Immunological Responses of Dogs Experimentally Infected with Dirofilaria immitis Kun-Ho Song, Mineo Hayasaki1, Chusnul Choliq2, Kyu-Woan Cho*, Hong-Ryul Han3, Bung-Hyun Jeong4, Moo-Hyung
Trang 1J O U R N A L O F
V e te ri n ary
S ci e n ce
A BS TRA CT8 )
T h r e e d o g s w e r e e x p e r i m e n t a ll y i n fe c t e d w i t h
D i r o f i l a r i a i m m i t i s A ll d o g s w e r e e u t h a n i s e d a t 3 0 , 3 6
an d 37 w e e k s afte r in o cu la tion o f D im m itis fo r th e
re c ov e ry o f ad u lt w orm s Th re e ca se s a cc ou n te d to
42.91 % re co ve ry of in oc u la te d w o rm s Se ru m s am p le s
from do gs e xp e rim e n tally in o cu late d w ith D im m it is
w e re an a lyze d by ELISA an d im m u n o blottin g
m e th od s An tibod y tite rs of d og s d e te cte d by ELIS A
pe ak e d be tw e e n 7 a n d 14 w e e k s th e n de cre a se d
be tw e e n w e e ks 15 to 24 follow e d by a n oth e r in c re as e
du rin g w e e ks 25 to 30 an d pe rsis te d th rou g h ou t th e
re m a in de o f th e e x pe rim e n t p e rio d An a ly sis of ad u lt
D im m it is p rote in s tain e d w ith Coo m as sie brillia n t
blu e R-250 in d ica te d se p arate ly m o re th an 10 ba n ds ,
an d th e m a jo r ban d s w e re 22, 40, 46, 56, 70, 72 an d 89
kD a An tig e n ic id e n tific atio n of e xtra cts a n tige n s of
ad u lts D im m it is by im m u n oblo ttin g an a ly sis
re v e ale d se ve ra l ban d s fro m p oo le d s e ra o f pa te n t
in fe ctio n (30 w e e k s afte r in o cu latio n ) Th e d e te c te d
ban d s w e re 24, 70, 80 a n d 110 kD a, 22, 72 a n d 84 kDa ,
an d 58 a n d 72 kDa in do gs 1, 2 an d 3, re s pe c tive ly.
Re su lts of an tibo dy tite rs re ac h e d h ig h le ve ls o n th e
4th m o ltin g sta ge a fte r in o cu la tion of in fe ctiv e la rv a
(L3), an d re in force d pre vio u s fin d in gs th at h ig h
m ole cu lar w e igh t re gio n s are de te c te d in yo u n g
an im a ls
Ke y w ord s: Dirofilaria im m itis , exp e r im en t a l in fe ct ion ,
E L I S A, im m u n ob lot t in g
＊Corresponding author:
Tel: +82-42-821-6788, Fax: +82-42-821-4216
E-mail: kyuwoancho@cnu.ac.kr
In tro d u ctio n
D ir ofi l a ri a i m m it is , com m on ly ca lled ca n in e h e a r t w or m ,
is t h e ca u s e of a s er iou s p a r a s it ic d is ea s e of d ogs e n d e m ic
in t em p er a t e , s u b t r op ica l a n d t r op ica l cou n t r ies [4 ] I n
ep izoot iologic s u r ve ys of d ir ofila r ia s is in d ogs , va r iou s
m et h od s for d et er m in in g in fe ct ion s t a t u s h a ve b ee n u s e d ,
in clu d in g m icr os cop ic exa m in a t ion of blood s m e a r s , b lood
s a m p le con cen t r a t ion t ech n iq u e s , K n ot t 's t e s t for d et e ct in g cir cu la t in g m icr ofila r ia e, e xa m in a t ion for id e n t ifica t ion of
a du lt wor m s a t n ecr op sy, a n d r a diogr a ph ic a n d a n giogr a p h ic eva lu a t ion s [2 ,2 3,2 6] A m a jor p r ob lem in m a k in g d ia gn os is
is th e dir ofila r ia sis wit hou t micr ofila r emia (occu lt d ir ofila r ia s is )
w h ich h a s b ee n s h ow n t o occu r in 1 0 % t o 67 % of d ogs t h a t
h a ve be en in fe ct e d n a t u r a lly T h is h a s b ee n a t t r ib u t ed t o
s in gle -s ex in fect ion , p r e s e n ce of im m a t u r e a d u lt s or im m u n e-media t ed clea r a n ce of micr ofila r iae [21] Th ese occu lt in fe ct ion s
a r e ve r y d ifficu lt t o d ia gn os e by m icr os cop ic e xa m in a t ion of blood , s o t h a t , t h e vet e r in a r y p r a ct it ion e r s u s u a lly m a k e t h e
d ia gn os is b a s e d on clin ica l s ign s , r a d iogr a p h ic evid e n ce , a n d
ot h e r in d ir e ct in d ica t ion of in fe ct ion [2 1]
T h e occu lt D i m m i t i s in fe ct ion s p os e t h e m os t s er iou s
d ia gn os t ic ch a lle n ge An d , s om e t im es , e ve n in p a t en t
in fect ion s , ob s t a cles cou ld b e a void e d in t h e d ia gn os is of
d ir ofila r ia s is d u e t o t h e low n u m be r s of m icr ofila r ia e or t h e
d ifficu lt y in d is t in gu is h in g m icr ofila r ia e of D im m i t i s fr om
t h os e of D i p en t a l on em a recon d i t u m [5 ] T o ove r com e t h e s e
p r oble m s , va r iou s im m u n ologica l t ech n iq u e s h a ve r ecen t ly
be en d eve lop e d [3,5 ,6 ,8 -12 ,1 4,15 ,18 ,1 9,28 ] Am on g t h es e
m et h od s, en zym e-lin k ed im m u n osor ben t a s sa y (E LI S A) a n d
im m u n oblot t in g a n a lys is h a ve be en r e cogn ized a s s im p le,
s en s it ive a n d p a r t icu la r ly s u it a b le for t h e p a r a s it ic d is ea s es
lik e D i m m it is in fect ion [5 ,6 ,9 ,11 ] Th e p r es e n t s t u d y w a s
p er for m e d t o e lu cid a t e t h e im m u n ologica l r e s p on s es of d ogs
exp e r im en t a lly in fe ct e d w it h in fe ct ive la r va e of D i m m it is
u s in g E LI S A a n d im m u n ob lot t in g
Immunological Responses of Dogs Experimentally Infected with Dirofilaria immitis
Kun-Ho Song, Mineo Hayasaki1, Chusnul Choliq2, Kyu-Woan Cho*, Hong-Ryul Han3, Bung-Hyun Jeong4, Moo-Hyung Jeon5, Bae-Kun Park6 and Duck-Hwan Kim
6D epartm ent of Veterinary Parasitology, C ollege of Veterinary M edicine, Chungnam N ational University, D aejeon 305-764, K orea 1Veterinary C linical C enter, School of Veterinary Medicine, Yam aguchi U niversity, Yam aguchi 753-8515, Japan
2Veterinary C linical C enter, Faculty of Veterinary M edicine, Bogor Agricultural U niversity, Bogor 16151, Indonesia
3D epartm ent of Veterinary Internal M edicine, College of Veterinary M edicine, Seoul N ational University, Seoul 151-742, K orea 4D epartm ent of Veterinary Internal M edicine, College of Veterinary M edicine, Kon-kuk U niversity, Seoul 143-701, K orea
Rece ived F eb 3, 2002 / Accep t ed Ma y 7, 2002
Trang 2M ate ri als an d M e th o d s
E x p e r i m e n t a l a n i m a ls
F ive h ea lt h y m on gr el ju ven ile d ogs of a p p r oxim a t ely
t h r ee m on t h s of a ge w e r e h ou s ed in m os qu it o-p r oof r u n s ,
a n d t r e a t ed w it h p ip e r a zin e for in t e s t in a l p a r a s it es p r ior t o
exp e r im en t a t ion
E x p e r i m e n t a l i n fe c t i o n
Th r e e d ogs w er e exp e r im en t a lly in fe ct ed w it h D i m m i t i s
by t h e p r oce d u r e s d es cr ibe d by H a ya s a k i [8] B r iefly,
m os q u it oe s , Aed es t ogoi, colle ct ed d u r in g t h e la r va l s t a ge
fr om t h eir n a t u r a l s p a w n in g a r ea s w e r e r e a r e d u n d er
la b or a t or y con d it ion s a n d in ocu la t ed b y fe ed in g on a d og
w it h a bou t 20 0 cir cu la t in g m icr ofila r ia l (M f) cou n t s p er 20㎕
of blood (Table 1) Two dogs were used as the negative
control Infective larvae (L3) of D im m itis were recovered
from the proboscis of the infected mosquitoes 10 to 14 days
microscopic observation Three dogs were subcutaneously
injected in the inguinal region with each dog receiving 228
(dog No 1), 278 (dog No 2) and 248 (dog No 3) infective
larvae (L3), respectively Experimental dogs were euthanised
at 30, 36 and 37 after inoculation for the recovery of adult
worms.
Se ru m sa m ple s
The blood was collected from the cephalic vein once a
analysis.
Circ u la tin g Mf co u n ts
Dogs were screened for Mf by concentration method, and
microfilarial density in 20㎕of blood was measured by
counting Mf on methylene blue stained smears [20].
P re pa ratio n o f D im m it is an tig e n
The crude extracts of D im m itis were prepared as
previously described [7] Briefly, the antigens used in this
study were extracted from adult worms of D im m itis by
phosphate buffered saline (PBS, pH7.2, 0 1 M ) T h e s e w or m s
w e r e h om oge n ize d a n d s on ica t e d b y t is s u e h om ogen ize r
(1 5m in , 4 ) a n d u lt r a s on ica t or (5 0 w a t t , 15 m in , 4 ),
r e s p e ct ively, a n d t h en a llow ed t o in cu ba t e over n igh t a t 4 Aft e r cen t r ifu ga t ion a t 1 8,0 00 g, t h e s u p er n a t a n t a s a n t igen
w a s colle ct e d a n d k ep t a t -8 0 Th e p r ot ein con ce n t r a t ion
of t h e a n t igen w a s d et er m in ed u s in g t h e m et h od s of L ow r y
et a l.[1 7]
E n z y m e -L i n k e d I m m u n o s o r b e n t A s s a y (E L I S A )
E LI S A w a s p e r for m ed by m et h od s of G r ie ve et a l.[6] D
im m i t i s a n t igen w a s d ilu t e d t o 1 0㎍/㎖ in PBS 100㎕ of antigen was dispensed into each well and incubated for 30 minutes at room temperature 200㎕ of 1% bovine serum albumin(BSA) solution in PBS was added and the plates were incubated at 4 overnight The wells were washed three times with 200㎕ of 0.1% Tween 20 in PBS (TPBS) in
3 minutes for each wash 100㎕ of the serum from the test subject was diluted to 1:100 in PBS and added to each well The microplates were incubated at 37 for 1 hour, washed
anti-dog IgG (ICN Pharmaceuticals, Inc., USA) diluted to 1:200 in PBS, was added to each well and incubated at 37 for 1 hour, then washed with TPBS three times (3 minutes for each wash) A fresh preparation of substrate working
methanol, 0.1M citrate buffer (pH4) and 3% hydrogen peroxide, and 100㎕ of the solution was added to each well and left at room temperature for 30 minutes The enzyme reaction was stopped by adding 50㎕ 4 N sulfuric acid and values of optical density (OD) were read at 498㎚ (Sanko
J unyaku co J apan) The specificity of the assay was
(Synbiotics co., San Diego, USA) using five of the known positive and ten known negative serum samples.
S od iu m do de c yl su lfa te -p olya cry la m ide g e l e le
ctro-p h ore sis (SD S-P AGE)
A minislab gel consisting of 12.5% acrylamide and 0.1% SDS was used as described by Laemmli [16] The serum samples were diluted to 500㎍ protein/㎖ in the sample buffer (0.0625 M Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.00125% bromophenol blue) and heated for 5 minutes in boiling water 10㎎ of protein was
Ta ble 1 W or m b u r d en of d ogs exp e r im en t a lly in ocu la t ed w it h D i m m it is
D o g N o N o o f L 3 i n je c t e d D u r a t i o n o f i n fe c t i o n
(w e e k s )
N o o f a d u l t s w o r m r e c o v e r e d
1
2
3
24 8
27 8
22 8
37 31 38
15 1(95 /9 6)
3 1(16 /1 5)
12 7(66 /6 1)
6 0.8 8
1 1.1 5
5 6.7 0
L 3: I n fe ct ive la r va e
F /M : F em a le /M a le
Trang 3t h e n loa d ed in e a ch w e ll, follow e d b y ele ct r op h or e s is on 20㎃
currents at 4 for 120 minutes The gels were subsequently
stained with Coomassie brilliant blue R-250 (Katayama
s e p a r a t e d ba n d s w er e e s t im a t e d u s in g m ole cu la r w e igh t
m a r k e r s (B I O -R AD , U S A)
Im m u n o b l o t t i n g
F ollow in g S D S -P AG E , t h e p r ot ein b a n d s in t h e
p olya cr yla m id e ge l w er e t r a n s fe r r e d ele ct r op h or et ica lly t o a
n it r ocellu los e m em br a n e s h e et s (p or e s ize 0.4 5㎛, BIO-RAD,
USA) at 80 volts at 4 for 120 minutes, with a Tris-glycine
electro-transfer buffer (0.025 M Tris, 0.192 M glycine, 20%
methanol and 0.1% SDS) The nitrocellulose sheets were
blocked for overnight in Tris-buffered saline (TBS) (0.02M
Tris-HCl, pH 7.5, 0.5M NaCl) containing 3% gelatin
(BIO-RAD, USA) The nitrocellulose sheets were cut into
strips and placed into pla s t ic t r a ys S t r ip s w e r e s e p a r a t e ly
r ea ct ed t o t e s t s a m p le s t h a t w er e d ilu t ed 1:50 0 in TB S
con t a in in g 1 % gela t in , a t r oom t e m p e r a t u r e for 2 h r s ,
per oxida se-conjugat ed goat a n ti dog IgG (ICN P h a r m a ceu t ica ls
I n c, U S A) s e r u m d ilu t e d t o 1 :5 00 in T B S con t a in in g 1 %
gela t in S t r ip s w e r e t h e n w a s h ed w it h T B S S u b s t r a t e
s olu t ion con s is t in g of 15㎎ of 4-chloro-1-naphtol dissolved in
5㎖ methanol, 15㎕ hydrogen peroxide, was added to each
well and allowed to develop at room temperature for 20
minutes The reactions were stopped by replacing the
substrate with TBS.
Results
Re c ov e ry o f w o rm s at n e c rop sy
Dogs were euthanised on week 30, 36 and 37 post inoculation for the recovery of adult worms At necropsy, 151 adult worms (95 females and 56 males) in Dog No.1, 31 adult worms (16 females and 15 males) in Dog No.2 and 127 adult worms (66 females and 61 males) in Dog No.3 were recovered from the right ventricle and pulmonary arteries These represent approximately 42.91% recovery of inoculated worms.
ELIS A of se ra from e xp e rim e n ta lly in fe c te d d og s
In the Dog No.1, No.2 and No.3, antibody titers in the sera of inoculated dogs were significantly detected 7 weeks post inoculation and persisted until 14 weeks Antibody titers of dogs detected by ELISA peaked between 7 and 14 weeks then decreased between weeks 15 to 24 followed by another increase during weeks 25 to 30 and persisted throughout the remainde of the experiment period.
An alys is o f a du lt D im m it is p rote in by Coo m as sie
brillian t blu e R-250 sta in in g
D im m itis proteins were separated into more than 10
bands stained with Coomassie brilliant blue R-250 (CBB) staining, the major bands were 22, 40, 46, 56, 70, 72 and 89 kDa.
Fig 1 Antibody responses of dogs to experimental D im m itis infection by ELISA using D im m itis antigens.
*The range of optical density of control group The arrow indicates the day of inoculation.
＊
Trang 4112 Kun-Ho Song, Mineo Hayasaki, Chusnul Choliq, Kyu-Woan Cho, Hong-Ryul Han, Bung-Hyun Jeong, Moo-Hyung Jeon, Bae-Kun Park and Duck-Hwan Kim
An tig e n ic ide n tific atio n o f cru de e xtrac t a n tige n s
of a du lt D im m it is by im m u n oblo ttin g
inoculation) from Dog No.1 revealed four antigenic bands
with 24, 70, 80 and 110 kDa Dog No.2 showed three
antigenic bands of 22, 72 and 84 kDa, while two antigenic
bands were detected in Dog No 3 with 58 and 72 kDa.
Discussion
Experimental D im m itis infections in dogs have been
performed by various investigators [6, 8, 27, 29] The recovery
rate of adult worms from dogs experimentally inoculated
with the infective larvae of D im m itis were 45%, Hayasaki
[8], 66%, Thrall et al.[27] and 50%, Wong et al.[29],
respectively In this study, three cases accounted to 42.91 %
recovery of inoculated worms Although this study did not
confirm the correlation between antibody titer and the
number of adult worm, Grieve et al.[6] reported that there
was no relationship between them.
The ELISA used in conjunction with a Knott's test,
exposure history, clinical signs, laboratory results, and
radiographic changes are useful for studying seroepizootiologic
pattern and risk factors of heartworm infection ELISA
testing has been shown to be capable of identifying prepatent
infections [6] Diagnosis of prepatent infection or asymptomatic occult infection is important to the practitioner who may be preparing to initiate a heartworm preventive program based
experimental studies of beagles have shown abnormalities of pulmonary arteriograms 6 months after inoculation with
infective D im m itis larvae, at which time the dogs were still
amicrofilaremic and results of indirect fluorescent antibody testing for antimicrofilarial antibodies were negative [22].
ELISA testing for the detection of D im m itis provide a
useful and improved assay for serological characterization and detection of infection Grieve et al.[6] reported that ELISA titers were not significantly increased until 11 or 16 weeks, and remained at maximum levels for the duration of the observation period However, the present study revealed that antibody levels were detected as early as 7 weeks post inoculation, and developed continuously to 14 weeks, and then diminished to 15 to 24 weeks, then climbed again to 25
to 30 weeks, and high titer levels persisted throughout later experiment time One explanation of these different results may be the different responses of individual dogs to the variable extent and duration of parasitic exposure [13] Another explanation may be that different antibodies are being detected by different protocols used [11].
Konno et al.[15] reported that protein bands in extracted
fig 2 Protein fractions of adult D im m itis were separated by SDS-PAG E s t a in e d w it h C oom a s s ie b r illia n t blu e
R -25 0(A), a n d D im m i t i s a n t ibod y r e s p on s es w it h D i m m i t i s a n t igen by im m u n ob lot t in g a n a lys is (B ) La n e
1:s t a n d a r d m a r k e r , la n e 2-4: n ega t ive con t r ol, la n e 5-7 : p a t e n t in fe ct ion (la n e 2 ,5: N o.1 , la n e 3,6: N o.2 , la n e 4,7: N o.3)
Trang 5a n t ige n of D im m i t i s w a s d et e ct ed by C oom a s s ie b r illia n t
blu e R -2 50 s t a in in g w e r e 44 ba n d s (1 4 t o 2 30 k D a ) T h e
pr esen t s t u dy r evea led t h a t D im m it is pr ot ein wa s s e p a r a t e d
in t o m or e t h a n 1 0 ba n d s (22 t o 8 9 k D a ) by C oom a s s ie
br illia n t blu e R -2 50 s t a in in g, t h e m a jor ba n d s d e t e ct e d w e r e
22 , 4 0, 4 6, 5 6, 70 , 72 a n d 89 k D a Alt h ou gh t h e n u m b er of
p r ot ein ba n d s w a s les s t h a n t h os e of Konno et al.[15],
similar protein bands were detected by Coomassie brilliant
blue R-250 staining Immunoblotting in the D im m i t i s
infection helps to clarify the relation of antigen- antibody in
immune response, a more effective application of immunologic
diagnosis [9,12,24] Tamashiro et al.[24] and Boto et al.[1]
reported that low molecular weight reactivity as more
prevalent in older and /or more heavily infected dogs in
experimental D im m itis infect ion by means of immunoblotting.
I n n a t u r a l D im m itis infection, Tanaka et al.[25] reported
the trends in the distribution of immunoblotting patterns
with either young animals and/or mild infection tended to
reveal antibodies with reactivity to antigen in the higher
molecular weight regions of immunoblotting (more than 80
kDa), he also reported that the majority of antibodies with
reactivity to antigens in the low molecular weight regions
(less than 37 kDa), occurred more frequently in older dogs
and/or heavier infections The results of Tamashiro et
al.[24], Tanaka et al.[25] and Boto et al.[1] suggested that
experimental and natural infections.
In the present st u d y, a n t ige n ic id e n t ifica t ion of cr u d e
ext r a ct a n t ige n s of a d u lt s D i m m it is b y im m u n ob lot t in g
a n a lys is r e ve a le d s eve r a l b a n d s fr om t h e p ooled s e r a of
p a t e n t in fe ct ion (30 w ee k s a ft er in ocu la t ion ) Th e d et ect ed
ba n d s w er e 2 4, 7 0, 8 0 a n d 11 0 k D a in D og N o.1 , 22 , 72 a n d
84 k D a in D og N o.2 , a n d 5 8 a n d 7 2 k D a in D og N o.3 T h e
r es u lt s p r e s en t ed h e r e a r e s im ila r t o t h os e of T a m a s h ir o e t
a l.[24 ] a n d B ot o et a l.[1 ], in w h ich t h e h igh e r m olecu la r
w e igh t r e gion s w e r e d e t e ct e d by im m u n oblot t in g in you n g
a n im a ls (m or e t h a n 70 k D a )
I n s u m m a r y, a n t ibod y t it e r s by E LI S A r e a ch h igh le vels
a t t h e 4 t h m olt in g s t a ge a ft e r in ocu la t ion of L3 , a n d t h e
h igh er m olecu la r w e igh t r e gion s a r e m a in ly d et e ct ed by
im m u n ob lot t in g in you n g a n im a ls F u r t h e r s t u d ie s by ot h er
t ech n iqu es a r e n e ed ed t o e lu cid a t e im m u n ologica l r es p on s es
in D i m m i t i s -in fe ct e d d ogs
A ck n o w le d g e m e n t
Th is s t u d y w a s fin a n cia lly s u p p or t ed b y 20 01 of Aca d em ic
R e s e a r ch & S ch ola r s h ip F ou n d a t ion , C h u n gn a m N a t ion a l
U n iver s it y
Re fe re n ce s
1 B o t o , W M , P o w e r , K G a n d L e v y , D A An t igen s
of D i rofi l a r ia i m m it is w h ich a r e im m u n oge n ic in t he
canine host: detection by immunostaining of protein
blots with the antibodies of occult dogs J I m m u n ol
1 98 4, 1 3 3 , 9 75 -9 80
2 C a r l i s le , C H C a n in e d ir ofila r ia s is I t s r a d iogr a p h ic
a p p ea r a n ce Vet R a d iol.1 98 0, 2 1 , 1 23 -13 0.
3 D z i m i a n s k i , M T , M c T e r , T L a n d M c C a ll, J W.
1 98 9 E va lu a t ion of t w o a d u lt h e a r t w or m a n t ige n
d ia gn os t ic t e s t k it s u s in g w ell-d e fin ed d og a n d ca t s e r a
I n : P r oc 3 4t h An n u M e et Am As s oc Vet P a r a s it ol
O r le n d o: Am As s oc Vet P a r a s it ol 3 3
4 E t t i n g e r , S J a n d F e ld m a n , E S T ext book of vet e r in a r y in t e r n a l m ed icin e: D isease of the dog and cat.
pp 937-939 5th ed WB Saunders, Philadelphia, 2000.
5 Glick m an , L.T., Grie ve , R B a n d B re its ch w e rdt, E.
B Serological pattern of canine heartworm (Di rof il a r ia
im m itis) Am J Vet Res 1984, 45, 1178-1183.
6 Grie ve , R B , J o h n so n , M M an d J ac obs on , R H.
Enzyme-linked immunosorbent assay for measurement
of an tibody responses to Dirofilaria im m itis in
experi-mentally infected dogs Am J Vet Res 1981, 42, 66-69.
7 Hay as ak i, M. Indirect hemagglutination test for
diagnosis of canine filariasis J pn Vet Sci 1981, 43,
21-26.
8 Hay as ak i, M Reaginic and hemmaglutinating antibody
production in dogs infected with Dirofilaria im m itis.
J pn J Vet Sci 1982, 44, 63-70.
9 Hay as ak i, M., Na ka m u ra, F an d Kats u h iko , K.
Immunoblotting analysis of somatic components of
Dirofilaria im m itis. J Vet Med Sci 1994, 56,
1181-1183.
10 Ho ov e r, J P , Fo x, J C an d Clay po ol, P L.
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