PCRを用いたHalicephalobus gingivalisの簡易検出方法の検討

誌名誌名 Japanese journal of nematology

著者著者 吉賀, 豊司

巻/号巻/号 37巻2号

掲載ページ掲載ページ p. 101-104

発行年月発行年月 2007年12月

農林水産省 農林水産技術会議事務局筑波産学連携支援センターTsukuba Business-Academia Cooperation Support Center, Agriculture, Forestry and Fisheries Research CouncilSecretariat

Vo1.37 NO.2 japanese joumal of Nematology December，2∞7

[SHORT COMMUNICATION]

Detection of Rαlicephαlobus gingivalis in soil nematode

samples using PCR

Toyoshi Yoshiga 1

Halicephalobus gingivalis is a panagrolaimid

nematode that白山田 fatalinfections in horses.

Once the nematode infects and propagates with-

in horses， it田 usesnasal， maxillary， and renal

granulomas as well as meningoencephalitis.

More than 50 cases of infection in horses have

been reported from all over the world (Anderson

et al.， 1998; Takai et al.， 2005a). In addition to

infecting horses， 3 cases of fatal infection of

Halice;りhalobusin humans have also been report-

ed in Canada and the USA. (Takai et al.， 2005a).

In ]apan， the first Halicephauフbusinfection in

horses was reported in 1981 in Tokyo

(Yoshihara et al.， 1985). In 2000 and 2003， 2

cases of infections in horses were reported in an

equestrian club in Ishikawa Prefecture

(Shibahara et al.， 2002; Takai et al.， 2005b). In

2006， an infected pony was reported in Ibaraki

Prefecture (Akagami et al.， 2007). Although

infections have been reported from different

places in recent years， there is no information

regarding the natural reservoir of the nematode

or its mode of infection.

H. gingivalis is a small nematode with a

maximum body length of approximately 400μm

(Andrassy， 1984). Due to the p∞r morphological

characterization and lack of information regard-

ing the distribution of the nematode， its detec-

'Laboratory of Nematology， Department of Applied Biologiα1 Scienc伐SagaUniverstiy， Saga 840-8502， Japan Tel./fax: +81 952 28 8746 E-mail addr間:tyoshiga@cc.saga-u.ac.jp

tion in the mixed soil nematode population is

laborious and time consuming. The establish-

ment of a simple， sensitive， and reliable method

for the identification of the nematode from the

soil nematode population is the key to determin-

ing the natural reservoir of the nematode and

preventing the Halicephalobus infection. In this

paper， I describe the method for identifying H.

gingivalis from the nematodes isolated企omsoil

MA TERIALS AND METHODS

Nematodes:

Nematodes were isolated from soil in a com-

post pile obtained from the equestrian club in

Ibaraki Prefecture where the HalicePhalobus

infection was reported in 2007 (Akagami et al.，

2007) using the Baermann funnel method at

270

C for 48 hr. Because H. gingivalis has been

isolated from compost pile in the USA (Nadler et

al.， 2003) the soil企omthe equestrian club was

chosen.

H. gingivalis juveniles were obtained from a

仕ozenrenal sample of a horse (Akagami et al.，

2∞η. A single juvenile was picked up from the

thawed renal sample by a dental file and was

used to confirm the efficiency of the primers

used. In order to evaluate the DNA extraction

method and perform the polymerase chain reac-

tion (PCR) analysis， the single or 5 juveniles

were added to about 3，000 nematodes isolated

from the soil in the compost pile.

Isolation of genomic DNA from the nematodes:

DNAex仕actionfrom a single nematode was

performed as described by Iwahori et al. (2000).

DNA from nematode mixtures was extracted

using the QIAamp DNA mini kit (Qiagen). The

nematodes were lysed in 200μ1 of the supplied

celllysis buffer containing proteinase K solution

at 650

C for 1 hr with oc臼 sionalvortexing. After

the nematode lysis was confirmed microscopi-

cally， DNA was purified by using the spin col-

umn provided， according to the manufacturer's

101-

instructions. DNA was e1uted from the spin c01-

umnint04∞μ1 of the supp1ied buffer and stored

at -30oC unti1 further use. The e1uent (20-30 ng

DNAIμ1) was di1uted with steri1e water (111， 1110

and 11100 dilution) and the diluted DNA s01ution

was used as a temp1ate for PCR.

PCR ana1ysis:

To amp1ify partia11arge剖 bunitribosoma1

DNA 江5UrDNA)， PCR was carried out accord-

ing to the procedure described by Nad1er et al.

(2003) using the Halicethalobus-specific primers

#632 (5' -GT AGCGT A T AGGAA T A T AC-

T A TGG-3') and #634 (5' -CTTCA TCCTGCT・

CAAGCA T AGA-3'). PCR was performed in a

10-μ1 reaction mixture containing 1μ1 of 10 x PCR buffer， 0.8μ1 of dNTP mixture， 0.05μ10f

TaKaRa Ex Taq@ (TaKaRa)， 1μM of each

primer， and 0.8μ1 of temp1ate DNA s01ution (1/1，

1110， or 1I100-di1uted DNA s01ution). The mix-

ture was incubated at 950C for 3 min followed

2∞7年12月

by 35 cydes of 94"C for 30 sec， 54"C for 30 sec，

and 720

C for 1 min， followed by post-amplifica-

tion extension at 720

C for 8 min. The PCR prod-

ucts were separated on 1.5% agarose ge1， and

DNA was visualized using 1μg/m1巴thidium

bromide s01ution. For DNA sequencing， a DNA

band measuring approximately 700 bp in 1ength

was excised from the ge1， and DNA was purified

using the Ge1-M Ge1 Extraction System

(Viogene).

日本線虫学会誌第2号第37巻

RESUL TS AND DISCUSSION

A DNA fragment-expected to be approxi-

mate1y 7∞bp 10ng-was suco自由llyamp1凶ed

by using a primer set (#632 and #634) and a

DNA temp1ate that was extracted from a sing1e

H gingivalis obtained from an equine samp1e

from Ibaraki Prefecture. Using this primer set，

the amp1ification of the partia1 LSU rDNA of H

gingivalis from nematode mixtures was per-

23 4

~O Q ~O 0 ~O 0 ーとどよさ o ...... ::!::: 0

、ー、、電. . : . . .... ・ 4守・・ マー でー

一OOF~F

41

一。亡F

一ごF

‘-

bp

1，000

500

Fig. 1. Amplification of partiall紅 gesubunit of ribosomal DNA. The arrow indiαtes出eposition of the expected PCR product. 1-3: DNA was ex仕actedfrom approximately 3，000 nematodes with a single H gingivaゐjuvenile.4: DNA was ex仕ヨctedfrom approximately 3，∞o nematodes with 5 juveniles of H gingiv，叫S.DNA solution of 111， 1/10， or 111∞dilution was used as a template. M

25/1∞-bp mixed DNA ladder.

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Vo1.37 NO.2 ]apanese ]ournal of Nernatology December，2∞7

formed. Following nematode isolation by

Baermann funnel method， the nematodes were

examined under a microscope， but HalicePha-

lobus nematodes were not detected. The nema-

tode sample was thus considered as a

Halicephalobus-free nematode sample. When the

DNA extracted from approximately 3，000 soil

nematodes except H. gingivalis was used for

PCR， no amplicon was detected (data not

shown). On the other hand， in the DNA extract-

ed from approximately 3，000 nematodes contain-

ing a single juvenile or 5 juveniles of H. gingi-

valis， an amplicon of approximately 700 bp was

detected (Fig. 1). This 700・bpamplicon was puri-

fied and was used for direct sequencing. The

DNA sequence was identical to the partial LSU

rDNA of H gingivalis from a frozen renal sample

from Ibaraki prefecture (data not shown). There

was no significant difference between the DNA

samples isolated from 1 or 5 H gingivalis juve-

nil白 in3，000 nematodes. These results demon-

S仕atethat this method is sufficient1y sensitive to

detect a single juvenile of H. gingivalis among

3，000 soil nematodes.

For the detection of nematodes， the method

used for nematode extraction from the soil is

critical. However， no information is available on

thee百iciencyof ex仕actionof H gingivalis; more-

over， live H gingivalis specimens are not avail-

able in ]apan. In the present study， the

Baermann funnel method was used to extract

nematodes because no information regarding the

dis仕ibutionof H gi昭ivalisis available， and the

processing of many soil samples is required to

mcr，回目thepossibility of detection. Unlike plant

parasitic nematodes， H gingiva1is is a bacterivo-

rous free-living nematode that generally exhibits

high motility. The Baermarm funnel method that

is based on nematode mobility is simple and

reproducible and appears to be suitable for the

present purpose of processing many samples.

Further study is necessary to improve the

ex仕actione妊iciencyof this method.

The nematode cuticle is a three-layered

structure covered with a trilaminar epicuticle

但irdand Bird， 1991)， which prot配 tsthe nema-

todes from physical and chemical s仕ess.In addi-

tion， nematodes are difficult to homogenize

using mechanical homogenizers such as a glass

homogenizer and Polytron骨 becauseof their

small size. To increase the reproducibility and

sensitivity along with the回 seof handling， the

QIAamp DNA mini kit (Qiagen) was selected for

DNA extraction. After incubation in the lysis

bu旺ercontaining proteinase K solution， most

nematodes were observed to be lysed in 1 hr. In

the present study， the yield of DNA from the

3，000 soil nematodes by this method was 6-12

μg which appears to be within the maximum

capacity of出iskit. This type of kit with an effi-

cient lysis buffer may be suitable for the isola-

tion of genomic DNA from nematodes.

By using the simple and reliable method

described in the prl白 entreport， the detection of

H. gingivalis from soil nematodes samples can

be simplified. Moreover， information regarding

its natural reservoir will also help in preventing

further infection.

ACKNOWLEDGEMENT

The author thanks Mr. Yuji Yaguchi of

Kennan Livestock Hygiene Service Center，

Ibaraki Prefecture， for providing compost sam-

ples， and Dr. Masataka Akagami of Kenhoku

Livestock Hygiene Service Center， Ibaraki

Prefecture， for providing a frozen horse renal

sample.

LITERA TURE CITED

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Tanaka， N.， Yaguchi， Y.， Kon叫ci，T.， Kondo，

T.， Yamanaka， T. and Kubo， M. (2007)

Granulomatous nephritis and meningoen-

cephalomyelitis caused by HalicePhalobus

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第37巻第2号 日本線虫学会誌 2007年12月

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Received August 6， 2∞7

Accepted November 16， 2007

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