158

The Use of PCR in Detecting Toxoplasma Parasites in the

Blood and Brains of Mice Experimentally

Infected with Toxoplasma gondii

Hiroshi SHIBATA", Shiba Kumar RAI2), Miyuki SATOH",

Keiko MURAKOSOn, Katsumi SUMP, Syoji UGA3),

Takeo MATSUMURA3) and Akira MATSUOKAn1)Department of Clinical Pathology, Hyogo College of Medicine

2)Department of Pathology, Institute of Medicine , Tribhuvan University Teaching Hospital3) Department of Medical Zoology, Kobe University

(Received: August 25, 1994)

(Accepted: October 27, 1994)

Key words: PCR, mice blood, mice brain , toxoplasmosis

Abstract

Polymerase chain reaction (PCR) has been extensively used for diagnosis recently because of itsvery high sensitivity and specificity . We studied the applicability of PCR to the early diagnosis oftoxoplasmosis in a murine model orally infected with Toxoplasma gondii (S-273) . PCR was performedusing EH24 and HE27 primers synthesized by the phosphoramidite method . Mice blood and brainscollected on various post infection days (PID) were analysed by PCR (35 cycles) . A portion of the braintissue from each mouse was examined microscopically for the presence of parasite cysts . Blood andbrain PCR were positive on the 9th and 12th day post-infection (DPI) . Toxoplasma cysts in braintissue appeared only on the 18th PID. The results showed that Toxoplsma parasites can be detected

earlier in the blood than in the brain during primary infection , indicating that blood PCR is the moreuseful procedure.

Introduction

Toxoplasma gondii causes various types of clinical syndromes in man including blindness,

congenital disorders, abortion and fatal toxoplasmic encephalitis (TE) . Lifethreatening infectionoccurs primarily among immunocompromised patients . Thus, T gondii is one of the most commonopportunistic pathogens that cause TE among patients with acquired immunodeficiency syndrome

(AIDSP-3', organ transplant recipients4,5) and others6m. An outbreak of toxoplasmic encephalitis hasalso been reported in Westren Hemisphere which is attributed to the spread of HIV infection andAIDS8'. TE in immunocompromised patients reportedly results from reactivation of latent infection9) .

The diagnosis of toxoplasmosis in immunocompromised patients is problematic . Serologicalmethods are not helpful because either no antibody is produced or it is delayed") . The isolation ofparasites by tissue culture or mouse inoculation are either insensitive or time consuming . Toovercome this problem, various investigators have used polymerase chain reaction (PCR) to diagnose

toxoplasmosis in different clinical samples11)-16) using various primers'347) PCR is reportedly verysensitive and specific for the diagnosis of toxoplasmosis"--17) . We evaluated the use of PCR in

Correspondence to: Hiroshi SHIBATA

Department of Clinical Pathology, Hyogo College of Medicine Hospital , 1-1, Mukogawa, Nishinomiya, 663 Japan

感染症学雑誌 第69巻 第2号

Use of PCR in Toxoplasma Parasite Detection 159

detecting the Toxoplasma parasite in the blood and brains of experimentally infected mice on various

post infection days (PID) along with the microscopic detection of parasitic cysts in brain tissues.

Materials and Methods

Mice and inoculation: Ten female ICR mice (Nippon CLEA Company, Japan) aged 4-5 weeks

were infected with T gondii (S-273) bradyzoites obtained from previously infected mice brains.

Briefly, mice brains containing approximately 20 cysts/4 mg brain tissue were homogenized in 5 mlof minimum essential medium (MEM) and mice were fed with 0.1 ml of the brain homogenate. The

infection day was designated as day 0.

Blood and brain samples: Blood and brain samples were obtained by sacrificing one infected

mouse on PID 2, 4, 6, 9, 12, 15, 18, 21, 24 and 27. The mice were killed by ether inhalation and the

abdomen was immediately exposed. Blood was collected from the abdominal aorta using a 1.0 mlsyringe. About 1.0 ml blood was collected from each of the mice, of which 0.6 ml was mixed with

ethylene diamine tetra-acetic acid (EDTA) anticoagulant and the remainder was tranferred to

Microtainers (45 X 8 mm) (Becton Dickinson, USA) for serum separation. Subsequently, the whole

brain was removed by opening the skull.DNA extraction: DNA from blood and brain tissue was extracted as follows. A portion of brain

tissue measuring about 2 mm3 was placed in a micro test tube (Eppendorf, Germany) and minced using

surgical blades. The minced tissue was washed in 300 ,ul of Tris-NaCl-MgC12 (TNM) buffer

centrifuged at 550 g. Then washed twice in Tris-NaCl-EDTA (TNE) buffer. The tissue precipitatewas resuspnded in 400 ,u 1, into which 3 ,u 1 of 20% SDS and 30 ,u1 of proteinase K (10 mg/ml) were

added. The mixture was agitated at room temperature until the tissue was completely digested. Sepa

Gene Reagent III (700 ,u1) and 400 ,u1 of Reagent IV (Sanko Junyaku Co. Ltd., Japan) were added tothe digest and mixed vigorously followed by centrifugation at 13,835 g for 10 min at 4C. The clear

supernatant was transferred into a micro test tube and 50 /21 Sepa Gene Reagent V and 500, u1

isopropanol were added followed by gentle mixing. After centrifugation at 13,835 g, the DNA

precipitate was washed twice in 500 ,u1 of 75% ethanol. The DNA was dried at 65C in thermal cycler(ASTEC Co., Japan) for 10 min, then dissolved in 100 ,ul of Tris-EDTA buffer heating at 70C for 15min.

Blood (500 ,ul) was mixed with 2.0 ml of haemolysin reagent (Tris-Sucrose-Triton X100) and

washed three times in the same reagent. EDTA-saline (2.0 ml), 200 ,u1 of 10% SDS and 100 ,u1 of

proteinase K were added to the precipitate and agitated until it was completely dissolved. Phenol-saturated Tris-EDTA buffer (2.0 ml) was added to this mixture, shaken vigorously, then separatedby centrifugation at 13,835 g. The supernatant was mixed with 2.0 ml of phenol-chloroform-isoamyl

alcohol for 15 min then clarified by centrifugation at 13,835 g.Primer: The primers Guay et al.17) reported were 5'-GGCACAACGAGCGCCACGC-3' (EH24) and

5'-TCATGAAGATCTGTTCATT-3' (HE27), were synthesized by phosphoramidite method.

PCR: The PCR mixture (95, u1) (Distilled water 64.75 ,u1, PCR Reagent buffer 10 ,u1, MgCl2 10, u1,10 mM dATP 2 ,u1, 10 mM dCTP 2/21, 10 mM dGTP 2, u 1, 10 mM dTTP 2, u1, primer EH24 1, u1, primer

HE27 1 ,u1, and DNA Taq polymerase 0.25 Ail) (Perkin Elmer Cetus, USA) and 5.0 ,u1 of DNA extract

were mixed and overlayed with a drop of mineral oil (Sigma Chemical Co., USA) and thirtyvive cycles

amplification were performed in PCR thermocycler (PC-700 ASTEC Co., Japan) along with positive

and negative controls.PCR product: The PCR product was mixed with loading buffer (4:1) and resolved by electrophor-

esis (Advance Co., Ltd., Japan: Mupid O2Z1) in 2% ultra pure agar (Life Technologies Inc., USA) geland Tris-Borate-EDTA (TBE) buffer at 100 V for 25 min along with molecular weight markers

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160 Hiroshi SHIBATA et al

(Toyobo Co., Ltd. Japan). The gel was then stained in ethidium bromide, visualised under UV light

(Mini Transluminator NTM-10, ASTEC Co., Japan) and photographed using polaroid camera (DS-300camera, FUNAKOSHI Co., Japan).

Results

Of the ten mice blood samples analysed by PCR, Toxoplasma gondii parasites were detected only

in the blood from PID 9 and not before or thereafter (Fig. IA). Brain tissue PCR revealed Toxoplasma

parasites from PID 12 until PID 27 (Fig. 1B). Direct microscopic examination of brain tissue revealedToxoplasma cysts only from PID 18 (Fig. 2) and thereafter. The size of the cysts varied from 15 to 35

,um. The parasite was detected in the blood by PCR three days earlier than in the brain tissue PCRand 9 days before cysts were microscopically detectable. Toxoplasma antibodies appeared from PID12 and remained thereafter (Fig. 3).

A: BloodPost infection days

B: BrainPost infection days

Fig. 1 Agarose gel (2.0 1,) electrophoresis of nucleic acid PCR products from

mice (A) blod sampli and (B) brain samples collected at various PID with

EH24 and HE27 primers. (A) MM: Molecular markers , 3 to 18: blood PCR andPc: Positive control. (B) MM: Molecular markers, 3 to 18 brain tissue samples

PCR and Pc: Positive control (497 bp).

Fig. 2 Mice brain wet preparation showing Texo-

plasma gondii (S273) cysts (x20) in light micros-copy.

感染症学雑誌 第69巻 第2号

Use of PCR in Toxoplasma Parasite Detection 161

Fig. 3 Comparision of findings of blood and brain tissue PCR, Direct micro-scopic examination of brain tissue and antibody detection in mice infected withT gondii (S-273) at various PID.

Discussion

In this study, we evaluated the use of PCR in detecting Toxoplasma parasite in blood and brain

tissue of experimentaly infected mice at various PID. The PCR findings were also compared with

direct microscopic examination of brain tissue for Toxoplasma cysts. We detected the parasite in the

blood earlier by PCR (on PID 9) than in brain tissue (on PID 12 and thereafter). PCR revealed that

the blood however, was positive only on PID 9 and not thereafter. This finding correlated with the

natural course of primary infection, where the parasite disseminates through bloodstream, causing

transient parasitemia before they localize in various tissues. However, the appearance of blood

parasitemia in primary infection differs among parasite strains and routes of infection. Weiss et al 18

detected Toxoplasma parasites by Southern blot hybridization within five days of infection in mice

peritoneally infected with the RH strain of T. gondii. Early detection of blood parasitemia in their

study is attributed to high virulence of the RH strain compared with S-273, which is of moderate.

Compared with blood and brain tissue PCR positivity at PID 9 and 12, direct microscopic

examination of brain tissue revealed Toxoplasma cysts only on PID 18 and thereafter. This appears

to be due to the time taken by parasite to form a recognisable tissue cyst. These findings illustrate

that blood PCR is more helpful in the early diagnosis of toxoplasmosis, as has been indicated by other

investigators16,9) particularly in AIDS patients, as parasitemia is a common feature of T gondii

infection during profound immunocompromisation. Though not helpful in the diagnosis of toxoplas-

mosis in AIDS patients, we also tested the mice serum samples for the detection of Toxoplasma

antibodies. Toxoplasma antibodies were evident on PID 12, the same day upon which brain tissue PCR

was positive.

As many investigators 11)•`17) have shown, our results also support the high sensitivity and

specificity of PCR. Hitt & Filice2) detected as low as 1-10 Toxoplasma parasites by means of PCR.

However, they found a significantly higher detection rate by mouse inoculation (62.0%) compared

with that by gene amplification (37.0%) and cell culture (25.0%). Their findings are in contrast to

many other reports showing the superiority of PCR over conventional diagnostic methods 11)•`17) PCR

however, is not cost-effective and cannot be performed in small laboratories. Moreover, the detection

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162 Hiroshi SHIBATA et al

of parasite in tissues by PCR cannot help to discriminate between active and latent infection 13).

Additionally, brain biopsy for the diagnosis of TE is not only difficult, but is also associated with high

morbidity. Therefore, detection of Toxoplasma parasites in blood appears to be useful in both early

diagnosis and in discriminating between active and latent infections, as parasitemia commonly occurs

as result of either primary or reactivated 9) infection.

References

1) Luft, B. J. & Remington, J. S.: Toxoplasmic encephalitis. AIDS commentary. J. Infect. Dis. 157: 1-6, 1988.2) Luft, B. J. & Remington, J. S.: Toxoplasmic encephalitis. Clin. Infect. Dis. 15: 211-222, 1992.3) Porter, S. B. & Sande, M. A.: Toxoplasmosis of central nervous system in the acquired immunodeficiency syndrome.

N. Eng. J. Med. 327: 1643-1648, 1992.4) Derouin, F., Gluckman, E., Beauvais, B., Devergie, A., Melo, R., Monny, M. & Lariviere, D.: Toxoplasma infection

after bone marrow transplantation: clinical and serological study of 80 patients. Bone Marrow Transplant. 1:67-73, 1986.

5) Pendry, K., Tait, R. C., McLay, A., Ho Yen, D., Baird, D. & Burnett, A. K.: Toxoplasmosis after BMT for CML.Bone Marrow Transplant. 5: 65-67, 1990.

6) Desmonts, G. & Couvreur, J.: Congenital toxoplasmosis: a prospective study of 378 pregnancies. N. Eng. J. Med.290: 1110-1116, 1974.

7) Rahman, M. S. & Rahman, J.: Toxoplasma in pregnancies. In. Infections in reproductive health, vol II. Uncommoninfections and special topics. Ed. Keith, L. G., Berger, G. S. & Edelman, D. A.: MTP press Ltd. Lancaster, England.

p 45-55, 1985.8) Luft, B. J., Remington, J. S. & Conley, F.: Outbreak of central nervous system toxoplasmosis in Western Europe and

North America. Lancet (i); 781-784, 1983.9) Frenkel., J. K. & Escajadio, A.: Cyst rupture as a pathogenic mechanism of toxoplasmic encephalitis. Am J. Trop.

Med. Hyg. 36: 517-522, 1987.10) Holliman, R. E.: The diagnosis of toxoplasmosis. Serodiagn. Immunother, Infect. Dis. 4: 83-93, 1990.11) Burg, J. L., Grover, C. M., Pouletty, P. & Boothroyd, J. C.: Direct and sensitivie detection of a pathogenic protozoan,

Toxoplasma gondii, by polymerase chain reaction. J. Clin. Microbiol. 27: 1787-1792, 1989.12) Grover, C. M., Thulliez, P., Remington, J. S. & Boothroyd, J. C.: Rapid prenatal diagnosis of congenital Toxoplasma

infection by using polymerase chain reaction and amniotic fluid. J. Clin. Microbiol, 28: 2297-2301, 1990.13) Van de Ven, E., Melchers, W., Galama, J., Camps, W. & Meuwissen, J.: Identification of Toxoplasma gondii

infections by BI gene amplification. J. Clin. Microbiol. 29: 2120-2124, 1991.14) Brezin, A. P., Eqwuagu, C. E., Silveria, C., Thulliez, P., Martins, M. C., Mandi, R. M., Belfer, Jr. R. & Nussenblat, R.

B.: Analysis of aqueous humour in occular toxoplasmosis. N. Eng. J. Med.(Letter) 324: 699, 1991.15) Parmley, S. F., Goebel, F. D. & Remington, J. S.: Detection of Toxoplasma gondii in cerebrospinal fluid from AIDS

patients by polymerase chain reaction. J. Clin. Microbiol. 30: 3000-3002, 1992.16) Dupouy-Camet, J., de Souza, S. N., Maslo, C., Paugan, A., Saimot, A. G., Benarous, R., Tourte-Schaefer, C. &

Derouin, F.: Detection of Toxoplasma gondii in venous blood from AIDS patients by polymerase chain reaction. J.Clin. Microbiol. 31: 1866-1869, 1993.

17) Guay, J. M., Dubois, C., Morency, M. J., Gagnon, S., Mercier, J. & Levesque, R. C.: Detection of pathogenic parasiteToxoplamsa gondii by specific amplification of ribosomal sequences using comultiplex polymerase chain reaction.J. Clin. Microbiol. 31: 203-207, 1993.

18) Weiss, L. M., Uden, S. A., Salgo, M., Tanowitz, H. B. & Wittner, M.: Sensitive and specific detection of toxoplasmaDNA in an experimental murine model: use of Toxoplasma gondii-specific cDNA and the polymerase chainreaction. J. Infect. Dis. 163: 180-185, 1991.

19) Filice, G. A., Hitt, J. A., Mitchell, C. D., Blackstad, M. & Sorenson, S. W.: Diagnosis of toxoplasma parasitemia inpatients with AIDS by gene detection after amplification with polymerase chain reaction. J. Clin. Microbiol. 31:2327-2331, 1993.

20) Hitt, J. A. & Filice, G. A.: Detection of Toxoplasma gondii parasitemia by gene amplification, cell culture, and mouseinoculation. J. Clin. Microbiol. 30: 3181-3184, 1992.

感染症学雑誌 第69巻 第2号

Use of PCR in Toxoplasma Parasite Detection 163

PCR法 を用いた トキ ソプラズマ感染実験マウスの血液お よび

脳組織か らの トキ ソプラズマ原虫の検 出

1)兵庫医科大学病院臨床病理学2)トリブヴァン大学教育病院病理学

3)神戸大学医学部医動物学

柴 田 宏1)Shiba Kumar RAI2)佐 藤 美 雪1)村 社 恵 子1)

住 勝 実1)宇 賀 昭 二3)松 村 武 男3)松 岡 瑛1)

要 旨

ポリメラーゼ連鎖反応(PCR法)は,高 感度で

特異性が高いことから近年臨床診断に広 く用い ら

れるようになった.筆 者 らは トキソプラズマ感染

症早期診断におけるPCR法 の有用性を検討する

目的でToxoplasma gondii (S-273株)経 口感染マ

ウスをモデル とした検討 を行った.PCR法 には

EH 24お よびHE27プ ライマーを用いそれぞれホ

スホア ミダイ ド法で合成 した.マ ウスの血液 と脳

は被感染時 より経時的に採取 し,35サ イクルの

PCR反 応 を実施 した.ま た,各 マウス脳組織の一

部 は トキソプラズマ原虫シス トの存在を見るため

に顕微鏡下に観察 した.血 液 と脳のPCR法 では

それぞれ感染9日 余 と12日後陽性 となった.脳 組

織中の トキソプラズマシス トは感染18日 後に検出

された.以 上の成績から,初 感染において トキソ

プラズマ原虫は脳よりも血液において早 く検出さ

れ,血 液 を用いたPCR法 はより有用な方法であ

ることが示された.

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