An immunofluorescence technique for the detection of Toxocara canis antibodies

Veterinary Parasitology, 1 (1976) 231--237 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

AN IMMUNOFLUORESCENCE TECHNIOUE FOR THE DETECTION OF TOXOCARA CANIS ANTIBODIES

E.J. RUITENBERG and JANNIE BUYS Laboratory o f Pathology, National Institute o f Public Health, P.O. Box 1, Bilthoven (The Netherlands) {Received January 19th, 1976)

ABSTRACT

Ruitenberg, E.J. and Buys, J., 1976. An immunofluorescence technique for the detection of Toxocara canis antibodies. Vet. Parasitol., 1: 231--237. An immunofluorescent (IF) test for the serodiagnosis of Toxocara canis infections in puppies is described. Frozen sections of male adult T. canis worms were used as antigen. A group of seven puppies, 6 weeks of age, was infected orally with 10 000 embryonated T. canis eggs each. In the sera of all animals IF antibodies could be detected from approximately 4 weeks after infection onwards. Titers were detectable until the end of the observation period (22 weeks). Two puppies of the same age were infected with 30 000 or 50 000 embryonated T. canis eggs respectively. Positive IF results were also obtained in the sera of these pups from week 4 post infection (p.i.) onwards. No correlation between titer and initial number of eggs administered was observed. Furthermore, no correlation was noticed between titer and number of adult worms recovered from the dogs. For comparison all sera were tested with the complement fixation (CF) test, using cuticle material of adult worms as antigen. Complement fixing antibodies could be detected in none of the serum samples. INTRODUCTION T h e m a j o r i t y o f p u p p i e s are n a t u r a l l y e x p o s e d t o i n f e c t i o n w i t h T o x o c a r a sp. T h e d i a g n o s i s o f t h i s i n f e c t i o n m a y be b a s e d on e i t h e r d i r e c t m e t h o d s { d e m o n s t r a t i o n o f eggs a n d a d u l t w o r m s in f a e c e s ) or i n d i r e c t (i.e. s e r o l o g i c a l ) m e t h o d s . A w i d e s p e c t r u m o f s e r o l o g i c a l t e c h n i q u e s has b e e n d e s c r i b e d u s i n g h u m a n a n d a n i m a l sera. T h e s e i n c l u d e c o m p l e m e n t f i x a t i o n t e s t , O u c h t e r l o n y agar gel p r e c i p i t a t i o n , m i c r o p r e c i p i t a t i o n t e s t o n living larvae, h a e m a g g l u t i n a t i o n t e s t as well as v a r i o u s i m m u n o f l u o r e s c e n c e t e s t s ( H o g a r t h - S c o t t , 1 9 6 6 ; Bisseru a n d W o o d r u f f , 1 9 6 8 ; A l j e b o o r i a n d Ivey, 1 9 7 0 ; L a m i n a , 1 9 7 0 a a n d b; R u i t e n b e r g e t al., 1 9 7 6 ) . O f t h e s e m e t h o d s t h e i m m u n o f l u o r e s c e n c e ( I F ) t e c h n i q u e s e e m s t o b e p r o m i s i n g , p a r t i c u l a r l y w i t h r e g a r d t o its s e n s i t i v i t y . I F t e s t s are u s u a l l y c a r r i e d o u t e m p l o y i n g i s o l a t e d s e c o n d s t a g e l a r v a e (Bisseru a n d W o o d r u f f , 1 9 6 8 ) , b e c a u s e m o s t i n v e s t i g a t o r s are i n t e r e s t e d in t h e d e t e c t i o n o f a n t i b o d i e s a g a i n s t t h e s e larval stages w h i c h m i g h t p r o d u c e t h e visceral larval m i g r a n s s y n d r o m e in m a n ( B e a v e r e t al., 1 9 5 2 ) . In t h e p r e s e n t investi-

232 gations we have tried the application of frozen sections of male adult T. canis worms as antigen in IF tests. The system was evaluated with sera from experimentally infected puppies. MATERIAL AND METHODS Experimental animals Beagle puppies, 6 weeks of age, were purchased from the Central Laboratory for the Breeding of Laboratory Animals, TNO, Zeist. The Netherlands. These pups were treated once with 200 mg/kg piperazine ® (Janssen Pharmaceutica, Beerse, Belgium) at the age of 4 weeks. They were housed indoors in steel cages on rasters which were thoroughly cleaned daily in order to prevent superinfection. The animals received a diet consisting of pellets (Hope Farms, Woerden, The Netherlands) and drinking water ad libitum. The parasites and inoculation The original culture of e m b r y o n a t e d T. canis eggs was kindly supplied by Dr Thienpont, Janssen Pharmaceutica, Beerse, Belgium. Subsequently, we maintained the infection in puppies from the same breed. Six weeks after initial infection, adult worms and eggs were collected from the faeces. Eggs, isolated directly from the faeces or from the uteri of female worms, were embryonated in 0.1 N H2SO4 solution in glass jars placed in a water bath at 30°C for 10--14 days. The eggs, thus embryonated, were used for experimental infection and maintenance of stock. In order to verify the infectivity of the embryonated eggs, mice were inoculated orally with 1 000 such eggs each. The livers of these mice were checked 3 days later for the presence of the larvae. Only eggs from batches which proved infective were used to inoculate puppies. Serum sampling Blood samples were collected by vena puncture at weekly intervals from the infected pups starting on day 0. The blood samples were directly centrifuged to obtain serum which was immediately stored at --20°C until the serological assays were performed. Faeces examination Faeces samples were checked qualitatively for the presence of eggs and mature worms starting 35 days post infection (p.i.) until the end of the observation period. In order to obtain all the worms from each animal, puppies were treated once with one dose of 200 mg/kg piperazine ® at 11 weeks p.i. and again with two doses in one day of 100 mg levamisole® (Janssen Pharmaceutica, Beerse, Belgium) per animal at 13 weeks p.i.

233

Immunofluorescence method Preparation of antigen Mature male T. canis worms were washed three times for 5 min each time in phosphate buffered saline (PBS) (0.01 M; pH 7.2). The worms were divided into suitable portions, embedded in Tissue Tek (Ames Co., Elkhart, Ind., U.S.A.), deep-frozen in isopentane and stored at --20 ° C. Frozen sections cut in a cryostat (7 p thick) were prepared. Sections were stored at --20°C without prior fixation.

Procedure Histological sections were fixed in acetone at --20°C for 60 min, then dried in an incubator at 37°C for 60 min. Two-fold serial serum dilutions in PBS were prepared. The sections were incubated with the serum dilutions at room temperature for 30 min. They were then washed three times in PBS for 5 min with gentle rotation after which the slides were dried. Fluorescein-conjugated rabbit anti-dog immunoglobulin prepared in rabbits (Nordic, Tilburg, The Netherlands), diluted 1 : 15, was used as conjugate. The sections were incubated with the conjugate at room temperature for 30 min and again washed three times in PBS for 5 min with gentle rotation. After washing, the sections were counterstained with ehriochrome black (Difco, Detroit, Mich., U.S.A.) diluted 1 : 40 for 30 sec. The slides were again washed twice in PBS for 5 min each washing, then air dried and covered with buffered glycerine as mounting medium. A Leitz Orthoplan microscope with an Osram B 200 bulb as light source (Excitation filter, BG 12; barrier filter, K 530) was used for examination.

Assessment of fluorescence Cuticle fluorescence was taken as criterion for specific fluorescence. Often the hypodermis, muscle layer and other internal structures showed fluorescence, whereas the cuticle did n o t fluoresce. This type of fluorescence was considered non-specific. When shrunken sections were used or when the cuticle was folded over the section, false positive reactions were occasionally obtained. Thus it is very important to use high quality sections only.

Complement fixation test (CF) Conventional CF was performed using cuticle material of the adult T. canis worm as antigen.

Experimental design In experiment I (groups 1--3) seven puppies were divided into one group of three animals (numbers 1, 2 and 3), one group of two animals (numbers 4 and 5) and another group of two animals (numbers 6 and 7). Puppies of the

234

first two groups were housed together in one cage per group while those of the third group were housed individually. Each puppy was infected orally with 10 000 embryonated T. canis eggs. Individual faecal samples were collected from the rectums of puppies 1--5 and checked for the presence of eggs from day 35 p.i. onwards, while faecal samples from puppies 6 and 7 were collected from the cage. Sera were collected and examined as described above. Sera and faeces of puppies 1--5 were examined for 22 weeks p.i. Sera of puppies 6 and 7 were examined until 9 weeks p.i. and the faecal samples of these animals were collected until the day after the levamisole treatment. In experiment II (group 4 ) t w o puppies (numbers 8 and 9), housed individually, were infected orally with 30 000 and 50 000 T. canis eggs, respectively. Serum sampling and examination was performed as in experiment I and the results compared with the seven pups given 10 000 eggs.

IF

1

2

IF

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132

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5

8

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]2

16 18 20 2J2 weeks

rF

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2

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8

10 12 I~. ~6 18 20 22

weeks P,

IF

4

32

0

2

4

6

8

10

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16

18

20

22

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2

4

6

8

10

12

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16

~8

weeks pl

20 22 wee~s D,

IF

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116

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12 14 16 18 20 22 week s p i

-o

18 '

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1214

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4

o--o ~--430,000eggs

o 50,000 eggs

6

8

10 12 weeksp

Fig.1. IF results in p u p p i e s numbers 1--5 ( 1 - - 5 ) , infected w i t h 10 0 0 0 T. canis eggs each. IF results in p u p p i e s numbers 8 and 9 (6), infected w i t h 30 0 0 0 and 50 0 0 0 T. canis eggs respectively. P -- piperazine; L = levamisole.

235

,oo. ,32

2

1

n u m b e r of worms

nurnber of worms

120

IF

, 2i? 16

18

12

0

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6

8

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weeks p t

~20

IF

100

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8O

60

IB

60

2C

12

20

t.O

2

I.

6

B

10 w e e k s pl

0

2

4

6

8 10 we e k s p

6

8

10 weeks p

Fig.2. Results of IF and worm counts in puppies numbers 6 (1) and 7 (2) infected with 10 000 T. canis eggs each. Piperazine treatment in week 10.

RESULTS Experiment I

In none o f the pre-infection sera were antibodies d e t e c t e d with either IF or CF. All animals responded to egg inoculation by uroducing antibodies detectable with IF, while the CF failed to show 11~, .sence of these antibodies (Figs 1 (1--5) and 2). Consistent positive results were obtained 4 weeks p.i., with the ex c e pt i on of animal n u m b e r 1, where the IF test was positive f r o m week 1 p.i. onwards. Antibodies remained detectable t h r o u g h o u t the observation period. Eggs were d e t e c t e d in all puppies; in animals 4, 6 and 7 from 5 weeks p.i. onwards, and in the ot her animals from week 6--7 p.i. onwards. Directly after anthelmintic t r e a t m e n t no change in titer was observed in animals numbers 1, 3, 4 and 5. In animal n u m b e r 2 a rise in titer was observed after 14 weeks p.i. In animal n u m b e r 5 IF titers decreased f r o m week 13 p.i. onwards. Before anthelmintic t r e a t m ent , adult worms were f o u n d in the p o o led faecal samples of bot h groups of animals (Table I). In t he faeces fr o m animals numbers 1, 2 and 3 two adult T. canis worms (one male and one female) were f o u n d on day 45 p.i. In the faeces from animals numbers 4 and 5 t wo adult T. canis worms (one male and one female) were f o u n d on day 45 p.i. and 25 adult worms were f o u n d on day 54 p.i. In the faeces from animal n u m b e r 6 adult worms were f o u n d f r om day 29 p.i. onwards, and in animal n u m b e r 7 f r o m day 42 p.i. onwards (Fig.2). After piperazine t r e a t m e n t , a total of 37 adult worms were f o u n d in the po ol ed faeces from animals numbers 1--5 within 3 days after t r e a t m e n t , whereas 1 day after the second anthelmintic t r e a t m e n t (levamisole) one adult w or m was isolated from t he pooled faeces samples o f these animals. In Fig.2 (1) and (2) b o t h IF titers and numbers o f adult worms recovered fr o m the faeces f r om puppies 6 and 7 are shown. In animal n u m b e r 6 the peak titer was 1 : 16 in weeks 6 and 7 p.i., and 171 adult worms (10t; females and 65 males) were recovered f r om this animal during the period

236 TABLE I Number of adult T. canis worms expelled prior to and after anthelmintic treatment Group

Dog number

Infection dose (number of eggs)

Treatment

1

1 2 3

10 000 10 000 10 000

P + L* P+L P+L

2

4 5

10 000 10 000

P+L P+L

3

6 7

10 000 10 000

4

8 9

30000 50 000

Number of worms expelled From infection until treatment

After treatment

Total

d

d

9

c~

9

20

18

34

3~

5 5

3 10

90 14

11 ¢, 5/

n.d. n.d.

+ +

+ +

.1]

9 1

1

13

14

P P

85 9

116 42

P P

+** +

J

+ +

f

n.d.*** n.d.

*P = piperazine; L = levamisole (for schedule see text). ** + = worms were present. ***n.d. = not done. 4--6 weeks p.i. In animal n u m b e r 7 titers varied b e t w e e n 1 : 16 and I : 32 in the p e r i o d 4 - - 9 weeks p.i., a n d 29 a d u l t w o r m s (26 females and 3 males) were r e c o v e r e d in t h e p e r i o d 6 - - 1 0 weeks p.i. Animals n u m b e r s 6 a n d 7 were t r e a t e d with p i p e r a z i n e alone in w e e k 10. O n e d a y a f t e r t r e a t m e n t , 8 adult w o r m s were f o u n d in the faeces f r o m animal 6, and 15 w o r m s in t h e faeces f r o m animal 7.

Experiment H In b o t h animals ( n u m b e r s 8 a n d 9) a n t i b o d i e s c o u l d be d e m o n s t r a t e d b y IF, b u t the CF tests were negative. C o n s i s t e n t positive results were o b t a i n e d f r o m w e e k 4 p.i. o n w a r d s (Fig.1 (6)). DISCUSSION With t h e I F m e t h o d described, T. canis a n t i b o d i e s c o u l d be d e t e c t e d . All s e r u m samples c o l l e c t e d o n d a y 0 were negative, suggesting t h a t even if pups were n a t u r a l l y i n f e c t e d at birth p r i o r t o p i p e r a z i n e t r e a t m e n t n o d e m o n s t r a b l e a n t i b o d i e s were p r e s e n t at t h e age o f 4 weeks. It is i m p o r t a n t t o stress t h a t o n l y cuticle f l u o r e s c e n c e was regarded as specific. In all cases n o CF a n t i b o d i e s c o u l d be d e m o n s t r a t e d , whereas I F a n t i b o d i e s were c o n s i s t e n t l y d e t e c t a b l e in all p u p p i e s f r o m a p p r o x i m a t e l y 4 weeks p.i. onwards. T h e a m o u n t o f

237

T. canis infective eggs administered did n o t seem to affect the a n t i b o d y levels. No differences in titers were observed between puppies infected with 10 000, 30 000 or 50 000 eggs. There was also no correlation between the IF titers and the numbers of adult worms recovered from the faeces. A wide variation both in antibody response and worm burden between the individual animals was observed, although each group of puppies originated from the same litter. Recently, Viens et al. (1975) described a similar IF technique for the serological diagnosis of toxocaral visceral larva migrans infection in man. Our own results in puppies and the results of Viens and co-workers in man certainly justify further research of this IF test, particularly with regard to its specificity. ACKNOWLEDGEMENTS

The authors are gratefully indebted to Mssrs Van Rheenen and Van Schaaik for zootechnical assistance and to Mr A. van Maarschalkerweerd for the preparation of the cryostat sections. REFERENCES Aljeboori, T.I. and Ivey, M.H., 1970. An improved hemagglutination technique for detecting antibody against T o x o c a r a canis. Am. J. Trop. Med. Hyg., 19: 244--248. Beaver, P.C., Snyder, C.H., Carrera, G.M., Dent, J.H. and Lafferty, J.W., 1952. Chronic eosinophilia due to visceral larva migrans; report of 3 cases. Pediatrics, 9: 7--19. Bisseru, B. and Woodruff, A.W., 1968. The detection of circulating antibody in human T o x o c a r a infections using the indirect fluorescent antibody test. J. Clin. Pathol., 21: 449--455. Hogarth-Scott, R.S.~ 1966. Visceral Larva Migrans -- an immunofluorescent examination of rabbit and human sera for antibodies to the ES antigens of the second stage larvae of T o x o c a r a canis, T o x o c a r a cati and Toxascaris leonina (Nematoda). Immunology, 10: 217--223. Lamina, J., 1970 a. Immunbiologischer Nachweis einer "Larva migrans visceralis-Infektion". Tierexperimentelle Ergebnisse. I. Mitteilung: Die Agar-Gel-Pr~izipitation nach Ouchterlony. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1: Orig., 215: 259--273. Lamina, J., 1970 b. Immunbiologischer Nachweis einer "Larva migrans visceratis-Infektion". Tierexperimentelle Ergebnisse. II. Mitteilung: Der Mikropr~izipitationstest an der lebenden Larve. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1: Orig., 215: 386--397. Ruitenberg, E.J., Panggabean, S.O., Geleijnse, M.E.M., Visser, A. and Sluiters, J.F., 1976. Onderzoek van kleuters op het voorkomen van hondespoelworminfecties. Ned. Tijdschr. Geneeskd., 120: 645--649. Viens, P., Strykowski, H., Richards, R. and Sonea, S., 1975. A modified immunofluorescent antibody technique for the serodiagnosis of human toxocaral larva migrans. Can. J. Public Health, 66: 237--240.