effect of parasite burden on the detection of fasciola hepatica antigens in sera and feces of...

Veterinary Parasitology 97 (2001) 101–112

Effect of parasite burden on the detection ofFasciola hepatica antigens in sera andfeces of experimentally infected sheep

Consuelo Almazán a, Guillermina Avila b, Héctor Quiroz c,Froylán Ibarra c,∗, Pedro Ochoa c

a Facultad de Medicina Veterinaria y Zootecnia, Departamento de Parasitologıa, Universidad Autónoma deTamaulipas, km. 5 carretera Victoria-Mante, Cd. Victoria, Tamaulipas CP 87000, Mexico

b Facultad de Medicina, Departamento de Microbiologıa y Parasitologıa, Universidad Nacional Autónoma deMéxico, México City, DF CP 04510, Mexico

c Facultad de Medicina Veterinaria y Zootecnia, Departamento de Parasitologıa, Universidad NacionalAutónoma de México, México City, DF CP 04510, Mexico

Received 25 October 1999; received in revised form 20 December 2000; accepted 21 December 2000

Abstract

The effect of Fasciola hepatica parasite burden on the detection of excretory/secretory (E/S) anti-gens in sera and feces of experimentally infected sheep was evaluated using a double antibody-basedcapture enzyme-linked immunosorbent assay (ELISA). Four groups of five sheep each were used.The first three groups were infected with 50, 100 and 200 metacercariae of F. hepatica, and thefourth group remained as non-infected control. On the day of infection and weekly thereafter, serumand fecal samples were taken. ELISA detected F. hepatica E/S antigen levels in serum from thefirst week post-infection (wpi) and in fecal supernatant from the fourth wpi, which were signifi-cantly (p < 0.05) higher than controls. F. hepatica eggs were not detected until after the eighthwpi. The correlation between absorbance of E/S antigens in serum with the fluke burden was 0.77(p < 0.0001) and in feces 0.76 (p < 0.0001) at 12th wpi. The sensitivity of the assay to detect E/Santigens in serum was 86.6% and in feces 93.3%. It is concluded that the ELISA technique usedin this study offers a diagnostic alternative for detecting early infections of F. hepatica in sheep.© 2001 Elsevier Science B.V. All rights reserved.

Keywords: Fasciola hepatica; Serum; Feces; E/S antigens; ELISA; Sheep

∗ Corresponding author. Tel.: +52-622-5899; fax: +52-622-5971.E-mail address: [email protected] (F. Ibarra).

0304-4017/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved.PII: S0 3 0 4 -4 0 17 (01 )00376 -4

102 C. Almazan et al. / Veterinary Parasitology 97 (2001) 101–112

1. Introduction

Traditionally, diagnosis of Faciola hepatica has been carried out through coproparasito-logical examination, however, in the acute phase and prepatent period of the disease, thepresence of the parasite cannot be determined due to the lack of egg output in feces.

Recently, immunodiagnosis has focused on the detection of antigens in feces of infectedhosts, since the parasite releases metabolic products and structural components which areshed in feces. These antigens can be detected by the capture ELISA technique widely usedfor protozoa (Randall et al., 1984; Stibbs et al., 1988), cestode (Allan et al., 1990; Deplazeset al., 1991; Baronet and Waltner-Toews, 1994) and nematode detection (Johnson and Benke,1996; Ellis et al., 1993; Sood et al., 1996). In the case of trematodes, some assays for F.hepatica have been done. Espino and Finlay (1994) developed a double antibody-basedcapture ELISA capable of detecting F. hepatica antigens in human feces. The assay wasalso tested in cattle (Castro et al., 1994) and rats (Espino et al., 1997). The objective of thepresent work was to evaluate the effect of fluke burden on the detection of fluke antigens inserum and feces of sheep experimentally infected with F. hepatica, using a double captureantibody ELISA assay. The sensitivity of the assay and the possible correlation betweenantigens in serum and feces with the parasite burden were also evaluated.

2. Materials and methods

2.1. Experimental infection

Twenty-three-month-old, crossbred sheep free of fluke and gastrointestinal nematodeswith an average weight of 30 kg were used. Sheep were obtained and kept at the Training,Production and Research Center for Ruminants at the National University. Prior to inocula-tion, nematode and fluke eggs were not detected in feces of any of the sheep as determinedby coproparasitoscopical examination (Quiroz, 1995). Animals were randomly allocated tofour groups of five animals each. Groups 1, 2, 3 were infected with 50, 100 and 200 metac-ercariae, respectively, of F. hepatica per sheep, given orally in a gelatin capsule. This wasdone as described by others (Boray et al., 1983). Group 4 remained as a non-infected controlgroup. At the time of infection, metacercariae were 1 month old and had 80% viability. It wasassessed by using the criteria of Boray et al. (1983) consisting in motility of the worms andclear observation of the excretory granules in some cysts under dissecting microscope. Theywere raised in Lymnaea cubensis experimentally infected in our laboratory with miracidiafrom eggs collected from gallbladders of cattle sacrificed at the local slaughterhouse. Ani-mals were kept on concrete and were fed dry herbage and some supplementary food. Waterwas provided ad libitum. Blood and fecal samples were collected at the beginning of theinfection and then at weekly intervals for 12 weeks. Serum was collected by centrifugationand stored at −20◦C, until used. Fecal samples were collected directly from the animal withproperly identified plastic bags, and transported to the laboratory where they were dividedinto two parts. One part was used to determine the presence of F. hepatica eggs (Quiroz,1995). The other part was used to perform the ELISA test for coproantigens. Samples werehomogenized in an equal quantity with PBS plus 0.3% tween 20 and bovine serum albumin

C. Almazan et al. / Veterinary Parasitology 97 (2001) 101–112 103

(BSA) at 0.1% (Sigma Chemicals), and the supernatant was obtained by centrifugation at10 000g for 25 min at 4◦C and stored at −20◦C until used. All animals were slaughtered at12 weeks post-infection (wpi), and adult flukes were recovered and counted.

2.2. Production of E/S antigen of F. hepatica

Condemned livers were obtained from the local slaughterhouse. Livers were dissectedto collect live adult flukes, which were washed five times with PBS (pH 7.2) to clean themup. Flukes were then incubated in RPMI 1640 culture medium containing 10 000 IU/ml ofpenicillin for 24 h at 37◦C. The supernatant was collected, the protein concentration wasdetermined according to Lowry et al. (1951), and the E/S antigen was aliquoted and storedat −20◦C.

2.3. Anti-F. hepatica polyclonal serum

To obtain anti-F. hepatica E/S antibodies, a 2.0 kg New Zealand rabbit was immunizedwith the antigen of F. hepatica. On day zero, 1 ml (0.8 mg/ml) of the E/S antigen of F.hepatica was emulsified with an equal quantity of Freund’s complete adjuvant and wasadministered subcutaneously. Four additional immunizations were given at 15 day intervals,using 1 ml of E/S antigen mixed with an equal quantity of Freund’s incomplete adjuvant.After the eighth week, the rabbit had an antibody titer >102 400 as determined by ELISA(Espino et al., 1987). Finally, blood was obtained by cardiac venipuncture and serum wascollected and adsorbed with bovine liver dust, and stored at −20◦C.

2.4. Anti-F. hepatica rabbit IgG

Precipitation of the anti-F. hepatica polyclonal serum was done using 50% ammoniumsulfate. Purification of the IgG in hyperimmune serum was undertaken by affinity chro-matography in a column of protein A sepharose CL 4B (Sigma Chemicals) according tothe manufacturer’s instructions. The IgG was eluted with glycine at 0.1 M, pH 2.5. Frac-tions were collected and optical densities (ODs) were determined with a spectrophotometer(Beckman DU-65) at 280 nm. The IgG was dialyzed for 12 h against PBS at 4◦C. The re-covered IgG from the column was pooled and concentrated by using a high-flow membranefilter (YM-10, Amicon). Protein concentration was measured using the method of Lowryet al. (1951) and stored at −20◦C. Afterwards, under reducing conditions an aliquot of con-centrated IgG was analyzed on a 12% acrylamide SDS-PAGE by the method of Laemmli(1970). Immunological recognition of the concentrated IgG was tested by ELISA (Espinoet al., 1987).

2.5. Preparation of anti-F. hepatica IgG conjugate

For conjugation of the peroxidase enzyme with the IgG antibody, the periodate methodwas used (Wilson and Nakane, 1978). The protein concentration of the IgG was adjustedto 5.2 mg/ml, being dialyzed with sodium bicarbonate (NaHCO3) 0.01 M, pH 8.0 for 12 hat 4◦C, then it was mixed with activated peroxidase; the ratio of IgG to peroxidase was 2:1.


The mixture was left to react in darkness at room temperature for 2 h under slow shaking.Then, sodium borohydrure was added to stop the reaction. The IgG was dialyzed againstPBS and stored at −20◦C.

2.6. ELISA for detection of F. hepatica E/S antigens

Sensitization of 96 flat-bottom microplates (Nunc-Immuno Plate F96, Cert, Maxisorp)was carried out using 25 �g/ml of rabbit anti-F. hepatica IgG diluted in carbonate buffer(0.05 M, pH 9.6). In each well, 100 �l of the dilution was placed, and then incubatedovernight at 4◦C. Blocking of excess-binding sites was performed by incubation with 200 �lof PBS 0.3% tween 20 containing 5% BSA for 1 h at 37◦C. Undiluted serum or fecalsupernatant were added (100 �l of each) in duplicate to the microplate wells, incubated for1 h at 37◦C. The secondary antibody (100 �l of a solution of 30 �g/ml of anti-F. hepatica IgGconjugated with peroxidase) was added and incubated for 1 h at 37◦C. Finally, 100 �l/wellof substrate solution (4 mg of ortho-phenylendiamine hydrochloride+10 ml of 0.1 M citratebuffer, pH 5.0 + 4 �l/ml of hydrogen peroxide, 30% (w/v)) were added and incubated indarkness at room temperature for 5 min. The reaction was stopped by addition of 50 �l/wellof 1 M sulfuric acid. Absorbance values were measured at 492 nm in an ELISA reader(Bio-rad, model 2550). Between each step, three washings were carried out with 200 �lof PBS 0.3% tween 20 and 0.1% BSA for 5 min each. For negative controls, serum andfecal supernatant from non-infected sheep were used. For positive controls 25 �g/ml of E/Santigen of F. hepatica in PBS were added to the corresponding wells. Positiveness of anindividual sample was determined when the absorbance value was equal or higher than thecutoff point of the assay. The cutoff point was obtained by the mean plus three times thestandard deviation (S.D.) of the absorbance values obtained in the control group.

2.7. ELISA for detecting known concentrations of F. hepatica E/S antigen

The efficacy of the assay to detect E/S antigens of F. hepatica was tested by titrationof the E/S antigen mixed with PBS 0.3% tween 20, serum and fecal supernatant fromnon-infected sheep. The initial concentration of the E/S antigens was 100 �g/ml which wasserially diluted to 0.012 �g/ml. These concentrations were tested in duplicate.

2.8. Detection of heterologous parasite antigens

In order to verify if antigens from other parasites could be detected by the assay, wholeworm extracts (25 �g/ml) from Paramphistomum spp., M. expansa, M. benedeni, T. solium,M. hirudinaceus, H. contortus, A. suum and T. spiralis in PBS, serum and fecal supernatantsof negative animals were tested.

2.9. Determination of sensitivity

Sensitivity was assessed by the use of the absorbance values obtained at ninth wpi whenall animals had positive fecal egg counts, using the formula described by Rosales (1989):

Sensibility = a

a + c× 100


where a is the number of sick animals which were positive to the test, and c the number ofsick animals which were negative to the test.

2.10. Statistical analysis

The absorbance values obtained with the four groups during the experiment as well as thenumber of flukes collected at necropsy were compared between groups and submitted to ananalysis of variance (ANOVA) using the Kruskall-Wallis and Nemenyi test (Zar, 1989). Todetermine the correlation between the absorbance of E/S antigens in serum and feces withthe fluke burden, a correlation analysis among assays was performed, using the Spearmancorrelation coefficients. All data were analyzed using the Statistical Analysis System(1982).

3. Results

On the eighth wpi, three animals in group 1, three animals in group 2 and two ani-mals in group 3, were positive for F. hepatica eggs. By the ninth wpi all infected animalswere positive for fluke eggs. The highest peak of egg shedding was observed on week12 post-infection, when necropsy was done (Table 1). There was a significant difference(p < 0.05) in the mean number of eggs between groups 1 and 3 from 10 wpi to the end ofthe study, and there were no differences between the others groups (p > 0.05).

Table 2 shows the recovery of flukes at necropsy. Animals in group 1 infected with 50metacercariae had a mean of 13.6 worms. Animals in group 2 infected with 100 metacer-cariae had a mean of 36.2 worms and animals in group 3 infected with 200 metacercariaehad a mean of 84.4 worms. There was only a significant difference (p < 0.05) betweengroups 1 and 3.

The absorbance values obtained with the ELISA in PBS, serum and fecal supernatantwere associated with the concentration of E/S antigen used (Fig. 1). The cutoff point forPBS was 0.046, for serum 0.240 and for fecal supernatant 0.348. The capability of the assayto detect F. hepatica E/S antigens was 12 ng/ml for PBS, 190 ng/ml for serum and 90 ng/mlfor fecal supernatant.

Table 1Mean ± standard deviation of F. hepatica eggs per gram of feces in the coprological examination of three infectedgroups of sheep

Group (n = 5)a Weeks post-infection

8 9 10 11 12

1 2 ± 1 3 ± 2.3 3.0 ± 1.5∗ 18.4 ± 4.7∗ 13.8 ± 7.3∗2 2.3 ± 1.15 4.12 ± 4.58∗ 12.4 ± 8.76 40 ± 18.7 91.6 ± 49.93 1.5 ± 0.70 1.8 ± 3.11 12.0 ± 8.3∗ 90.8 ± 59.1∗ 194.2 ± 167.8∗

a Groups 1, 2 and 3 were infected with 50, 100 and 200 metacercariae of F. hepatica, respectively.∗ Indicates significant difference (p < 0.05).


Table 2Number of F. hepatica recovered at necropsy from three experimentally infected groups of sheep

Group (n = 5)a Recovered parasites

Average S.D. Minimum Maximum Percentage of inoculation

1 13.6∗ 3.43 11 19 27.22 36.2 16.2 17 56 36.23 84.4∗ 2.96 80 88 42.2

a Groups 1, 2 and 3 were infected with 50, 100 and 200 metacercariae of F. hepatica, respectively.∗ Indicates significant difference (p < 0.05).

There was no cross-reactivity in the double antibody capture ELISA for F. hepatica whenwhole worm antigens of Paramphistomum spp., M. expansa, M. benedeni, T. solium, M.hirudinaceus, H. contortus, A. suum and T. spiralis were tested (Fig. 2).

Results on the detection of F. hepatica E/S antigens in serum showed absorbance valuesover the cutoff point (≥0.240) from the first wpi in some animals from the three infectedgroups (Fig. 3). Groups 1 and 2 showed similar optical values, starting with one positiveanimal from group 1 and two positive animals from group 2 at the first wpi. At week 9, fourout of five animals of each group were positive and maximum detection occurred in positiveanimals in this week. Three animals from group 3 were positive from the first wpi, and atweek 4, all animals in this group were positive and remained so during the whole experiment.Mean absorbance values of the control group were significantly different (p < 0.05) fromgroup 3 at the second wpi, from group 1 at the third wpi and from group 2 at the fourthwpi. No statistical differences were observed (p > 0.05) between the three infected groupsduring the whole experiment.

Fig. 1. Titration of E/S antigen of F. hepatica in different diluents by the capture ELISA.


Fig. 2. Different parasite whole worm antigens tested by the capture-ELISA for cross-reactivity with F. hepatica.

For fecal E/S antigens, mean absorbance values of all infected groups were over thecutoff point (≥0.348) from the fourth wpi, and remained over the cutoff point until necropsy(Fig. 4). At week 4, five animals were negative, three animals from group 1 and the other twofrom groups 2 and 3. All animals from group 3 were positive from the fifth wpi. There was

Fig. 3. Serum E/S antigen values obtained by capture ELISA in experimentally infected sheep with metacercariaeof F. hepatica. Groups 1, 2 and 3 were infected with 50, 100 and 200 metacercariae of F. hepatica, respectively.


Fig. 4. Coproantigen (E/S) values obtained by capture ELISA in experimentally infected sheep with metacercariaeof F. hepatica. Groups 1, 2 and 3 were infected with 50, 100 and 200 metacercariae of F. hepatica, respectively.

no significant difference (p < 0.05) in mean absorbance values of the four groups duringthe first three wpi; however, there was significance (p < 0.05) from the fourth wpi, inthe absorbance obtained in the three infected groups when compared with the non-infectedcontrol group. There was no significant difference in mean absorbance values between thethree infected groups (p > 0.05).

At the ninth wpi, 13 animals out of 15 infected animals were positive to serum antigensand 14 out of 15 were positive to coproantigens. The sensitivity in serum and feces, was 86.6and 93.3%, respectively. The correlation coefficient between absorbance of E/S antigensin the serum and feces with the parasite burden was of 0.77 and 0.76 (p < 0.0001),respectively.

4. Discussion

All sheep were successfully infected with gelatin capsule containing either 50, 100 or 200metacercariae of F. hepatica raising an implantation percentage of 27.2, 36.2 and 42.2%,respectively. This number of worms collected is similar to the data obtained by others (Borayet al., 1983).

Detection of parasite antigens in feces has been reported for different parasitic dis-eases (Deplazes et al., 1990; Vinayak et al., 1991; Allan and Craig, 1992; Baronet andWaltner-Toews, 1994). At the present time, there are several immunological methods ca-pable of detecting antigens of F. hepatica with high sensitivity. Espino and Finlay (1994)detected up to 15 ng/ml in feces of humans and Abdel-Rahman et al. (1998) detected 300 pgof coproantigen/ml of fecal supernatant in calves. In the present study, the E/S antigens ofF. hepatica were detected in quantities of 12 ng/ml in PBS, 190 ng/ml in serum and 90 ng/mlin feces of sheep. The values obtained with PBS are in line with those reported in a previousstudy (Espino et al., 1990). However, the detection values in serum and feces was lowerthan that reported by Espino and Finlay (1994) and Abdel-Rahman et al. (1998). These


differences may be due to the capture antibody used, since in our work a polyclonal serumwas used whereas in the two other studies two different monoclonal antibodies were used.The absorbance values obtained in the titration curve of the E/S antigen in PBS, serum andfecal supernatant, showed a proportional relationship to the concentration of antigen usedand the absorbance values of the E/S antigen in the serum and in the feces were higher whencompared with PBS. Also, the cutoff point and the detection limits for the E/S antigens weredifferent between diluents, and this may be attributable to components present in the serumand in feces which resulted in an elevation of the non-specific limit of the assay (Viscidiet al., 1984). The presence of fecal proteases as desorbing interfering factors in human fecalsamples has been reported. The use of 50% fetal calf serum or 5% BSA in diluting buffersimproved antigen detection by ELISA (Viscidi et al., 1984). In the present study, fecal sam-ples were homogenized in PBS 0.3% tween 20 containing 0.1% BSA, while sera were usedundiluted in the ELISA for E/S antigens; the addition of BSA to the diluting buffer mayexplain the difference in the detection limits of the assay, when the E/S F. hepatica antigenwere added to fecal supernatants of non-infected animals.

The ELISA permitted detection of E/S F. hepatica antigens from the first wpi in theserum of six infected sheep, and from the fourth wpi the absorbance values were signif-icantly different (p < 0.05) in all three groups with reference to the control group. Thehighest detection of F. hepatica serum E/S antigens was recorded at ninth wpi, and this is inaccordance with that reported by Rodrıguez and Hillyer (1995), who detected antigens ofthe parasite in serum of sheep from the second wpi, recording the highest absorbance valuesat eighth wpi. This is also in accordance with natural infections with F. hepatica in humanswhere fluke antigens were detected in the serum of patients before egg shedding in feces(Espino et al., 1987, 1990). Duménigo et al. (1999) also detected circulating E/S antigensof F. hepatica in the serum of experimentally infected sheep in the first wpi. High variationregarding the absorbance values was observed. The S.D. at the beginning of the infectionwas small, being the highest S.D. in infected groups at ninth wpi. These increments of theS.D. are in coincidence with the time in which parasites becomes adults, which may releasehigh quantities of antigen into circulation and also this may be attributable to the physiolog-ical activity of the parasites in the bile ducts (Hanna, 1980). A decrease in antigenemia wasobserved after the ninth wpi, and this was also observed by Rodrıguez and Hillyer (1995),in F. hepatica infected sheep at 10th wpi and by Duménigo et al. (1999), who reportedthat infected sheep showed negative results at seventh wpi. The fact that absorbance valuesdecreased after the ninth wpi could be attributed to the establishment of the parasite in thebile duct, with reduction of antigen available to circulation (Langley and Hillyer, 1989;Abdel-Rahman et al., 1998).

The detection of E/S F. hepatica antigens in feces of experimentally infected sheep waspossible in some animals starting at fourth wpi and OD values increased continuously untileighth wpi when the highest value was observed. From the fourth wpi, the mean absorbancevalues were significantly different (p < 0.05) from controls. This is in accordance withthat reported by Duménigo et al. (1999), who detected antigens of the parasite in feces frominfected sheep at fifth wpi and with Abdel-Rahman et al. (1998), who detected coproantigenin calves infected with more than 10 flukes by the sixth wpi. This coincides with the arrivalof most of the flukes in the bile ducts and release of E/S products into the bile (Quiroz,1995). F. hepatica E/S antigens in feces would not be detectable prior to 4 weeks because


flukes are outside the bile ducts. The higher detection observed at eighth wpi may beattributed to the rapid growth and maturation of the flukes associated with a higher metabolicrate and increased shedding of antigens (Abdel-Rahman et al., 1998). The decrease ofcoproantigen observed at ninth wpi has also been reported by Rodrıguez and Hillyer (1995)and Abdel-Rahman et al. (1998), and this may be due to the fact that after the establishmentof flukes in the bile duct, shedding of the surface antigens is continuous, although at aslightly slower rate (Hanna, 1980).

No cross-reactions occurred when whole worm antigen of parasites such as Paramphis-tomum spp., H. contortus, M. expansa, M. benedeni, M. hirudinaceus, T. spiralis, A. suum,and T. solium were used in the assay. It would be suitable to use E/S antigens of differentparasites, but due to the time in which this study was done, it was not possible to do it.However, previous reports have been demonstrated that a polyclonal antibody against E/Santigen of tape worms can also detect whole worm antigen from different parasites of thesame genus (Deplazes et al., 1990, 1991). In the work reported by Espino et al. (1990),the specificity was 100%. Therefore, the possibility of false positives would be very low,thus making the test highly specific. The coefficient of correlation for E/S antigens in theserum with the fluke burden was 0.77, this indicates a positive linear correlation. As thefluke burden increases, the quantity of E/S antigens in the serum also increases. Espinoand Finlay (1994) demonstrated a correlation between the absorbance of antigens with thenumber of immature flukes migrating to the bile ducts of humans, which contrasted withthe results of Espino et al. (1997) where there was no correlation between antigenemia andfluke burden in rats experimentally infected with F. hepatica. Likewise, Duménigo et al.(1999) did not find any correlation between adult worms, number of eggs and E/S antigensin serum of sheep. The correlation coefficient for absorbance of E/S antigens in feces withthe fluke burden (0.76) suggests that there is also positive correlation between the excretedE/S antigens in feces and the fluke burden. This agrees with data previously reported byCastro et al. (1994), who found a positive correlation between the number of F. hepaticaeggs and the detection of antigens in the feces of cattle using the sandwich-ELISA method.Duménigo et al. (1996) also found a positive correlation between the fluke burden and theconcentration of coproantigens in 100 cattle sacrificed at the slaughterhouse. In calves withexperimentally induced infection, Abdel-Rahman et al. (1998) found a strong correlation(r = 0.96) between the OD values for the fecal ELISA and number of flukes. Duménigoet al. (1999) also found a positive correlation between fecal antigens in sheep experimen-tally infected, and egg output and adult worm burden. Other studies with cestodes (Avila,1992; Sakai et al., 1995) have also shown that detection of coproantigens is dependent onthe parasite burden. However, Allan and Craig (1994) did not find any relationship betweendetection of coproantigens and parasite burden in mice infected with H. diminuta.

In previous studies, it was demonstrated that F. hepatica shares antigens with S. mansoniand F. gigantica (Hanna and Hillyer, 1984), which can produce a cross-reaction when testsfor detection of antibodies are done (Rodrıguez and Hillyer, 1995). In this assay, no antigensfrom S. mansoni, F. gigantica and D. dendriticum, were tested. However, in Mexico thereis no information regarding the presence of the first two parasites and with regard to D.dendriticum, there are just two reports on its presence (Basurto and Peláez, 1898; Quirozet al., 1997). Therefore, possible cross-reactions with antigens of these trematodes probablyis not a factor that would affect the use of this test to diagnose fasciolosis in Mexico.


E/S antigens in serum were detected during the first wpi, and decreased after the prepatentperiod. However, E/S antigens in the feces were detected during the prepatent period andthey were still present after the patent period. The combined use of both assays increasesthe capability of diagnosis of fasciolosis using this capture ELISA.

Acknowledgements

The authors are indebted to Biol. Lauro Trejo from the Centro Nacional de ParasitologıaAnimal for the kind donation of metacercariae, to Dr. Ana Flisser and staff, from theDepartment of Microbiology and Parasitology, Faculty of Medicine-UNAM and personalfrom CEPIER-UNAM for all given support, to Canadian International Development Agency(CIDA) and to Dirección General de Intercambio Académico (UAT-UNAM) by a grantgiven to the first author to carry out M. Sc. studies. Study partially supported by the projectPAPIIT-IN218996 DGAPA-UNAM.

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effect of parasite burden on the detection of fasciola hepatica antigens in sera and feces of...

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