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Using indirect ELISA to assess different antigens for the serodiagnosis of Fasciola gigantica infection in cattle, sheep and donkeys
Research in Veterinary Science 86 (2009) 466–471
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Using indirect ELISA to assess different antigens for the serodiagnosis of Fasciola gigantica infection in cattle, sheep and donkeys W.S. Awad a,c,*, A.K. Ibrahim b,c, F.A. Salib a a
Department of Internal Medicine and Infectious Diseases, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt Department of Clinical Pathology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt c Biotechnology Center for Services and Research, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt b
a r t i c l e
i n f o
Article history: Accepted 21 August 2008
Keywords: iELISA Fasciola gigantica Cattle Sheep Donkeys Crude worm antigen Excretory–secretory antigen Glutathione S-transferase antigen
a b s t r a c t An indirect enzyme-linked immunosorbent assay (iELISA) was evaluated for its diagnostic capability in detecting antibodies against Fasciola gigantica infection in cattle, sheep and donkeys sera using crude worm, excretory–secretory and glutathione S-transferase antigens prepared from adult liver fluke. Presence of F. gigantica worms at post-mortem examination of cattle, sheep and donkey’s livers was taken as a gold standard for the evaluation of the assay. The diagnostic sensitivity, specificity and accuracy percentages of iELISA were determined for each antigen. Excretory–secretory antigen gave the best results for the serodiagnosis of F. gigantica infection in cattle, sheep and donkeys using iELISA with diagnostic sensitivity percentages of 93.3%, 94.9% and 93.3%, respectively, while the specificity percentages were 96.7%, 97.2% and 96.3%, respectively, whereas the accuracy percentages were 95%, 96% and 95.7%, respectively. The diagnostic sensitivity percentages of iELISA using crude worm antigen were 96.7%, 100% and 93.3%, respectively, while the specificity percentages were 80%, 83.3% and 85.2%, respectively, whereas the accuracy percentages were 88.3%, 86.7% and 87%, respectively. The diagnostic sensitivity percentages of iELISA using glutathione S-transferase antigen were 66.7%, 71.8% and 60%, respectively, while the specificity percentages were 70%, 77.8% and 77.8%, respectively, whereas the accuracy percentages were 68.3%, 74.7% and 73.9%, respectively. Conclusively, excretory–secretory antigen dependent iELISA can be used as a reliable serodiagnostic test for F. gigantica infection in cattle, sheep and donkeys. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Fasciolosis is a parasitic disease caused by the digenetic trematodes, Fasciola gigantica (tropical liver fluke) and Fasciola hepatica (temperate liver fluke), leads to a significant economic loss (FAO, 1994; Torgerson and Claxton, 1999). Fasciolosis reduces the production of meat, milk and wool, in addition to losses due to mortalities, liver condemnation and expenditures for anthelmintics using (Mas-Coma et al., 1995; Hillyer and Apt, 1997). Recently fasciolosis recognized as an important pathogen of human worldwide (Mas-Coma et al., 1999). The Nile Delta considered one of the endemic areas in the world for human fasciolosis (Safar et al., 2005; Curtale et al., 2007). Human cases with liver cirrhoses and ectopic infection in vital organs as brain, lung and eye had been recorded (Mas-Coma et al., 1999; Dalimi and Jabarvand, 2005).
* Corresponding author. Address: Department of Internal Medicine and Infectious Diseases, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt. Tel.: +20 2 25916083; fax: +20 2 35725240. E-mail address: [email protected] (W.S. Awad). 0034-5288/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2008.08.009
In Egypt, the disease is highly enzootic affecting cattle, sheep, donkeys, goats and buffaloes (Haridy et al., 2002, 2006; Mazyad and El-Nemr, 2002; Awad et al., 2004; El-Shazly et al., 2005; Morsy et al., 2005). Traditionally, diagnosis based on observation of Fasciola eggs in feces (Boray, 1985), while the pathological findings of immature fasciolosis occurs as early as 3 weeks post-infection, whereas coprological diagnosis can be performed only at 2 months post-infection in cattle (Hillyer et al., 1985) or 3 months post-infection in sheep (Zimmerman et al., 1982). This method of diagnosis has a poor sensitivity during the patent period due to the relatively low number of eggs shed, particularly in cattle (Happich and Boray, 1969). The early diagnosis of fasciolosis during prepatent period using antibody detection tests make immunodiagnosis an essential method for arresting its negative impact on productivity and preventing economic loss (Fagbemi et al., 1997; Sanchez-Andrade et al., 2000; Dixit et al., 2004). To improve diagnosis during both early and chronic infections, an increasing interest directed towards the serodiagnosis of parasitic diseases as a rapid, simple to perform and inexpensive technique (Arriaga et al., 1989; Castro et al., 1995).
W.S. Awad et al. / Research in Veterinary Science 86 (2009) 466–471
ELISA-based antibody detection systems are excellent for the early diagnosis of fasciolosis in animals and are suitable for epidemiological studies (Clery et al., 1996; Bossaert et al., 2000). Several studies had been performed on immunodiagnosis of F. hepatica infection in cattle and sheep (Zimmerman et al., 1985; Hillyer and Galanes, 1988; Santiago and Hillyer, 1988), while fewer studies had been done on serodiagnosis of F. gigantica infection in ruminants (Swarup et al., 1987; Fagbemi and Obarisiagbon, 1990). The purpose of this study is to determine the diagnostic sensitivity, specificity and accuracy of the indirect ELISA using three extracted F. gigantica (crude, excretory–secretory and glutathione Stransferase) antigens for serodiagnosis of fasciolosis in cattle, sheep and donkeys, using post-mortem examination of their livers as a gold-standard for the evaluation of the assay.
2. Materials and methods 2.1. Animals Sixty cattle and 75 sheep slaughtered at El-Monieb and El-Warrak slaughterhouses and 69 donkeys slaughtered at Giza zoo (as a food for carnivorous animals) were examined for the presence of mature and immature fasciola flukes in their livers and bile ducts according to the method of Anderson et al. (1999). Each liver was placed in a large basin, all the flukes in the gall bladder and the major bile ducts were collected into a small plastic cup. The liver was then sliced into strips of about 1 cm in thickness and soaked in normal saline for about 1 h. Flukes emerging from the cut bile ducts were put into the plastic cup and each sliced strip was thoroughly squeezed from end to end, washed in saline and discarded. The contents of the basin sieved, put into a Petrie dish and the adult, immature and cut pieces of flukes were added to the cup. Identification of the live flukes performed according to Andrews (1999). Examined animals were classified into fasciola infected and fasciola free groups. 2.2. Samples Whole blood samples without anticoagulant were collected from fasciola infected and fasciola free animals for separation of sera. Blood samples were allowed to clot on bench at room temperature in inclined position for few hours, centrifuged at 3000 rpm for 20 min and serum samples were obtained and stored at 20 °C until tested. 2.3. Preparation of F. gigantica antigens 2.3.1. Preparation of crude worm antigen Fasciola gigantica crude worm antigen (Fg-Cr Ag) was prepared as described by Oldham and Williams (1985). Briefly, adult F. gigantica flukes collected from the bile ducts of cattle were washed three times in phosphate buffered saline (PBS), drained and freezedried for 24 h at 80 °C. The dried flukes were ground into a fine powder and suspended in PBS, then homogenized in a high-speed blender for 15 min. at maximum speed and allowed to extract overnight at 4 °C. After centrifugation at 3000 rpm for 15 min, the supernatant was filtered and sterilized by passing through 0.45 lm and 0.22 lm Millipore filters, aliquotted and stored at 80 °C until assayed. 2.3.2. Preparation of F. gigantica excretory/secretory antigen Fasciola gigantica excretory/secretory antigen (Fg-ES Ag) was prepared according to Simsek et al. (2006). Briefly, adult F. gigantica worms were collected from the bile ducts of cattle, washed several times in 0.01 M PBS, pH 7.4 to remove all traces of blood and bile.
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The worms were incubated in PBS (5 worms/10 ml) at 37 °C for 6 h. Worms were removed by sieving and sieved PBS containing E/S products was centrifuged at 10,000 rpm for 30 min at 4 °C, then the supernatant was filtered from a 0.2 lm Millipore filter. Subsequently, these products were dialyzed against distilled water for 24 h, aliquotted and stored at 80 °C until assayed. 2.3.3. Preparation of F. gigantica glutathione S-transferase (GST) antigen Fasciola gigantica glutathione S-transferase antigen (Fg-GST Ag) was prepared according to Estuningsih et al. (1997) using the affinity chromatography method. A pre-packed Glutathione Sepharose 4-B (Pharmacia Biotech., Sweden) was used for purification of GST. The column washed with 20 ml PBS (pH 7.3) to remove the preservatives, the gel equilibrated with 6 ml PBS + 1% Triton X100, the F. gigantica crude antigen was applied diluted in PBS + 1% Triton X-100 to the column, the effluent discarded, then the column washed with 2 10 ml of PBS, the bound material was eluted with 10 ml of elution buffer (5 mM Glutathione in 50 mM Tris–HCl, pH 8.0) and 1–2 ml fractions were collected and stored at 80 °C until assayed. 2.3.4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) The SDS–PAGE analysis of Fg-Cr, Fg-ES and Fg-GST antigens of F. gigantic was performed according to Laemmli (1970) and the obtained protein bands were stained with Coomassie blue R-250. 2.4. Measurement of protein concentration in prepared antigens Protein concentration of each prepared antigens (Fg-Cr, Fg-ES and Fg-GST) was determined according to Lowry et al. (1951). 2.5. Application of indirect enzyme-linked immunosorbent assay (iELISA) The iELISA was carried out according to Zimmerman et al. (1982) and the optimal antigen, serum and conjugate concentrations were determined after preliminary checkerboard titration according to (Catty and Raykundalia, 1989). In this study, the optimum conditions were 5 lg/ml coating buffer for Fg-Cr and Fg-ES antigens, 10 lg/ml coating buffer for Fg-GST antigen, 1:100 serum dilution, 1:3000 alkaline phosphatase labeled rabbit anti-bovine, anti-ovine and anti-horse IgG (Sigma) as conjugate and 1 mg pnitrophenyl phosphate (p-NPP) dissolved in 1 ml substrate buffer as substrate. The iELISA was performed in 96 wells microtiter plates (NuncImmuno Plate MaxiSorp, Intermed). The wells were coated with 100 ll of antigen (at optimal conc.) diluted in 0.1 M carbonate buffer (pH 9.6) and incubated overnight at 4 °C. Washed twice with PBS containing 0.01% Tween-20 (PBS/Tween) and blocked with 100 ll per well of 0.1% bovine serum albumin in 0.01 M PBS (pH 7.4) for 1.5 h at 37 °C. After blocking, plates were washed three times with PBS/Tween. A 100 ll of cattle, sheep or donkey sera (diluted 1:100 in PBS) were added to the wells, followed by incubation for 1.5 h at 37 °C. The plates were washed again (five times) and 100 ll of 1:3000 anti-bovine, anti-ovine or anti-horse (according to the animal species) alkaline phosphatase labeled rabbit IgG (diluted in PBS) was added to the wells and the plates were incubated at 37 °C for 2 h. Finally, after another five washes, 50 ll of p-NPP diluted in substrate buffer was added to each well, and after incubation in the dark for 15 min at 37 °C the enzymatic reaction was stopped with 50 ll/well of 3 N NaOH. The absorbance of each well was read at a wavelength of 405 nm using a titertek multiskan ELISA reader and the results were expressed as optical density (OD).
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In each ELISA plate, three negative control sera and one positive control serum were included for each animal species. Negative control sera were obtained from six young animals less than 4 months old with two successive negative (3 months intervals) fecal examination for fasciola eggs, while the positive control serum was a pool of sera obtained from four slaughtered animals with adult liver fluke in their livers. Positive values were assigned according to Rodriguez-Peiez and Hillyer (1995) as those values with absorbance readings greater than the cut off value, which calculated as mean OD of negative sera plus three standard deviations. 2.6. Diagnostic evaluation of indirect-ELISA Results of iELISA using different antigens of F. gigantica for detecting antibodies against fasciola in sera of cattle, sheep and donkeys were evaluated against the results of presence or absence of flukes in their livers which taken as a gold standard and the diagnostic sensitivity, specificity and accuracy of the assay were calculated according to Timmreck (1994) and Smith (1995). Sensi Tþ 100 means the ability of a test to correctly identivity ¼ ðTþÞðFÞ tify the percentage of those who have the disease; while specificity T ¼ ðTÞþðFþÞ 100 means the ability of a test to correctly identify the percentage of those who do not have the disease; whereas accuracy ¼ ðTþÞþðTÞ 100 describe the degree to which measureTN ment reflects the true status of what is being measured and it is used to express the overall performance of a diagnostic test. NB; T+ (=true positive), T (=true negative); F+ (=false positive), F (=false negative) and TN (= total number). 3. Results Livers of 60 cattle, 75 sheep and 69 donkeys were examined for the presence of mature and immature F. gigantica in their bile ducts. Cattle were classified into 30 infected and 30 non-infected animals, while sheep were classified into 39 infected and 36 noninfected animals, whereas donkeys were classified into 15 infected and 54 non-infected animals. Sera of these animals were collected and stored at 20 °C until be tested by iELISA.
Fig. 1. Migration pattern of Fg-Cr (A), Fg-ES (B) and Fg-GST (C) antigens in SDS– PAGE gel stained by Coomassie blue.
Antigens (Fg-Cr, Fg-ES and Fg-GST) were prepared from adult F. gigantica collected from the bile ducts of cattle and their protein content was determined. The migration pattern of Fg-Cr, Fg-ES and Fg-GST antigens in SDS–PAGE gel (Fig. 1) was analyzed using Gel-ProÒ analyzer v4. Fg-Cr antigen revealed 17 protein bands with molecular weight of 13.8, 15.7, 17.4, 20.8, 23.2, 25.6, 27.4, 30.1, 31.3, 32.4, 33.5, 35, 42.9, 53.3, 92.4, 178.4 and 262.3 kDa. Fg-ES antigen revealed seven protein bands with molecular weight of 15, 28, 31.6, 32.9, 39.4, 83.3 and 101.7 kDa. Fg-GST antigen revealed one protein band with a molecular weight of 24 kDa. Goat antiserum to Fg-GST (obtained from research teamwork studying vaccination of goats against fasciolosis using Fg-GST) cross-reacts with the prepared Fg-GST, confirming it is indeed GST. The results of both examination of livers for the presence or absence of liver fluke and in same time corresponding results of iELISA using Fg-Cr, Fg-ES and Fg-GST antigens were obtained and illustrated in Table 1. The diagnostic sensitivity, specificity and accuracy percentages of iELISA using Fg-Cr, Fg-ES and Fg-GST antigens for diagnosis of F. gigantica infection in cattle, sheep and donkeys were calculated and recorded in Table 2.
4. Discussion and conclusion For several decades, F. gigantica infection was diagnosed by fecal examination which has several disadvantages such as it is time and effort consuming, needs a suitable volume of fecal sample, cannot detect early infection with high-false negative rate in chronic infection due to the intermittent shedding of eggs in feces and cases with ectopic infection. Examination of 5 g of feces had a sensitivity of 66.7% from cattle whose infection status had been confirmed at necropsy examination (Anderson et al., 1999) with higher sensitivity obtained by three sub-samples leading to traditional coproscopy methods can have a superior test performance over other test systems. The main limitation of this approach is the analysis of large volumes of feces is labour intensive with possible sources of misidentification of eggs include the presence of paramphistomum eggs which have similar morphology but in a different color. Fecal examination is also unable to diagnose ectopic fasciolosis where fasciola worm develop in tissues other than liver. All of these encourage us to study another method for diagnosis of fasciolosis. Recent studies on fasciolosis focused on the importance of ELISA in diagnosis of animal fasciolosis and it becomes the most widely used test, because of its simplicity, reliability and easy mechanization. Several attempts to purify different fasciola antigens used in the assay to improve its diagnostic sensitivity and specificity. In this study, three F. gigantica antigens (Fg-Cr, Fg-ES and FgGST) were used to evaluate the diagnostic sensitivity, specificity and accuracy of iELISA for the diagnosis of F. gigantica infection in cattle, sheep and donkeys. Results of iELISA were evaluated in comparison to the results of post-mortem findings (presence of fluke in liver of examined animal) which used as a gold standard. True positive, false positive, true negative and false negative cases were determined (Table 1), as well as sensitivity, specificity and accuracy percentages were recorded for each antigen and animal species (Table 2). False positive cases were detected in examined cattle (60), sheep (75) and donkeys (69) sera by iELISA using Fg-Cr Ag as 6, 6 and 8 cases in percentages of 10%, 8% and 11.6%, respectively, while by using Fg-ES Ag they were 1, 1 and 2 cases in percentages of 1.7%, 1.3% and 2.9%, respectively, whereas by using Fg-GST Ag they were 9, 8 and 12 cases in percentages of 15%, 10.7% and 17.4%, respectively. False positive cases detected by iELISA may be attributed to recent administration of effective fasciolicides which eliminated
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W.S. Awad et al. / Research in Veterinary Science 86 (2009) 466–471 Table 1 Results of examination of cattle, sheep and donkeys livers for liver flukes and iELISA on their sera using Fg-Cr, Fg-ES and Fg-GST antigens Results of iELISA using
Results of liver examination in Cattle (60)
Fg-Cr Ag
+
Fg-ES Ag
+
Fg-GST Ag
+
Sheep (75)
Donkey (69)
+ (30)
(30)
+ (39)
(36)
+ (15)
(54)
29 (48.3%) 1 (1.7%)
6 (10%) 24 (40%)
39 (52%) 0 (0%)
6 (8%) 30 (40%)
14 (20.3%) 1 (1.5%)
8 (11.6%) 46 (66.7%)
28 (46.7%) 2 (3.3%)
1 (1.7%) 29 (48.3%)
37 (49.3%) 2 (2.7%)
1 (1.3%) 35 (46.7%)
14 (20.3%) 1 (1.5%)
2 (2.9%) 52 (75.4%)
20 (33.3%) 10 (16.7%)
9 (15%) 21 (35%)
28 (37.3%) 11 (14.7%)
8 (10.7%) 28 (37.3%)
9 (13%) 6 (8.7%)
12 (17.4%) 42 (60.9%)
Table 2 The diagnostic sensitivity, specificity and accuracy percentages of iELISA using Fg-Cr, Fg-ES and Fg-GST antigens for diagnosis of F. gigantica infection in cattle, sheep and donkeys Antigen type
Fg-Cr Ag
Animal spp. Sensitivity (%) Specificity (%) Accuracy (%)
Cattle 96.7 80 88.3
Fg-ES Ag Sheep 100 83.3 86.7
Donkey 93.3 85.2 87
Cattle 93.3 96.7 95
adult flukes from the biliary ducts but specific antibodies still persist in serum for 2–7 months (Zimmerman et al., 1982; Santiago and Hillyer, 1988; Levieux et al., 1992; Castro et al., 2000) and also this may be due to the possession of common antigens by different helminths (Pelley and Hillyer, 1978; Fagbemi and Obarisiagbon, 1991). These two phenomena present a problem in the serodiagnosis of fasciolosis and this means that any serodiagnostic test used to diagnose infection in individual animals must be interpreted with care. In this study five cases of donkeys were infected with hydatid cysts in their livers, two of them gave positive result with iELISA using Fg-Cr Ag only, one gave positive result with both iELISA using Fg-Cr and Fg-GST antigens, another one gave positive result with both iELISA using Fg-Cr and Fg-ES antigens, while last one do not react with any antigen. These findings showed a clear crossreaction was detected between fasciola antigens (especially Fg-Cr Ag) and hydatid cysts antigens that clarify some false positive cases in donkeys. False negative cases detected in cattle, sheep and donkeys sera by iELISA using Fg-Cr Ag were 1, 0 and 1 in percentages of 1.7%, 0% and 1.5%, respectively, while by using Fg-ES Ag 2, 2 and 1 cases were recorded in percentages of 3.3%, 2.7% and 1.5%, respectively, whereas by using Fg-GST Ag 10, 11 and 6 cases were recorded in percentages of 16.7%, 14.7% and 8.7%, respectively. The false negative results may be attributed to modulation of the host immune response by liver flukes as reported by Sandeman and Howell (1981) and Zimmerman et al. (1983) who recorded a significant immune suppression during F. hepatica infection. Sensitivity percentages of iELISA using Fg-Cr Ag were calculated for diagnosis of fasciolosis in cattle, sheep and donkeys as 96.7%, 100% and 93.3%, respectively, while by using Fg-ES Ag were 93.3%, 94.9% and 93.3%, respectively, whereas by using Fg-GST Ag were 66.7%, 71.8% and 60%, respectively. It was clear that, the highest sensitivity (100%) was obtained by using Fg-Cr Ag for diagnosis of F. gigantica infection in sheep, whereas the lowest sensitivity (60%) was obtained by using Fg-GST Ag for diagnosis of F. gigantica infection in donkeys. Specificity percentages of iELISA were also calculated with highest specificity (97.2%) was recorded when Fg-ES Ag was used in iELISA for diagnosis of F. gigantica infection
Fg-GST Ag Sheep 94.9 97.2 96
Donkey 93.3 96.3 95.7
Cattle 66.7 70 68.3
Sheep 71.8 77.8 74.7
Donkey 60 77.8 73.9
in sheep, while the lowest specificity (70%) was recorded when Fg-GST Ag was used in iELISA for diagnosis of F. gigantica infection in cattle. From these results, it was clear that, sensitivity of iELISA using Fg-Cr Ag (93.3–100%) was less high than that when using Fg-ES Ag (93.3–94.9%), but in the same time, its specificity (80–85.2%) is obviously lower than that of Fg-ES Ag (96.3–97.2%). This may be attributed to the numerous antigenic components of Fg-Cr Ag than those of Fg-ES Ag leading it to be more sensitive and less specific than Fg-ES Ag. The high sensitivity (more than 93.3%) and high specificity (more than 96.3%) of iELISA using Fg-ES Ag for the diagnosis of F. gigantica infection in cattle, sheep and donkeys support the use of this antigen in the serodiagnosis of fasciolosis in different animal species as described by Zimmerman et al. (1982), Santiago and Hillyer (1988), Mousa (1994), Hillyer et al. (1996), Ibarra et al. (1998), Reichel (2002), Awad et al. (2004), Molloy et al. (2005), and Charlier et al. (2008). It must be taken in consideration that, the sensitivity and specificity of tests will vary according to the population on which they are tested (Whiting et al., 2004; Leeflang and Bossuyt, 2005) and the season at which samples being tested (Charlier et al., 2008). Furthermore, no tests currently available can be considered as having both 100% sensitivity and 100% specificity. Glutathione-S-transferases are present as several isoenzymes in Fasciola and are important for the detoxification of endogenous or exogenous toxic compounds (Mitchell, 1989; Brophy and Barrett, 1990). In spite of the effort, time and cost needed to purify FgGST Ag, it gives low sensitivity and specificity when used as a coating antigen in the iELISA for the diagnosis of F. gigantica infection in cattle, sheep and donkeys. This may be attributed to that, GST is located at the surface or just below the surface of the adult flukes, which are thought to continually, shed their teguments to evade the host immune system and thus GST is of very low concentration resulting in low-antibody response against it (Morphew et al., 2007). On the other hand, by using this antigen as a vaccine, a good protection rate against infection was recorded (Morrison et al., 1996; Spithill and Dalton, 1998).
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The accuracy percentages were recorded with the highest accuracy (96%) obtained when using iELISA with Fg-ES Ag for diagnosis of fasciolosis in sheep and the lowest one (68.3%) obtained when using iELISA with Fg-GST Ag for diagnosis of fasciolosis in cattle. Using Fg-ES Ag in iELISA gives high-accuracy rates (more than 95%) in cattle, sheep and donkeys. Conclusively, Fg-ES Ag was the best coating antigen in iELISA for the serodiagnosis of fasciolosis in cattle, sheep and donkeys due to its high sensitivity, specificity and accuracy rates. Acknowledgements We would like to express our deepest thanks to Professor Dr. Wahid Mousa, Department of Parasitology, Faculty of Veterinary Medicine, Cairo University for his great cooperation and assistance during the whole work, also we would like to express our sincere thanks to the National Academy of Science that funded us to complete our study.
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Farm animals’ fascioliasis in Ezbet El-Bakly (Tamyia Center) Al-Fayoum Governorate. J. Egypt. Soc. Parasitol. 35, 825–832. Mousa, W.M., 1994. Evaluation of specific F. Gigantica antigens for diagnosis of fascioliasis in experimentally and naturally infected sheep by ELISA. Vet. Med. J. 42, 77–81. Oldham, G., Williams, L., 1985. Cell mediated immunity to liver fluke antigens during experimental Fasciola hepatica infection of cattle. Parasite Immunol. 7, 503–516. Pelley, R.P., Hillyer, G.V., 1978. Demonstration of a common antigen between Schistosoma mansoni and Fasciola hepatica. Am. J. Trop. Med. Hyg. 27, 1192– 1194. Reichel, M.P., 2002. Performance characteristics of an enzyme-linked immunosorbent assay for the detection of liver fluke (Fasciola hepatica) infection in sheep and cattle. Vet. Parasitol. 107, 65–72. Rodriguez-Peiez, J., Hillyer, G.V., 1995. Detection of excretory-secretory circulating antigens in sheep infected with Fasciola hepatica and with Schistosoma mansoni and Fasciola hepatica. Vet. Parasitol. 56, 57–66. Safar, E., Mikhail, E., Bassiouni, G., El-Bassiouni, S., El-Kholy, H., 2005. Human fascioliasis in some areas in Cairo and Giza Governorates, Egypt. J. Egypt. Soc. Parasitol. 35, 181–192. Sanchez-Andrade, R., Paz-Silva, A., Suarez, J., Panadero, R., Diez-Banos, P., Morrondo, P., 2000. Use of sandwich enzyme linked immunosorbent assay for the diagnosis of natural Fasciola hepatica infection in cattle, Galicia (NW Spain). Vet. Parasitol. 93, 39–46. Sandeman, R.M., Howell, M.J., 1981. Response of sheep to challenge infection with Fasciola hepatica. J. Parasitol. 74, 818–820. Santiago, N., Hillyer, G.V., 1988. Antibody profile by EITB and ELISA of cattle and sheep infected with F. Hepatica. J. Parasitol. 74, 810–818. Simsek, S., Koroglu, E., Utuk, A.E., Altay, K., 2006. 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Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Using indirect ELISA to assess different antigens for the serodiagnosis of Fasciola gigantica infection in cattle, sheep and donkeys W.S. Awad a,c,*, A.K. Ibrahim b,c, F.A. Salib a a
Department of Internal Medicine and Infectious Diseases, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt Department of Clinical Pathology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt c Biotechnology Center for Services and Research, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt b
a r t i c l e
i n f o
Article history: Accepted 21 August 2008
Keywords: iELISA Fasciola gigantica Cattle Sheep Donkeys Crude worm antigen Excretory–secretory antigen Glutathione S-transferase antigen
a b s t r a c t An indirect enzyme-linked immunosorbent assay (iELISA) was evaluated for its diagnostic capability in detecting antibodies against Fasciola gigantica infection in cattle, sheep and donkeys sera using crude worm, excretory–secretory and glutathione S-transferase antigens prepared from adult liver fluke. Presence of F. gigantica worms at post-mortem examination of cattle, sheep and donkey’s livers was taken as a gold standard for the evaluation of the assay. The diagnostic sensitivity, specificity and accuracy percentages of iELISA were determined for each antigen. Excretory–secretory antigen gave the best results for the serodiagnosis of F. gigantica infection in cattle, sheep and donkeys using iELISA with diagnostic sensitivity percentages of 93.3%, 94.9% and 93.3%, respectively, while the specificity percentages were 96.7%, 97.2% and 96.3%, respectively, whereas the accuracy percentages were 95%, 96% and 95.7%, respectively. The diagnostic sensitivity percentages of iELISA using crude worm antigen were 96.7%, 100% and 93.3%, respectively, while the specificity percentages were 80%, 83.3% and 85.2%, respectively, whereas the accuracy percentages were 88.3%, 86.7% and 87%, respectively. The diagnostic sensitivity percentages of iELISA using glutathione S-transferase antigen were 66.7%, 71.8% and 60%, respectively, while the specificity percentages were 70%, 77.8% and 77.8%, respectively, whereas the accuracy percentages were 68.3%, 74.7% and 73.9%, respectively. Conclusively, excretory–secretory antigen dependent iELISA can be used as a reliable serodiagnostic test for F. gigantica infection in cattle, sheep and donkeys. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Fasciolosis is a parasitic disease caused by the digenetic trematodes, Fasciola gigantica (tropical liver fluke) and Fasciola hepatica (temperate liver fluke), leads to a significant economic loss (FAO, 1994; Torgerson and Claxton, 1999). Fasciolosis reduces the production of meat, milk and wool, in addition to losses due to mortalities, liver condemnation and expenditures for anthelmintics using (Mas-Coma et al., 1995; Hillyer and Apt, 1997). Recently fasciolosis recognized as an important pathogen of human worldwide (Mas-Coma et al., 1999). The Nile Delta considered one of the endemic areas in the world for human fasciolosis (Safar et al., 2005; Curtale et al., 2007). Human cases with liver cirrhoses and ectopic infection in vital organs as brain, lung and eye had been recorded (Mas-Coma et al., 1999; Dalimi and Jabarvand, 2005).
* Corresponding author. Address: Department of Internal Medicine and Infectious Diseases, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt. Tel.: +20 2 25916083; fax: +20 2 35725240. E-mail address: [email protected] (W.S. Awad). 0034-5288/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2008.08.009
In Egypt, the disease is highly enzootic affecting cattle, sheep, donkeys, goats and buffaloes (Haridy et al., 2002, 2006; Mazyad and El-Nemr, 2002; Awad et al., 2004; El-Shazly et al., 2005; Morsy et al., 2005). Traditionally, diagnosis based on observation of Fasciola eggs in feces (Boray, 1985), while the pathological findings of immature fasciolosis occurs as early as 3 weeks post-infection, whereas coprological diagnosis can be performed only at 2 months post-infection in cattle (Hillyer et al., 1985) or 3 months post-infection in sheep (Zimmerman et al., 1982). This method of diagnosis has a poor sensitivity during the patent period due to the relatively low number of eggs shed, particularly in cattle (Happich and Boray, 1969). The early diagnosis of fasciolosis during prepatent period using antibody detection tests make immunodiagnosis an essential method for arresting its negative impact on productivity and preventing economic loss (Fagbemi et al., 1997; Sanchez-Andrade et al., 2000; Dixit et al., 2004). To improve diagnosis during both early and chronic infections, an increasing interest directed towards the serodiagnosis of parasitic diseases as a rapid, simple to perform and inexpensive technique (Arriaga et al., 1989; Castro et al., 1995).
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ELISA-based antibody detection systems are excellent for the early diagnosis of fasciolosis in animals and are suitable for epidemiological studies (Clery et al., 1996; Bossaert et al., 2000). Several studies had been performed on immunodiagnosis of F. hepatica infection in cattle and sheep (Zimmerman et al., 1985; Hillyer and Galanes, 1988; Santiago and Hillyer, 1988), while fewer studies had been done on serodiagnosis of F. gigantica infection in ruminants (Swarup et al., 1987; Fagbemi and Obarisiagbon, 1990). The purpose of this study is to determine the diagnostic sensitivity, specificity and accuracy of the indirect ELISA using three extracted F. gigantica (crude, excretory–secretory and glutathione Stransferase) antigens for serodiagnosis of fasciolosis in cattle, sheep and donkeys, using post-mortem examination of their livers as a gold-standard for the evaluation of the assay.
2. Materials and methods 2.1. Animals Sixty cattle and 75 sheep slaughtered at El-Monieb and El-Warrak slaughterhouses and 69 donkeys slaughtered at Giza zoo (as a food for carnivorous animals) were examined for the presence of mature and immature fasciola flukes in their livers and bile ducts according to the method of Anderson et al. (1999). Each liver was placed in a large basin, all the flukes in the gall bladder and the major bile ducts were collected into a small plastic cup. The liver was then sliced into strips of about 1 cm in thickness and soaked in normal saline for about 1 h. Flukes emerging from the cut bile ducts were put into the plastic cup and each sliced strip was thoroughly squeezed from end to end, washed in saline and discarded. The contents of the basin sieved, put into a Petrie dish and the adult, immature and cut pieces of flukes were added to the cup. Identification of the live flukes performed according to Andrews (1999). Examined animals were classified into fasciola infected and fasciola free groups. 2.2. Samples Whole blood samples without anticoagulant were collected from fasciola infected and fasciola free animals for separation of sera. Blood samples were allowed to clot on bench at room temperature in inclined position for few hours, centrifuged at 3000 rpm for 20 min and serum samples were obtained and stored at 20 °C until tested. 2.3. Preparation of F. gigantica antigens 2.3.1. Preparation of crude worm antigen Fasciola gigantica crude worm antigen (Fg-Cr Ag) was prepared as described by Oldham and Williams (1985). Briefly, adult F. gigantica flukes collected from the bile ducts of cattle were washed three times in phosphate buffered saline (PBS), drained and freezedried for 24 h at 80 °C. The dried flukes were ground into a fine powder and suspended in PBS, then homogenized in a high-speed blender for 15 min. at maximum speed and allowed to extract overnight at 4 °C. After centrifugation at 3000 rpm for 15 min, the supernatant was filtered and sterilized by passing through 0.45 lm and 0.22 lm Millipore filters, aliquotted and stored at 80 °C until assayed. 2.3.2. Preparation of F. gigantica excretory/secretory antigen Fasciola gigantica excretory/secretory antigen (Fg-ES Ag) was prepared according to Simsek et al. (2006). Briefly, adult F. gigantica worms were collected from the bile ducts of cattle, washed several times in 0.01 M PBS, pH 7.4 to remove all traces of blood and bile.
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The worms were incubated in PBS (5 worms/10 ml) at 37 °C for 6 h. Worms were removed by sieving and sieved PBS containing E/S products was centrifuged at 10,000 rpm for 30 min at 4 °C, then the supernatant was filtered from a 0.2 lm Millipore filter. Subsequently, these products were dialyzed against distilled water for 24 h, aliquotted and stored at 80 °C until assayed. 2.3.3. Preparation of F. gigantica glutathione S-transferase (GST) antigen Fasciola gigantica glutathione S-transferase antigen (Fg-GST Ag) was prepared according to Estuningsih et al. (1997) using the affinity chromatography method. A pre-packed Glutathione Sepharose 4-B (Pharmacia Biotech., Sweden) was used for purification of GST. The column washed with 20 ml PBS (pH 7.3) to remove the preservatives, the gel equilibrated with 6 ml PBS + 1% Triton X100, the F. gigantica crude antigen was applied diluted in PBS + 1% Triton X-100 to the column, the effluent discarded, then the column washed with 2 10 ml of PBS, the bound material was eluted with 10 ml of elution buffer (5 mM Glutathione in 50 mM Tris–HCl, pH 8.0) and 1–2 ml fractions were collected and stored at 80 °C until assayed. 2.3.4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) The SDS–PAGE analysis of Fg-Cr, Fg-ES and Fg-GST antigens of F. gigantic was performed according to Laemmli (1970) and the obtained protein bands were stained with Coomassie blue R-250. 2.4. Measurement of protein concentration in prepared antigens Protein concentration of each prepared antigens (Fg-Cr, Fg-ES and Fg-GST) was determined according to Lowry et al. (1951). 2.5. Application of indirect enzyme-linked immunosorbent assay (iELISA) The iELISA was carried out according to Zimmerman et al. (1982) and the optimal antigen, serum and conjugate concentrations were determined after preliminary checkerboard titration according to (Catty and Raykundalia, 1989). In this study, the optimum conditions were 5 lg/ml coating buffer for Fg-Cr and Fg-ES antigens, 10 lg/ml coating buffer for Fg-GST antigen, 1:100 serum dilution, 1:3000 alkaline phosphatase labeled rabbit anti-bovine, anti-ovine and anti-horse IgG (Sigma) as conjugate and 1 mg pnitrophenyl phosphate (p-NPP) dissolved in 1 ml substrate buffer as substrate. The iELISA was performed in 96 wells microtiter plates (NuncImmuno Plate MaxiSorp, Intermed). The wells were coated with 100 ll of antigen (at optimal conc.) diluted in 0.1 M carbonate buffer (pH 9.6) and incubated overnight at 4 °C. Washed twice with PBS containing 0.01% Tween-20 (PBS/Tween) and blocked with 100 ll per well of 0.1% bovine serum albumin in 0.01 M PBS (pH 7.4) for 1.5 h at 37 °C. After blocking, plates were washed three times with PBS/Tween. A 100 ll of cattle, sheep or donkey sera (diluted 1:100 in PBS) were added to the wells, followed by incubation for 1.5 h at 37 °C. The plates were washed again (five times) and 100 ll of 1:3000 anti-bovine, anti-ovine or anti-horse (according to the animal species) alkaline phosphatase labeled rabbit IgG (diluted in PBS) was added to the wells and the plates were incubated at 37 °C for 2 h. Finally, after another five washes, 50 ll of p-NPP diluted in substrate buffer was added to each well, and after incubation in the dark for 15 min at 37 °C the enzymatic reaction was stopped with 50 ll/well of 3 N NaOH. The absorbance of each well was read at a wavelength of 405 nm using a titertek multiskan ELISA reader and the results were expressed as optical density (OD).
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In each ELISA plate, three negative control sera and one positive control serum were included for each animal species. Negative control sera were obtained from six young animals less than 4 months old with two successive negative (3 months intervals) fecal examination for fasciola eggs, while the positive control serum was a pool of sera obtained from four slaughtered animals with adult liver fluke in their livers. Positive values were assigned according to Rodriguez-Peiez and Hillyer (1995) as those values with absorbance readings greater than the cut off value, which calculated as mean OD of negative sera plus three standard deviations. 2.6. Diagnostic evaluation of indirect-ELISA Results of iELISA using different antigens of F. gigantica for detecting antibodies against fasciola in sera of cattle, sheep and donkeys were evaluated against the results of presence or absence of flukes in their livers which taken as a gold standard and the diagnostic sensitivity, specificity and accuracy of the assay were calculated according to Timmreck (1994) and Smith (1995). Sensi Tþ 100 means the ability of a test to correctly identivity ¼ ðTþÞðFÞ tify the percentage of those who have the disease; while specificity T ¼ ðTÞþðFþÞ 100 means the ability of a test to correctly identify the percentage of those who do not have the disease; whereas accuracy ¼ ðTþÞþðTÞ 100 describe the degree to which measureTN ment reflects the true status of what is being measured and it is used to express the overall performance of a diagnostic test. NB; T+ (=true positive), T (=true negative); F+ (=false positive), F (=false negative) and TN (= total number). 3. Results Livers of 60 cattle, 75 sheep and 69 donkeys were examined for the presence of mature and immature F. gigantica in their bile ducts. Cattle were classified into 30 infected and 30 non-infected animals, while sheep were classified into 39 infected and 36 noninfected animals, whereas donkeys were classified into 15 infected and 54 non-infected animals. Sera of these animals were collected and stored at 20 °C until be tested by iELISA.
Fig. 1. Migration pattern of Fg-Cr (A), Fg-ES (B) and Fg-GST (C) antigens in SDS– PAGE gel stained by Coomassie blue.
Antigens (Fg-Cr, Fg-ES and Fg-GST) were prepared from adult F. gigantica collected from the bile ducts of cattle and their protein content was determined. The migration pattern of Fg-Cr, Fg-ES and Fg-GST antigens in SDS–PAGE gel (Fig. 1) was analyzed using Gel-ProÒ analyzer v4. Fg-Cr antigen revealed 17 protein bands with molecular weight of 13.8, 15.7, 17.4, 20.8, 23.2, 25.6, 27.4, 30.1, 31.3, 32.4, 33.5, 35, 42.9, 53.3, 92.4, 178.4 and 262.3 kDa. Fg-ES antigen revealed seven protein bands with molecular weight of 15, 28, 31.6, 32.9, 39.4, 83.3 and 101.7 kDa. Fg-GST antigen revealed one protein band with a molecular weight of 24 kDa. Goat antiserum to Fg-GST (obtained from research teamwork studying vaccination of goats against fasciolosis using Fg-GST) cross-reacts with the prepared Fg-GST, confirming it is indeed GST. The results of both examination of livers for the presence or absence of liver fluke and in same time corresponding results of iELISA using Fg-Cr, Fg-ES and Fg-GST antigens were obtained and illustrated in Table 1. The diagnostic sensitivity, specificity and accuracy percentages of iELISA using Fg-Cr, Fg-ES and Fg-GST antigens for diagnosis of F. gigantica infection in cattle, sheep and donkeys were calculated and recorded in Table 2.
4. Discussion and conclusion For several decades, F. gigantica infection was diagnosed by fecal examination which has several disadvantages such as it is time and effort consuming, needs a suitable volume of fecal sample, cannot detect early infection with high-false negative rate in chronic infection due to the intermittent shedding of eggs in feces and cases with ectopic infection. Examination of 5 g of feces had a sensitivity of 66.7% from cattle whose infection status had been confirmed at necropsy examination (Anderson et al., 1999) with higher sensitivity obtained by three sub-samples leading to traditional coproscopy methods can have a superior test performance over other test systems. The main limitation of this approach is the analysis of large volumes of feces is labour intensive with possible sources of misidentification of eggs include the presence of paramphistomum eggs which have similar morphology but in a different color. Fecal examination is also unable to diagnose ectopic fasciolosis where fasciola worm develop in tissues other than liver. All of these encourage us to study another method for diagnosis of fasciolosis. Recent studies on fasciolosis focused on the importance of ELISA in diagnosis of animal fasciolosis and it becomes the most widely used test, because of its simplicity, reliability and easy mechanization. Several attempts to purify different fasciola antigens used in the assay to improve its diagnostic sensitivity and specificity. In this study, three F. gigantica antigens (Fg-Cr, Fg-ES and FgGST) were used to evaluate the diagnostic sensitivity, specificity and accuracy of iELISA for the diagnosis of F. gigantica infection in cattle, sheep and donkeys. Results of iELISA were evaluated in comparison to the results of post-mortem findings (presence of fluke in liver of examined animal) which used as a gold standard. True positive, false positive, true negative and false negative cases were determined (Table 1), as well as sensitivity, specificity and accuracy percentages were recorded for each antigen and animal species (Table 2). False positive cases were detected in examined cattle (60), sheep (75) and donkeys (69) sera by iELISA using Fg-Cr Ag as 6, 6 and 8 cases in percentages of 10%, 8% and 11.6%, respectively, while by using Fg-ES Ag they were 1, 1 and 2 cases in percentages of 1.7%, 1.3% and 2.9%, respectively, whereas by using Fg-GST Ag they were 9, 8 and 12 cases in percentages of 15%, 10.7% and 17.4%, respectively. False positive cases detected by iELISA may be attributed to recent administration of effective fasciolicides which eliminated
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W.S. Awad et al. / Research in Veterinary Science 86 (2009) 466–471 Table 1 Results of examination of cattle, sheep and donkeys livers for liver flukes and iELISA on their sera using Fg-Cr, Fg-ES and Fg-GST antigens Results of iELISA using
Results of liver examination in Cattle (60)
Fg-Cr Ag
+
Fg-ES Ag
+
Fg-GST Ag
+
Sheep (75)
Donkey (69)
+ (30)
(30)
+ (39)
(36)
+ (15)
(54)
29 (48.3%) 1 (1.7%)
6 (10%) 24 (40%)
39 (52%) 0 (0%)
6 (8%) 30 (40%)
14 (20.3%) 1 (1.5%)
8 (11.6%) 46 (66.7%)
28 (46.7%) 2 (3.3%)
1 (1.7%) 29 (48.3%)
37 (49.3%) 2 (2.7%)
1 (1.3%) 35 (46.7%)
14 (20.3%) 1 (1.5%)
2 (2.9%) 52 (75.4%)
20 (33.3%) 10 (16.7%)
9 (15%) 21 (35%)
28 (37.3%) 11 (14.7%)
8 (10.7%) 28 (37.3%)
9 (13%) 6 (8.7%)
12 (17.4%) 42 (60.9%)
Table 2 The diagnostic sensitivity, specificity and accuracy percentages of iELISA using Fg-Cr, Fg-ES and Fg-GST antigens for diagnosis of F. gigantica infection in cattle, sheep and donkeys Antigen type
Fg-Cr Ag
Animal spp. Sensitivity (%) Specificity (%) Accuracy (%)
Cattle 96.7 80 88.3
Fg-ES Ag Sheep 100 83.3 86.7
Donkey 93.3 85.2 87
Cattle 93.3 96.7 95
adult flukes from the biliary ducts but specific antibodies still persist in serum for 2–7 months (Zimmerman et al., 1982; Santiago and Hillyer, 1988; Levieux et al., 1992; Castro et al., 2000) and also this may be due to the possession of common antigens by different helminths (Pelley and Hillyer, 1978; Fagbemi and Obarisiagbon, 1991). These two phenomena present a problem in the serodiagnosis of fasciolosis and this means that any serodiagnostic test used to diagnose infection in individual animals must be interpreted with care. In this study five cases of donkeys were infected with hydatid cysts in their livers, two of them gave positive result with iELISA using Fg-Cr Ag only, one gave positive result with both iELISA using Fg-Cr and Fg-GST antigens, another one gave positive result with both iELISA using Fg-Cr and Fg-ES antigens, while last one do not react with any antigen. These findings showed a clear crossreaction was detected between fasciola antigens (especially Fg-Cr Ag) and hydatid cysts antigens that clarify some false positive cases in donkeys. False negative cases detected in cattle, sheep and donkeys sera by iELISA using Fg-Cr Ag were 1, 0 and 1 in percentages of 1.7%, 0% and 1.5%, respectively, while by using Fg-ES Ag 2, 2 and 1 cases were recorded in percentages of 3.3%, 2.7% and 1.5%, respectively, whereas by using Fg-GST Ag 10, 11 and 6 cases were recorded in percentages of 16.7%, 14.7% and 8.7%, respectively. The false negative results may be attributed to modulation of the host immune response by liver flukes as reported by Sandeman and Howell (1981) and Zimmerman et al. (1983) who recorded a significant immune suppression during F. hepatica infection. Sensitivity percentages of iELISA using Fg-Cr Ag were calculated for diagnosis of fasciolosis in cattle, sheep and donkeys as 96.7%, 100% and 93.3%, respectively, while by using Fg-ES Ag were 93.3%, 94.9% and 93.3%, respectively, whereas by using Fg-GST Ag were 66.7%, 71.8% and 60%, respectively. It was clear that, the highest sensitivity (100%) was obtained by using Fg-Cr Ag for diagnosis of F. gigantica infection in sheep, whereas the lowest sensitivity (60%) was obtained by using Fg-GST Ag for diagnosis of F. gigantica infection in donkeys. Specificity percentages of iELISA were also calculated with highest specificity (97.2%) was recorded when Fg-ES Ag was used in iELISA for diagnosis of F. gigantica infection
Fg-GST Ag Sheep 94.9 97.2 96
Donkey 93.3 96.3 95.7
Cattle 66.7 70 68.3
Sheep 71.8 77.8 74.7
Donkey 60 77.8 73.9
in sheep, while the lowest specificity (70%) was recorded when Fg-GST Ag was used in iELISA for diagnosis of F. gigantica infection in cattle. From these results, it was clear that, sensitivity of iELISA using Fg-Cr Ag (93.3–100%) was less high than that when using Fg-ES Ag (93.3–94.9%), but in the same time, its specificity (80–85.2%) is obviously lower than that of Fg-ES Ag (96.3–97.2%). This may be attributed to the numerous antigenic components of Fg-Cr Ag than those of Fg-ES Ag leading it to be more sensitive and less specific than Fg-ES Ag. The high sensitivity (more than 93.3%) and high specificity (more than 96.3%) of iELISA using Fg-ES Ag for the diagnosis of F. gigantica infection in cattle, sheep and donkeys support the use of this antigen in the serodiagnosis of fasciolosis in different animal species as described by Zimmerman et al. (1982), Santiago and Hillyer (1988), Mousa (1994), Hillyer et al. (1996), Ibarra et al. (1998), Reichel (2002), Awad et al. (2004), Molloy et al. (2005), and Charlier et al. (2008). It must be taken in consideration that, the sensitivity and specificity of tests will vary according to the population on which they are tested (Whiting et al., 2004; Leeflang and Bossuyt, 2005) and the season at which samples being tested (Charlier et al., 2008). Furthermore, no tests currently available can be considered as having both 100% sensitivity and 100% specificity. Glutathione-S-transferases are present as several isoenzymes in Fasciola and are important for the detoxification of endogenous or exogenous toxic compounds (Mitchell, 1989; Brophy and Barrett, 1990). In spite of the effort, time and cost needed to purify FgGST Ag, it gives low sensitivity and specificity when used as a coating antigen in the iELISA for the diagnosis of F. gigantica infection in cattle, sheep and donkeys. This may be attributed to that, GST is located at the surface or just below the surface of the adult flukes, which are thought to continually, shed their teguments to evade the host immune system and thus GST is of very low concentration resulting in low-antibody response against it (Morphew et al., 2007). On the other hand, by using this antigen as a vaccine, a good protection rate against infection was recorded (Morrison et al., 1996; Spithill and Dalton, 1998).
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The accuracy percentages were recorded with the highest accuracy (96%) obtained when using iELISA with Fg-ES Ag for diagnosis of fasciolosis in sheep and the lowest one (68.3%) obtained when using iELISA with Fg-GST Ag for diagnosis of fasciolosis in cattle. Using Fg-ES Ag in iELISA gives high-accuracy rates (more than 95%) in cattle, sheep and donkeys. Conclusively, Fg-ES Ag was the best coating antigen in iELISA for the serodiagnosis of fasciolosis in cattle, sheep and donkeys due to its high sensitivity, specificity and accuracy rates. Acknowledgements We would like to express our deepest thanks to Professor Dr. Wahid Mousa, Department of Parasitology, Faculty of Veterinary Medicine, Cairo University for his great cooperation and assistance during the whole work, also we would like to express our sincere thanks to the National Academy of Science that funded us to complete our study.
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