17
The serological diagnosis of canine echinococcosis by an enzyme immunoassay useful for epidemiological surveys. Benito, 1; Carmen a, D.t; Spinelli. de la Cuesta, 4 & Guisantes, J.A. 1•
, Postigo, I.t; Martinez, J.1; Estibalez, J.J. 3 ; Martín
JDepartment
Irnmunology, u"'",-!u,", Country. P.O. Box 450. 01
[email protected] of 3Department of Health and 4Department Basque '-'
Fax: 34.945.01
LHU.',Á.
UlllUUillV1V5
Received: 18.12.00
Accepted: 29.05.01
Abstract: An enzyme immunoassay method (ELISA) for the diagnosis of canine echinococcosis was in our laboratory. For the of the method, three different prepared from Echinococcus of ovine origin prnnlf,uPr! for coating the salid whole antigen from hydatid eyst fluid somatic antigen [roro sonicated and worm excretorylseeretory antigens obtained by eulture (PxES). The results described herewith were obtained employing PxSM sensitivity and of the test was determined 36 sera from dogs infected with E. and 126 sera from ínfected with other eommon helminths (cesto the determined cut-off a of 80.5% and a specificity of 91.7% was obtained and control sera, The predictive value for positive result was 592% and 97.1 % for the negative was 90.7%. On the bas!s ofthe results obtained, we conclude that the ELISA method is a CHabll\J~" of canine echinococcosis and may be applied for epidemiological surveys. ELISA,
words; Echinococcosis, Echinococcus granulosus, miology
Resumen: Se ha desarrollado en nuestro laboratorio un enzimoinmunoensayo (ELISA) para el dla:gné~StH;O serológico de la equinococosis canina. Para la de la técnica se prepararon tres de antígenos diferentes de Echinococcus obtenidos a partir de ovinos: total de hidatídico antígeno somático de (PxSM) y antígeno de excreciónisecreción de obtenido mediante cultivo Todos ellos fueron inicialmente evaluados en la PxSM como el más adecuado. La sensibilidad y especificidad del método fue deterfase sólida del ensayo, eligiéndose el minada analizando 36 sueros de perros infectados naturalmente con E. granulosus, 127 sueros de perros libres de helmintos y 126 sueros de canes infectados con otros cestodos y nematodos. De acuerdo a la línea de cOlte determinada, con este método ELISA se una sensibilidad del y una del 91,7%. El valor predictivo positivo fue del 59,2%, mientras que el negativo fue del 97,1 %. La eficacia diagnóstica fue de! En base a estos podemos afirmar que el enzimoinmunoenpara el de la canina, que ser Palabras clave: epidemiología.
LAl"UWJvVvV~
parasitaria, lO'" =j.'VO,j~ caninas, ¡ml1Uniod.lagnó:;tico, ELISA,
Echínococcus granulosus,
1. Introduction caused
by
the
Cyclophyllidea from the genus Echinococcus is one of the major zoonotic helminthíasis both animal and human health. It is widely distributed al! over the world and reports trom the last few years show Ihat it is over hydatidosis-free geographícal areas (Schantz et E. is
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18 Benito, A. et al., Serological diagnosis of canine echinococcosis.
of the unilocular disease known as classic 1982). It is transmitted among domestic which harbour the adult tapeworm (canine echiand other which act as intermediate hosts for the Human and eattle may be infected after the AUM~V"~ of infective eggs, in the faeces of ronmental contaminatíon. and
show prevalence rates that reach 22.5 per 100,000 inhabitants la I Prevalence estimates for livestoek areas the pereento determine the granulosus in dogs to determine the cal situation in a country or 1984). The of the variability of results employed should be considered 1992). The search and identification of E. sus eggs in dog faeces presents certain difficulties due to their morphological similarities wíth other eggs of the genus Taenia. Another method relies on the identification of the adult cestode by coprology after treatment with arecoline bromhydrate. Besides the risks in the use of this technique both for the field and personnel as well as for the dog, the results obtained may not be accurate for a correct
under certain circumstances disease control campaigns. For the last few years research has concentrated on immunoenzymatic methods of detectíon of antibodíes in blood el al., I Jenkins el 1990; Craig, 1 as well as of circulating antigens (Spinelli el 1996). The detectíon of parasite-specífic antigen faeces was developed antigens of adult E. (Deplazes et al., 1992) or somatic antigens (Allan el al., 1992). The results obtained indicated variability within the accuracy rates established by the different authors for E. granulosus diagnosis in faeces (Deplazes el al., 1992; Allan et al., 1992; Craig et al., 1995).
Difficulties in faeces as well as the risk for the personnel involved should be considered. Conversely, in the blood sampIes are easier to obtain due to a collaboration of the owners. Although neither method is of a serological test may have many epidemiological studies in our For this reason, the objective of the work was to a of canine echitechnique for the suitable for live dogs, which allows the of a large animal popuJation, without risk for neither field and laboratory personnel nor the environment, and with good sensitivity and This aim ¡neludes the employment of E. granulosus antigens from sheep of North of Spain and sera from uninfected or naturally helminth infected and sed by necropsy at the Basque Country.
2. Materials and Methods 2.1) Antigens Three different antigens were prepared: whole antigen from ovine hydatid cyst fluid (WHF), somatic antigen from sonicated protoscoleces (PxSM) and worm excretory/secretory antigen obtained by culture with the purpose of estimating their efficíency on the sensitisatíon of the ELISA plates. The WHF antigen was obtained as described Varela-Diaz and Coltorti (1976). PxES antigen were obtained from in vi/ro cultures of protoscoleces of E. granulosus as described by Carmena et al. (1999). Briefly, once obtained by asepfrom hydatid cysts of ovine origin, the were washed with phosphate buffer and their viability was assessed (Smyth 1 Howell, 1986). Then, they were maintained in a culture media with PBS, glucose and at 37° C and 5% C02 (Carmena el al., 1999). The culture medium was collected every 8 hours and concentrated. The obtained were stored at -25° C. from The PxSM ~ .... ,""'" from of ovine were washed with PBS and at -25 0 C in PBS with enzy2 mM and EDTA 5 mM). Far were thawed and sonicated in order to break the cellular structures and solubilise the nic The material was 25° C for 12 h, then thawed and obtained was susminutes at 3500 r.p.m. The pended in Triton X-114 Chemical St Louis) and sonicated for a new extraction of
19 Benito, A.
el
al., Serological diagnosis of caoine echinococcosis.
soluble Finally, the obtained were divided in aliquots and at -25° C. The proteín eoncentration of the PxSM (2.95 mgiml) was determined by the bicinchoninic acid assay (Sigma Chemical St. sera
Sera from 289 dogs distributed in three groups were studied: 1 (E. 36 with natural E. granulosus infeetions. granulosus-uninfected): 22 sera from U"'E;llVO,vU by necropsy which were with Taeniídae and 104 sera from dogs tized with other helminths, including Dipylidium caninum, Toxocara canis, Toxascaris leonina, caninum, Uncinaria and Trichuris vulpis. Group 3 (negative control sera): 127 sera from without worm burdens at necropsy. Feral dogs were obtained from the Animal Proteetion Centre belonging to the Town Couneil of Vitoría Spain). Blood was taken from the cephalie vein, allowed to clot and the sera was sto red for varof time at -25 0 C. all dogs were ying helminth burdens recorded, identified and sto red at -25 0 C. 2.3) ELISA
The assay was as for diagnosing human hydatidosís 1981), with sorne added modifications for the sis of caníne eehinococcosis. The optímum antígen eoncentration (10 mg/mI) was determíned the saturatíon method et al., 1984), and used to sensitize polystyrene microtiter plates (Nunc, with 100 mi per well. pIates were incubated for 1 hour at 37° C. The may be stored by freezing at -25° C without a decrease in reaeti vi ty. The sensitized were then washed three times with distílled water eontaining 0.05% Tween 20 and bloeked with 200 ¡.tI per well of PBS with 1% of BSA seroalbumin, Chemical St Louis) for 1 hour at 37° C. The sera were diluted with PBS 7,2 containing 0.5% of BSA and tested in duplicate in dilutions ranging from 1:50 to 1:400. The sera were then incubated for 1 hour at 37° C. Then, were washed as described aboye. 100 ¡.ti of a rabbit IgG con(Sigma Chemícal St Louis) diluted 1: 1000 in PBS with 4% BSA and 0,05% Tween 20 was plaeed for 1 in each and the pi ates were ineubated hour at 37" C.
a further washing, 100 !-tI of substrate, a solution of aeid (5AS) and were plaeed in each well. The substrate was prepared by díssolving 80 mg of 5AS in 100 mI of warm distilled water and adjusting the pH to 6.0 with NaOH (l N). befare 0.05% H202 was added to thc 5AS solutíon at arate of 1 mi for every 9 mi of 5AS solution. Plates wcre incubated at room temperature in the dark for 1 after whieh the reaetíon was by 0.025 mi per well ofNaOH IN. The absorbance was determined at 450 nm (A450).
3. Results 3.1)
selection
While WHF-ELISA aehieved poor results, sinee the differences in absorbanee values between homologous and control sera were poor, a good differentÍation between and control sera was obtained by PxSM and PxES Results obtained are shown in Figure 1. The results described herePxSM because with were obtained it is easier to obtain in suítable quantities. ELISA
The results were m according to a po oled control
Units (A.U.) serum (8000
A calibratíon curve was made this serum. The absorbance cut-off value was determined from the mean of the A450 values of the sera from Groups 2 and 3 two standard deviations (s.d.), that gave more than 6089 A.U. was considered 2).
A 450nm
0.35
_ _ WHF --B- P"ES _ _ PxSM
- Logaritlun of sera diluliotl
Fig. l. Differential vallles between positíve and negatíve control sera usíng differenl antígens in the adsorption of th" plates. WHF: Whole antigen from ovine hydatid cyst fluid, l'xES: Worm tory/secretory antigen, PxSM: Somatic antigen from protoscoleces.
20 Benito, A. el al., Serologieal diagnosis of eanine echinocoecosis.
curve, were To determine the within-observer each semm was tested 20 times in each in order to obtain the between-observer consecutive assays were carríed out with both sera several the mean the standard deviatíons and the variatíon coefficients were detennined. These results are shown in Table 2. The assay is
