COMPARISON OF TWO TECHNIQUES FOR DIAGNOSIS OF GIARDIASIS IN DOGS
DECOCK C.*, CADIERGUES M.C.**, LARCHER M.*, VERMOT S.** & FRANC M.**
Summary:
Giardiasis is an intestinal parasitosis affecting dogs and able to infect human beings. Its diagnosis can not be done with the only clinical signs, the main of which is non characteristic diarrhoea. It implicates to perform further tests to detect the parasite. The zinc sulfate concentration technique |ZSCT| is the more effective one if performed on two or three successive days. Fecal ELISA kits have been developed to detect Giardia in humans and were found to be less sensitive than the ZSCT in dogs. In this study, we used 30 infected Beagles to compare the sensitivity of one, two or three fecal examinations following Z S C T and one or two ELISA tests. W e conclude that if a single Z S C T is insufficient, two or three ZSCT and one or two ELISA using the commercial kit ProSpecT® Giardia have almost the same sensitivity.
KEY WORDS : dog, giardiasis, diagnosis.
Résumé : COMPARAISON D E D E U X TECHNIQUES D E DIAGNOSTIC D E LA GIARDIOSE CANINE
La giardiose est une parasitose intestinale qui touche notamment le chien et est susceptible de se transmettre à l'homme. Le diagnostic de cette maladie ne peut pas se faire grâce aux seuls signes cliniques dont le principal est une diarrhée non caractéristique. Il nécessite le réalisation de teste destinés à détecter le parasite. La coproscopie microscopique selon la technique de flottation en solution de sulfate de zinc est la plus efficace si elle est réalisée deux ou trois jours consécutifs. Les kits ELISA fécaux,
commercialisés pour l'homme, donnent de moins bons résultats.
Lors de cette étude, réalisée sur 30 Beagles infectés, nous avons comparé les sensibilités d'une, deux ou trois coproscopies et celles d'un ou deux teste ELISA utilisant le kit commercial ProSpecT®
Giardia. Nos résultats montrent que si une seule coproscopie est insuffisante, la réalisation de deux ou trois coproscopies, comme celle d'un ou deux teste ELISA présentent des sensibilités proches.
MOTS CLES : chien, giardiose, diagnostic.
G
iardia duodenalis is a protozoan parasite found in the small intestine o f mammals including dogs and human beings. In canine populations, the prevalence o f this parasite reaches 13 % to 17 % (Beu- gnet et al, 2000; Villeneuve et al., 2 0 0 0 ) but puppies in kennels are more affected than adults. T h e infestation occurs by ingestion o f feces or water containing cysts.This disease is often considered as a zoonosis (Avedis- sian, 1988; Faubert, 1988; T h o m p s o n et al, 2000). Many infections o f dogs with Giardia are probably not asso
ciated with clinical signs (Barr et al., 1994; Bourdeau, 1993; Bourdoiseau, 2000; Leib et al., 1999; Zajac, 1992).
W h e n symptoms occur, diarrhoea is the more frequent one. F e c e s are pale, soft and smelling. T h e y may have a greasy appearance reminding o f steatorrhea.
T h e most c o m m o n l y used treatment in dogs is metro
nidazole (Zimmer et al., 1986; J a y a g o p a l a et al., 1 9 9 2 ) . Other drugs as tinidazole (Zimmer et al., 1 9 8 6 ) , fura
zolidone (Kirkpatrick et al., 1 9 8 4 ) , ipronidazole (Abbitt et al., 1 9 8 6 ) and quinacrine (Zimmer et al., 1 9 8 6 ) are
* U n i t é d e D e r m a t o l o g i e - P a r a s i t o l o g i e , É c o l e N a t i o n a l e V é t é r i n a i r e d e T o u l o u s e , F r a n c e .
** U M R 1 8 1 , É c o l e N a t i o n a l e V é t é r i n a i r e d e T o u l o u s e , 2 3 , c h e m i n d e s C a p e l l e s , 3 1 0 7 6 T o u l o u s e C e d e x 3 , F r a n c e .
C o r r e s p o n d e n c e : M i c h e l F r a n c . E-mail: m . f r a n c @ e n v t . f r
also effective. Benzimidazole anthelmintics have also b e e n r e c o m m e n d e d for their activity against Giardia.
F e n b e n d a z o l e (Barr et al, 1994; Zajac et al, 1 9 9 8 ) , oxfendazole (Villeneuve et al., 2 0 0 0 ) , febantel (asso
ciated with praziquantel and pyrantel p a m o a t e ) (Barr et al., 1 9 9 8 ) and albendazole (Barr et al., 1 9 9 3 ) w e r e generally investigated with success w h e n they w e r e used at the anthelmintic dosage.
T o b e effective, treatment must b e associated with hygienic m e a s u r e s in order to avoid recontamination.
Giardiasis can not b e diagnosed with the only clinical signs. It implicates to perform tests to prove the pre
s e n c e o f the parasite. T h e zinc sulfate concentration t e c h n i q u e (ZSCT) is the m o r e effective o n e if per
formed o n two or three successive days (Zimmer &
Burrington, 1986). Other methods have practical or sen
sitivity p r o b l e m s as peroral string test (Barr et al., 1 9 9 2 ) , e n d o s c o p i c duodenal aspiration (Pitts et al., 1983; Zajac et al, 1 9 9 2 ) and fecal smear ( Z i m m e r &
Burrington, 1 9 8 6 ) . Fecal ELISA kits, that detect Giardia specific antigen (GSA 6 5 ) , have b e e n d e v e l o p e d to detect Giardia in h u m a n s and w e r e found to b e less sensitive than the ZSCT in dogs (Barr et al, 1992; Hop
kins et al, 1 9 9 3 ) .
In this study, w e have c o m p a r e d the sensitivity o f o n e , two or three fecal e x a m i n a t i o n s following ZSCT and
P a r a s i t e , 2 0 0 3 , 10, 6 9 - 7 2
Note de recherche 69
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/2003101p69
DECOCK C . CADIERGUES M.C., LARCHER M . VERMOT S. & FRANC M.
o n e or t w o c o m m e r c i a l ELISA tests ( P r o S p e c T ® Giar
dia) that detects also GSA 6 5 antigena.
MATERIAL AND METHODS
T
hirty B e a g l e s ( 1 5 males and 15 females), betw e e n 16 and 17 w e e k s old, w e r e included. All dogs w e r e c o m i n g from a k e n n e l dedicated to laboratory sale. T h e y w e r e v a c c i n a t e d against dis
temper, hepatitis, parvovirus infection, leptospirosis and rabies virus infection. All w e r e clinically in g o o d health at the b e g i n n i n g o f the trial. T h e y lived in b o x e s o f six d o g s w h i c h w e r e daily w a s h e d with water u n d e r pressure and w e e k l y disinfected with quaternary a m m o n i u m sb. T h e day before e a c h fecal sample collection they w e r e placed in individual cages.
T h e y w e r e fed with c o m m e r c i a l dry dog foodc. T h r e e e x a m i n a t i o n s using the ZSCT o n fresh fecal samples w e r e m a d e o n day 0. day 1 and day 2. In the s a m e time, detection o f GSA65 antigen with ELISA tests was performed o n day 1 and day 2 using the manufacturer's r e c o m m e n d e d p r o c e d u r eb. 0.1 g o f feces w e r e m i x e d with 0.4 ml o f dilution Buffer. T h e n , it was placed in wells and added with 0.1 ml o f dilution Buffer. After o n e hour incubation period, wells w e r e rinced three times with 0.4 ml o f w a s h Buffer solution, then four drops o f e n z y m e conjugate w e r e added. 3 0 minutes later, wells w e r e rinced five times. T h e n , four drops o f c o l o r substrate w e r e added before a ten minutes incubation period. O n e drop o f stop solution was m i x e d in e a c h well. T h e microplate was read spectro- photometrically at a 4 5 0 nm O . D . within 10 minutes.
Concerning the ZSCT, w e mixed o n e gram o f feces in 10 ml o f 3 3 % zinc sulfate solution and let it during 10 minutes before the observation o f 10 m i c r o s c o p i c fields with the X 4 0 objective.
Later, both m e t h o d s w e r e applied to the feces on day 2 0 and day 2 1 .
T o c o m p a r e the two methods o f diagnosis w e used the χ2 test. Comparisons w e r e c o n s i d e r e d as significant if P < 0 . 0 5 .
RESULTS
I
ndividuals results are shown in Table I and Table IV.T h e r e was n o significant difference b e t w e e n the n u m b e r o f positive animals detected by three s u c cessive ZSCT ( 1 0 0 % ) , two successive ZSCT (94 % ) . o n e ELISA ( 9 3 % ) or two successive ELISA (97 % ) (Tables II
a P r o S p e c T ® Giardia M i c r o p l a t e A s s a y . A l e x o n - T r e n d , I n c . R a m s e y , MN 5 5 3 0 3 - D i s t r i b u t i o n : Elitech, ZI d e P e r b o s t 3 1 8 0 0 L a b a r t h e Inard.
b A s e p t o ® B L D 8 0 L a b o r a t o i r e s M é r i e l S.A., F - 0 2 3 7 0 V a i l l y - s u r - A i s n e .
c M e d i u m J u n i o r . R o y a l C a n i n , F - 3 0 4 7 0 A y m a r g u e s .
and III). However, a single ZSCT ( 7 2 % ) is sensibly less effective that the other tested methods (P < 0.05). W h e n c o m p a r e d to two ZSCT, o n e ZSCT is still less sensitive
ZSCT ELISA
D o g s DO D1 D 2 D1 D 2
1 3 1 - 1 4 6 - 1 4 2 - 1 2 1 - 1 2 6 - 1 5 2 - 1 4 5 - 1 2 0 - 1 4 4 + + + + +
1 3 9 + + + + —
1 5 1 - 1 4 3 + + - + +
1 5 6 - 1 4 1 - 1 3 8 - 1 1 9 - 1 7 0 + - + + +
1 3 6 - 1 4 7 - 1 6 2 - 1 3 3 - 1 4 9 - 1 2 9 + + + +
1 4 8 + + - -
161 + - - - +
1 3 0 - 1 6 4 - 1 7 3 + - + +
122 - + + +
1 4 0 - - + +
P o s i t i v e d o g s 1 8 2 2 2 3 2 8 2 8
T a b l e I. - R e s u l t s o f t h e tests for d e t e c t i o n o f Giardia in 3 0 d o g s o n d a y 0 , day 1 a n d d a y 2 ( + : p o s i t i v e : - : n e g a t i v e ) .
3 Z S C T 2 Z S C T 1 Z S C T
N u m b e r o f positive d o g s 2 9 2 8 . 2 8 . 2 6 18, 2 2 , 2 3 M e a n p e r c e n t a g e o f positive d o g sa 100 9 4 72*
T a b l e II. - C o m p a r i s o n o f t h e s e n s i b i l i t y o f 1. 2 o r 3 Z S C T for d e t e c tion of Giardia. a W e c o n s i d e r e d that a d o g w a s p o s i t i v e if at least o n e of t h e t h r e e Z S C T w a s p o s i t i v e . * Significant d i f f e r e n c e b e t w e e n this result a n d 1 0 0 % ( P < 0 . 0 5 ) .
T e s t s r e s u l t s N u m b e r of d o g s 3 ZSCT
(DO a n d / o r D1 ELISA 1 ELISA 1 ELISA 2 ELISA
a n d / o r D 2 ) ( D l a n d / o r D 2 ) ( D l ) ( D 2 ) ( D l a n d / o r D 2 )
Positive Positive 27 27 2 8
Positive Negative 2 2 1
Negative Positive 1 1 1
Negative Negative 0 0 0
Sensitivity 9 3 % 9 3 % 9 7 %
T a b l e III. - C o m p a r i s o n o f 3 Z S C T a n d 1 o r 2 ELISA for d e t e c t i o n o f Giardia in d o g s .
Z S C T E L I S A D o g s D 2 0 D21 D 2 0 D 2 1 1 3 1 - 1 5 1 - 1 4 8 - 1 4 3 - 1 6 4 - 1 6 1 - 1 6 2 - 1 4 5 - 1 2 0 + - + +
141 + + - +
1 5 6 - 1 4 6 - 1 4 0 - 1 2 1 + - + +
1 3 0 - 1 3 8 - 1 3 6 - 1 4 7 - 1 7 3 - 1 4 9 - 1 2 9 - 1 4 4 - + + +
1 2 2 - 1 3 9 - 1 3 3 - 1 5 2 - - - +
1 1 9 - 1 7 0 - - - +
1 2 6 - - - -
P o s i t i v e d o g s 14 18 2 5 2 8
T a b l e IV. - R e s u l t s of t h e tests for d e t e c t i o n o f Giardia in 2 9 d o g s o n d a y 2 0 a n d d a y 21 ( + : p o s i t i v e : - : n e g a t i v e ) .
70 Note de recherche P a r a s i t e . 2 0 0 3 . 10, 6 9 - 7 2
D I A G N O S I S O F G I A R D I A S I S IN D O G
T a b l e V. - C o m p a r i s o n b e t w e e n 1 a n d 2 Z S C T u s i n g t h e results o n d a y s 1, 2, 2 0 a n d 2 1 . a W e c o n s i d e r e d that a d o g w a s p o s i t i v e if at least o n e o n t h e t w o Z S C T w a s p o s i t i v e . * S i g n i f i c a n t d i f f e r e n c e b e t - w e e n this result a n d 1 0 0 % ( P < 0 . 0 5 ) .
T e s t s r e s u l t s N u m b e r of d o g s
2 ZSCT ELISA 2 ELISA
( D 2 0 a n d / o r ( D 2 0 a n d / o r 1 ELISA 1 ELISA ( D 2 0 a n d / o r
D 2 1 ) D 2 1 ) ( D 2 0 ) ( D 2 1 ) D 2 1 )
Positive Positive 4 8 4 8 4 9
Positive Negative 2 2 1
Negative Positive 5 8 8
Negative Negative 4 1 1
Sensitivity 9 6 % 9 6 % 9 8 %
T a b l e V I . - C o m p a r i s o n o f 2 Z S C T a n d 1 o r 2 ELISA for d e t e c t i o n o f Giardia in d o g s .
(77 % ) w h e r e a s o n e ELISA ( 9 6 % ) or two ELISA ( 9 8 % ) are equivalent ( T a b l e s V and V I ) .
DISCUSSION
T
h e zinc sulfate c o n c e n t r a t i o n t e c h n i q u e during three c o n s e c u t i v e days is often described as the r e f e r e n c e m e t h o d to d e t e c t Giardia in d o g f e c e s ( Z i m m e r & Burrington, 1 9 8 6 ; Barr et al., 1 9 9 2 ) . In this study, w e s h o w e d that t w o ZSCT at 24 hours give g o o d results w h i c h are not sensibly different from t h o s e o b t a i n e d with three ZSCT. O n the o t h e r hand, w e confirmed that a single ZSCT is not suffi- cient to m a k e a g o o d s c r e e n i n g o f the parasite. This m a y b e due to the intermittent s h e d d i n g o f cysts b y infected animals. Moreover, the m i c r o s c o p i c e x a m i - nation o f the slide d o e s not assure to detect cysts w h e n present unless watching the w h o l e slide what is taking time.The use o f the ELISA test (ProSpecT® Giardia, Alexon- T r e n d ) gives sensitivity results near from that o f two or three ZSCT. It m a k e s it a reliable t e c h n i q u e for dia- gnosis in dogs, but the practicality o f the ELISA w h i c h is m o r e c o m p l i c a t e ( e v e n if results c a n b e r e a d visually), the long time test required (about two hours) and its high cost restrict the u s e o f this m e t h o d to par- ticular c a s e s . It m a y b e advantageous for the control of a great n u m b e r o f animals or w h e n the collection of fecal samples during t w o or three days is not p o s - sible. It appears necessary, c o m b i n e d with ZSCT for evaluation o f treatment.
ACKNOWLEDGEMENTS
T
h e authors would like to express their gratitude to Cécile Caubet, Martine R o q u e s and Dr Greg Martin for their assistance.REFERENCES
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Reçu le 15 octobre 2002 Accepté le 12 décembre 2002
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