, Yoshiya Yoshida, Mitsunobu Imai,
Yasutomo Arashima. , Kimitoshi Katol , Motohiko Ogawa2 and Toshikatsu Hagiwara2
Kanagawa Prefectural Public Health Laboratory,
Nakao 1‑1‑1, Asahi‑ku, Yokohama, Kanagawa 241‑0815,

JNihon UniversiO, School ofMedicine,Oyaguchi‑Kamimachi 30‑1, Itabashi‑ku, Tokyo 1 7310032 and2National Institute of lnfectious Diseases,

Toyama 1‑23‑1, Shinjuku‑ku, Too)0 162‑8640
Communicated by Kunihiko Masukawa(Accepted April 17, 2001)

Q fever is caused by a zoonotic pathogen, Coxiella bumetii.
Infected humans exhibit innuenza‑like resplratOry Symptoms.
The antibody‑positive population is high among animal farm

Table 1. Detection ofanti‑C. burnetii antibody byIFl and ELISA2

workers including veterinary doctors ( 1). It was suggested

lF titer negative positive

ELISA (phase II)that some patients diagnosed as having atyplCal pneumonia< 1 1 つJ 6or other resplratOry infectionsactually have C. burnetii′0 ′ムU 2 430

つJ 4 /ムU 1tJ I 0infections (2,3).

This report deals with the detection of anti‑C. burnetii
antibodies among 48 patients with resplratOry infections. All
the patients showed prolonged fever, cough and resplratOry'IF was perfTormed by using C. burneEii Nine Mile strain phase II cul‑

excretion with unknown etiology. Though anti‑C. burnetii
antibody isusually detected by meansof the immunofluores‑

tured in Vero cells as an antigen. The suspension of the cultured Cburnetii was spotted on a slideglass. Sera were serially diluted by PBSO

cent antibody technique (IF) employing phase II antigen (4),2‑fold and spotted ontothe antlgCn On the slide glass. After incubationfor 1 h, the shdc glass was dried, overlaid withFITC‑Iabeledanti‑humanIgG (Biosource lntemational, lnc., Tago products, Camarille, Calif.,

this method requlreS expertise. Therefore, we performed theIF in combination with ELISA by uslng phase I and phase IIUSA), incubated fわr 40 min at 37oC, washed with PBS(‑), dried, andantlgenS.

then examined using a f一uorescent microscope.

Positive control serum from a convalescent Q fever patient

2ELISA wasperformcd by uslng Phase I and phase II C. burnetii Nine(5) showed a titer of x512 in IF and X16,000 in ELISA (6)for both phase 1 and II antlgenS. C. burnetii TK‑1 strain wasisolated &om the patient.

ml were distributed in a volume of 100 JLl into each welt of961WelI

Three among seven sera with IF titers x16 were negative

were diluted seriany two‑foldwithPBS(‑) containing 0.05% Tween

Mile strain as antigcns (6). The antigens at concentrations of 50 ′上g/micro‑plates (Greiner Labortcchnik Ltd., Frickcnhausen, Germany) andsolidified. Two percent skim milk was used as a blocking rcagent. Sera

as detemined by ELISA. However, among eight sera with

20 and 0.1% bovine serum albumin (PBS‑TB). An aliquot or 100 ′上t

was added to each well. The plates were incubated for 2.5 h at 37oC.

lF titers higher than x32, Only one was negative (Table 1).

Aftcr 3min ofwashingwithPBS‑TB three times, peroxidase‑labeledlF titer X 16 0r lower was probably due to non‑speciBc reaction

anti‑human lgG (Biosourcc International) was added in a volume of
100 LEI and incubated for 1 h at 37oC. A触r 3 min ofwashingwith PBS‑

or to cross‑reaction. Actually, it has been陀pOrtedthat Bartonella

TB three times,the color developing agent (1 mg of3･31,5･5‑tetramethyl‑or Legionella antlgenS Cross‑reacted with C. burnetii antigen

benzidine, 9 ml of phosphate‑cltrate buffer, and 2 LEI of 30% H202)(7,8). Relatively higher titers, >×32, probably reflect speciBc

was added in a volume or 100 〟.I and incubated fわr 15 min at room

reactions because the IF and ELISA data matched well.

tcmpcraturc. The reaction was stopped by adding 50 fLI 4N H2SO4.
The absorbance at 450 nm was measured. An absorbancc higherthan

Positivlty tended to be higher fわr phase ll antlgen than fわr

2. 1 ‑fold or more rclativc tothe negative control wasjudged as a positive

phase I antigen (data not shown).

indication

ELISA‑positive sera with lF titers higher than x32 WerePeen in 7 of48 (14･6%) respiratory infections(Table 1 ), while
lt W.aP Seen in 1 of200 (0･5%) in a healthy population･ Theand chronic resplratOry infections.

posltlVe rate Was about 301fold higher in the resplratOry

infections. This may indicate the involvement of C. burnetii

Phase I and Phase lI C. burnetii (Nine Mile strain) werein resplratOry infections. However, the involvement of otherobtained from Dr. E. Kovacova, Czechoslovakia Academy

antlgen‑Cross‑reactive pathogens could not be excluded.

or Science.

Further studies with paired sera from more patients will beneeded for elucidating the causal relation between C. burnetii

REFERENCES*Corresponding author: Tel: +81 ‑45‑36311030, Fax: +8 1 145‑363‑

1. Derrick, E. H. (1973): The course of infection withCoxiella burnetii. Med. J. Aust., 1, 1051‑1057.

1037

75

reactlOnS between Coxiella burnetii and Legionella

nuorescence. Clin. Diagn. Lab. Immuno1., I, 189‑196･
5. Oda, H. andYoshie, K. (1989): Isolation ofa Coxiellamicdadei. Clin. Diagn. Lab.lmmuno1., 4, 2081212.

76