Early detection of Fasciola gigantica infection in ... - Springer Link

Parasitol Res (2008) 103:141–150 DOI 10.1007/s00436-008-0941-4

ORIGINAL PAPER

Early detection of Fasciola gigantica infection in buffaloes by enzyme-linked immunosorbent assay and dot enzyme-linked immunosorbent assay Niranjan Kumar & S. Ghosh & S. C. Gupta

Received: 12 February 2008 / Accepted: 20 February 2008 / Published online: 16 March 2008 # Springer-Verlag 2008

Abstract In an attempt to develop a suitable serological test for early detection of Fasciola gigantica infection in buffaloes, a group of proteins were isolated from the somatic antigen of the parasite by immunoaffinity chromatography. The process of isolation of the proteins has been standardized and significant level of repeatability was achieved. To test the diagnostic potentiality of the antigens, two serological tests, viz., enzyme-linked immunosorbent assay (ELISA) and dot enzyme-linked immunosorbent assay, were standardized using the sera from experimentally noninfected (group A) and infected (group B) animals. Further, the sensitivity and the specificity of the tests were evaluated employing the field sera from animals of different parasitic load viz., F. gigantica positive (group C), F. gigantica and Gastrothylax crumenifer positive (group D), F. gigantica and Gigantocotyle explanatum positive (group E), a group of sera without F. gigantica but other trematode infection (group F), only G. crumenifer positive (group G), only G. explanatum positive (group H), G. crumenifer and G. explanatum positive (group I), and PM negative (group J) collected from slaughterhouses of Bareilly (Uttar Pradesh, India) and Patna (Bihar, India). In plate ELISA, the sensitivity of the antigen and the test was 75.75% while the specificity was 97%, 95%, and 98%, respectively, against G. crumenifer, G. explanatum, and mixed infection of G. crumenifer and G. explanatum, respectively. In the case of dot ELISA the sensitivity was 86.5% and specificity was 92.3%, 94.7%, and 90%, respectively, against G. crumenifer, G. explanatum, and mixed infection of G. crumenifer and G. explanatum, respectively. The potentialN. Kumar : S. Ghosh (*) : S. C. Gupta Division of Parasitology, Indian Veterinary Research Institute, Izatnagar, 243122 Bareilly, India e-mail: [email protected]

ity of the antigen in the diagnosis of field infection is discussed.

Introduction Fasciola gigantica and Fasciola hepatica are recognized as the two most economically important helminth parasites of production animals (Spithill et al. 1997). The prevalence rate of infection is often high in sheep, cattle, and buffaloes probably because of close contact of animals with infective metacercariae (mc) on pasture and high susceptibility to infection. The worldwide losses in animal productivity due to fasciolosis were estimated as over US$ 3.2 billion per annum (Spithill et al. 1997). Chowdhuri (1994) documented that 14 Indian states and Union territories are affected by the disease. It has been realized that of the several parasitic diseases prevalent in India, fasciolosis has been a disease of prime concern on account of large animal population at risk and frequent outbreaks of the disease in the country. In contrast, global demand for animal-origin food in developing countries is predicted to grow by 2.8% per annum from 1993 to 2020 and hence the control of fasciolosis could contribute significantly to improve animal production. In fasciolosis, detection of fluke eggs in feces is considered as the most reliable diagnostic method. However, coprological confirmation of the disease prior to application of strategic antifluke medication seems of a little or no significance to avoid heavy economic losses, while advantages of immunodiagnostic techniques lie in the detection of early infection during the prepatent stage and in mild infection (Santiago and Hillyer 1988; Fagbemi and Guobadia 1995; Anderson et al. 1999; Rokni et al. 2003; Ghosh et al. 2005a, b).

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Sustained efforts have been directed towards the development of specific serodiagnostic tests for early detection of this infection in animals for limiting the negative impact of F. gigantica infection on the livestock productivity (Hillyer et al. 1996; Sanchez-Andrade et al. 2000; ElKerdany et al. 2002). In the division of Parasitology of the Indian Veterinary Research Institute, concerted attempts have been made to identify the diagnostic antigens and tests for specific diagnosis of F. gigantica with partial success (Dixit et al. 2002; Yadav et al. 2005; Raina et al. 2006). In our earlier efforts, we have purified infection-induced antigen of F. gigantica (Gupta 2001) and tested it against experimental infection of cattle and buffaloes. Realizing the potentiality of the antigen in the present study, we evaluated the diagnostic potentiality of the antigen and two serological tests viz., enzyme-linked immunosorbent assay (ELISA) and dot ELISA, to detect field infection in buffaloes.

Materials and methods Laboratory animals Six New Zealand white rabbits of 1kg in weight were procured from the small animal section of the institute and each was maintained in the disinfected cage of 14 × 12 × 10in. in size. The animals were given food and water ad libitum. Animals were maintained as per the guidelines of the Committee for the Purpose of Control and Supervision of Experimentation on Animals. Animal infections In the Division of Parasitology we are continuously maintaining mc of F. gigantica for different type of experiments. About 100 mc were orally administered to each of the four New Zealand white rabbits. Two rabbits were kept as uninfected control. Rabbits were bled periodically and anti-Fasciola antibodies were detected by double immunodiffusion (DID) test (Ouchterlony 1964). On 10weeks postinfection (PI), blood samples were collected by cardiac puncture and sera were separated for isolation of immunoglobulins. Preparation of somatic antigen of F. gigantica The detail of the preparation of somatic antigen has been elaborated in Ghosh et al. (2005a). Briefly, mature F. gigantica were dried off and fluke powder was prepared. The fluke powder was suspended in chilled phosphatebuffered saline (PBS; pH 7.2) containing a cocktail of protease inhibitors (1mM of ethylene diamine tetraacetic

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acid, 1mM of ethylene glycol bis-NNNN-tetraacetic acid, 1mM of N-ethylmaleimide, and 1mM of phenyl methyl sulfonyl fluoride) and stirred for extraction. The extracted antigen was centrifuged at 1,780RCF for 30min at 4°C. Supernatant was collected and centrifuged at 21,910RCF for 45min. The collected supernatant was equilibrated against 20mM Tris, 0.5M NaCl, pH 8.0, filtered by 0.45µm syringe filter (Sartorius) and kept as somatic antigen of F. gigantica (FgSAg). The protein concentration was determined (Lowry et al. 1951). Purification of antigen and SDS-PAGE The detail has been given in Ghosh et al. (2005b). Briefly, total immunoglobulins were precipitated by 40% ammonium sulfate (Fey et al. 1976). Immunoglobulins G (IgGs) were isolated by anion exchange chromatography (Talwar and Gupta 1992). The purified IgGs were used as ligands for binding with CNBr-activated sepharose 4B (Sigma Chemical Company, USA). The FgSAg was dialyzed in equilibrating buffer, filtered and then loaded on preequilibrated column. The column was washed with excess equilibrating buffer and the bound fractions were eluted using elution buffer (0.1M glycine HCl, pH 2.2). The protein concentration of the eluted fraction was determined (Aiken and Learmoth 1996). Both FgSAg and F2 were electrophoretically resolved on 1.0-mm-thick gel using a discontinuous system (Laemmli 1970). The stacking gels constituted of 3% acrylamide in 0.5M Tris, pH 6.8 with 0.25% sodium dodecyl sulfate (SDS) and resolving gels were 12% acrylamide in 1.5M Tris, pH 8.8 with 0.25% SDS. Gels were stained with Coomassie Brilliant Blue R250 to identify the marker proteins in the range of 14 to 100kDa (Banglore Genei, India), FgSAg and F2. Gels were scanned by gel documentation and analysis system (Syngene, UK) using Genesnap and Genetool programs. For characterization of the protein, the fractions were also resolved by native polyacrylamide gel electrophoresis (PAGE) after removal of SDS and β-mercaptoethanol from the buffers. Collection of sera Sera from experimentally infected, noninfected, F. gigantica postmortem (PM) positive, F. gigantica and Gastrothylax crumenifer positive, F. gigantica and Gigantocotyle explanatum positive, a group of sera without F. gigantica but other trematode infection, only G. crumenifer positive, only G. explanatum positive, G. crumenifer and G. explanatum positive, and PM-negative sera were collected from slaughterhouses of Bareilly (Uttar Pradesh, India) and Patna (Bihar, India). A group of sera of unknown parasitic load were also collected from the slaughterhouses and from

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in each well and incubated at 37°C for 1h. Wells were washed again three times with PBS-T and 100μl of antibovine IgG horseradish peroxidase (HRPO) conjugate (Sigma Chemical Company, USA) in 1:5,000 dilution prepared in 1% skimmed milk in PBS was added to each well and incubated at 37°C for a period of 1h. After washing, 100µl of O-phenylendiamine dihydrochloride (OPD; Sigma Chemical Company, USA; 40mg OPD in 100ml of phosphate citrate buffer, pH 5.0 and 40µl H2O2) was added to each well and kept in dark for color development. The reaction was stopped after 2min using 50μL of 3N HCl in each well. The absorbance readings were taken at 492nm on an ELISA reader (Biorad, USA). The data were expressed as the mean of the optical density (OD) recorded for paired samples. The sera samples collected from healthy, uninfected animals were used as negative controls, while sera samples collected from experimentally infected animals were used as positive controls. Negative cutoff value was decided from the mean ELISA OD (492nm) values of sera of the uninfected buffaloes of experimental negative group, n = 20 (buffaloes reared in dairy farm, Livestock Production and Management Section, Indian Veterinary Research Institute). A total

the field of Bareilly and Patna. The details about the animals from which the sera were collected, parasitic load in these animals, and the geographical origin of the slaughtered buffaloes are given in Table 1. ELISA Indirect ELISA was performed as per the method described by Engvall and Perlman (1971) with some modifications. The optimum concentration of the antigen, conjugate, and sera were determined by a series of checkerboard titrations. The 96-well flat-bottomed polystyrene microtiter plates (Greneir, Germany) were coated with 100µl containing 2.5μg/ml of F2 in ELISA coating buffer in each well. The plates were incubated overnight at 4°C (Harlow and Lane 1988). The wells were washed twice for 10min with PBS-T (PBS pH 7.2, 0.05% Tween-20) using platform rocker mixer (Bangalore Genei, India). The plates were blocked with blocking buffer (3% skimmed milk in PBS) and incubated for 1h at 37°C to avoid nonspecific binding. The plates were again washed three times with PBS-T. Sera samples collected from different sources were diluted (1:100) in 1% skimmed milk prepared in PBS (pH 7.2) and 100μl of each diluted serum was dispensed as duplicate

Table 1 Showing details of the group of animals from which sera were collected Buffalo Origin of group animals

Species of parasites

A

Experimental Nil infection(s)

B

Experimental infection(s) Postmortem infection(s) Postmortem infection(s) Postmortem infection(s) Postmortem infection(s) Postmortem infection(s) Postmortem infection(s) Postmortem infection(s) Postmortem infection(s)

C D E F G H I J K a b

Number of buffaloes

Geographic origin

20

Dairy farm of Livestock Production and Management

8

Section, Indian Veterinary Research Institute, Izatnagar Division of Parasitology, Indian Veterinary Research Institute, Izatnagar Bareilly (Uttar Pradesh) and Patna (Bihar) slaughterhouses

F. gigantica and G. crumenifer

15

Bareilly (Uttar Pradesh) and Patna (Bihar) slaughterhouses

F. gigantica and G. explanatum

6


Miscellaneous group without F. gigantica infection 3 G. crumenifer

9


36


G. explanatum

18


G. crumenifer and G. explanatum

39


102


F. giganticaa and P. epiclitumb F. gigantica

Nil Unknown

Animals were infected with 200 mc Animal was infected with 1,000 mc

4 and 1, respectively

66 (field=52, Bareilly (Uttar Pradesh) and Patna (Bihar) slaughterhouses slaughterhouse=14) and field

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of 103 OD values were taken for estimation of cutoff value. The cutoff value was calculated from the two mean OD of uninfected buffaloes sera + standard deviation. The cutoff value was 0.376. Western blotting The test was performed as described by Towbin et al. (1979). The FgSAg and F2 antigens were transferred to polyvinyl difluoride (PVDF) by a semidry blot cell (Atto Corporation, Japan). The transfer efficiency was checked by staining the PVDF with 0.1% Ponceau solution. The membranes were blocked in 5% nonfat milk (NFM) in PBS. Blocked membranes were washed in PBS containing 0.05% Tween-20 for 30min and then the individual strips were cut for processing in individual serum. The optimum dilution of primary antibody was standardized (1:50) and the strips were incubated in respective serum for 1h at 37°C followed by washing for 15min in PBS-T. The optimum concentration of secondary antibody (antibovine alkaline phosphatase, Sigma Chemical Company, USA) was standardized at 1:1,000 dilution and the PVDF strips were probed in secondary antibody for 1h at 37°C followed by washing in PBS-T for 15min. Enzyme substrate reaction was visualized by reacting the processed strips with 10-ml substrate buffer (pH 9.5) containing 5-bromo-4-choloro-3indolyl phosphate p-toluidine (33µl) and nitro blue tetrazolium (44µl). Finally, the reaction was stopped after appearance of visible band with several changes of distilled water. The result was analyzed by gel documentation and analysis system using Genesnap and Genetool programs (Syngene, UK). Dot ELISA The dot ELISA (Dipstick ELISA) has been defined as a simple screening method (qualitative analysis) for the field as well as laboratory sera using dipstick (Pappas et al. 1983). Nitrocellulose paper sticks (Mdi, India) were used for standardization of the test. A series of checkerboard titrations were initially performed to determine the optimum quantum of the antigen, conjugate, and sera. Each dipstick was coated with 450ng of F2 and was incubated at 37°C for 1h before storing at room temperature (RT) for further use. Dipsticks were first washed three times with PBS-Tween-20 (0.05%) for 15min and then blocked with 5% NFM PBS for 1h at RT. The sticks were thoroughly washed three times in PBS-T and then each stick was incubated separately for 1h at RT in respective serum diluted 1:50 in 1% NFM in PBS. After three washes in PBS-T, the sticks were dipped in antibovine HRPO (Bangalore Genei, India) conjugate at a dilution rate of 1:1,000 in 1% NFM in PBS for 1h at RT. The sticks were

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washed and then were dipped in chromogenic substrate buffer (10mg diaminobenzidine per 50-ml 50-mM Tris buffer pH 7.6 and 30μl of 30% v/v H2O2) for color development. After 10min of incubation in the dark at RT, the sticks were thoroughly washed in several changes of distilled water to stop the reaction and dried on filter paper. Positive reaction was characterized by the formation of a distinct colored dot. Determination of sensitivity and specificity The sensitivity and specificity of antigen vis-à-vis test was calculated (Mandal et al. 1998): Sensitivity % = (true positive − false negative) / true positive × 100 and Specificity % = (true negative − false positive) / true negative × 100

Results Purification of FgSAg and SDS-PAGE profile A total of 1.44-g FgSAg was loaded in batches on the column and 60-mg bound antigen (F2) was recovered after single-step purification. The recovery percent of F2 antigen with respect to FgSAg was 4.16%. Electrophoretic separation of FgSAg in reducing condition on 12% polyacrylamide gel resolved into 16 proteins with the molecular weight of 14, 16, 21, 23, 25.8, 27, 29, 32, 37, 39, 42, 43, 47, 52, 57, and 76kDa when stained with 0.25% Coomassie Brilliant Blue R 250. Following purification, a total of five proteins of molecular weight of 23, 25.8, 27, 32, and 37kDa were isolated as F2 (Fig. 1a). Electrophoretic separation of the antigens in native (nonreducing) condition resolved F2 into two major proteins of 35–37 and 27–29kDa (Fig. 1b). ELISA All the sera of group A (negative control) gave significantly lower OD values than the cutoff value, while the sera number (1, 2, 3, and 4) of group B gave higher and Paramphistomum-epiclitum-infected serum (number 5) gave lower OD value(s) than the cutoff value (Fig. 2). The results of the study confirmed the highest level of sensitivity of the antigen and the test in detecting F. gigantica infection in experimental condition without any cross-reactivity with P. epiclitum. When the ELISA data obtained from the sera of group C animals were analyzed, it was observed that all the animals which were PM positive to F. gigantica were found serologically positive (Fig. 2). Among the 15 sera of group D, sera number 9, 14, and 15 gave OD values of 0.39, 0.4, and 0.42, respectively, which

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Fig. 1 a SDS-PAGE (12%) analysis of FgSAg (lane 1) and F2 (lane 2) in reducing condition, M = molecular weight marker b SDS-PAGE (12%) analysis of F2 (lane 1) in nonreducing condition, M = molecular weight marker

were very close to cutoff value (Fig. 2). Similarly, of the six animals in group E, sera number 1 and 4–6 gave OD values which are significantly higher than the cutoff values while the other two sera of the group gave OD values close to cutoff values (Fig. 2). Among the sera of group F, although there was a little variation in the PM status of the animal number 2, 6, and 8, significantly higher OD value was recorded in the serum number 8 (Fig. 2); this is due to the presence of early stage of infection of F. gigantica as detected by western blot (Fig. 4, strip number 3). All the

Fig. 2 ELISA reactivity pattern of buffaloes sera of Group A–F

other sera of the group gave OD values which were below cutoff value. When the sera of G.-crumenifer-positive group of animals (group G) were tested, it was observed that significant anti-F2 antibodies were detected only in serum number 23 by ELISA (Fig. 3) and in western blot (Fig. 4, strip number 4), thus confirming early stage of F. gigantica infection in the animal. Similarly, the high level of specificity of the antigen was confirmed against G. explanatum because none of the sera (group H) gave significant anti-F2 antibody response (Fig. 3). In all the sera except sera number 10, 11, and 29 of group I, the significant anti-F2 antibody response could not be detected (Fig. 3). However, in the western blot format the serum number 10 was found negative of F. gigantica infection (Fig. 4, strip number 9). The specificity and sensitivity level of the antigen and the test were further confirmed when all the sera of group J (except sera number 36 and 82) failed to detect significant anti-F2 antibodies. The serum number 36 which gave higher OD value was also found positive in western blot (Fig. 4, strip number 5). Thus, confirming the presence of early F. gigantica infection in the animals which was not visible in liver at necropsy. Finally, F. gigantica prevalence rate of 20% was recorded in the buffaloes of the studied area (group K; Fig. 3). Dot ELISA After checkerboard titration, the optimum concentration of the antigen for coating was determined as 450ng per dot. The primary and secondary antibody concentration was optimized at 1:50 and 1:1,000 levels, respectively. The sera of experimentally negative group of animals (group A) gave negative reaction (Fig. 5, dot 1, 2) while the serum of group B animal reacted strongly in dot ELISA

1.4

OD at 492 nm

1.2 1 0.8 0.6 0.4 0.2 0 0

5

10

15

20

Number of animals (ordinal) Cut-off line

Group A

Group B

Group D

Group E

Group F

Group C

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Fig. 3 ELISA reactivity pattern of buffaloes sera of Group G–K

1.4

OD at 492 nm

1.2 1 0.8 0.6 0.4 0.2 0 0

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Number of animals (ordinal) Cut-off line

(Fig. 5, dot 3), while the serum no. 5 collected from the experimentally P. epiclitum-infected animal gave no reaction (dot 4). All the tested sera of group C animals were found strongly positive (dots 5–7). Of the 13 sera of group D tested by dot ELISA, sera number 3–5 and 13 reacted negatively (dots 8–20) while other reacted strongly. It was observed that dot ELISA could not detect infection at the low level of three to five parasites. When the test was applied with the sera of group E animals, it was observed that the two animals in the group gave ELISA OD values very close to cutoff value (Fig. 2) and both the animals possess very low parasite load. Consequently, both the sera reacted negatively in dot ELISA (dots 22, 24) while the other sera gave strong reaction in dot ELISA (dots 21, 23, 25, 26). It is interesting to note that the sera no. 8 of group F gave positive reaction in both the test formats while other gave negative reaction (dots 27–35). Of the 36 sera of group G, 26 were tested in dot ELISA and the results are presented in Fig. 5, dots 36–61. Serum number 23 which gave positive reaction in ELISA reacted

Group G

Group H

Group I

Group J

Group K

positively in dot ELISA (dot 61), confirming the sensitivity of the test. Among the 18 sera of group H, except the serum number 3, all the sera reacted negatively in dot ELISA, thus confirming a very high level of specificity of the test. The result indicated that there was a strong possibility of having F. gigantica infection in animal number 3 which was not detectable in PM. The sera of G.-crumenifer- and G.-explanatum-positive group of animals (group I) reacted negatively to F2 antigen except the sera nos. 21, 23, 24, and 36 (dots 101, 103, 104, 116). Although the sera of group J were collected from PMnegative animals, the sera nos. 27, 57, and 58 reacted positively (dots 147, 164, 165). Sensitivity and specificity of the tests In plate ELISA, the sensitivity of the antigen and test was 75.75% while the specificity against G. crumenifer was 97%, against G. explanatum 95%, and against mixed infection of G. crumenifer and G. explanatum 98%. In the case of dot ELISA, the sensitivity was 86.5% and specificity against G. crumenifer 92.3%, against G. explanatum 94.7%, and against G. crumenifer and G. explanatum 90%.

Discussion

Fig. 4 Immunoblot employing buffalo sera of different PM status. Legends: 1, F. gigantica positive (experimental); 2, F. gigantica positive (PM positive); 3, hydatid cyst and very light G. crumenifer; 4, G. crumenifer positive; 5, PM negative; 6, experimental negative; 7, PM negative; 8, G. explanatum positive; 9: G. crumenifer and G. explanatum positive

Purification of F. gigantica functional antigens by affinity chromatography is expected to remove host components, which if present are liable to cross react with the conjugate and elicit false results in ELISA and dot ELISA. Affinity chromatography has been shown to be a very effective tool for isolation of candidate diagnostic and vaccine molecule (Willadsen et al. 1989; Fagbemi et al. 1995, 1997) and so in the present study the principle of the affinity chromatographic method has been utilized for isolation of F2 antigen which was recovered at the level of approximately 4.16% of total protein loaded.

Parasitol Res (2008) 103:141–150 Fig. 5 Reactivity pattern of different sera in dot ELISA. Group A, dots 1, 2; group B, dots 3, 4; group C, dots 5–7; group D, dots 8–20; group E, dots 21–26; group F, dots 27–35; group G, dots 36–61; group H, dots 62– 80; group I, dots 81–120; groups J, dots 121–174

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Electrophoretic separation on 12% gel resolved F2 antigen into five protein bands with the molecular weight of 37, 32, 27, 25.8, and 23kDa. The F2 antigen was specifically isolated using infection-induced rabbit Igs. However, in earlier studies, Velusamy et al. (2004) isolated 54-kDa F. gigantica-specific protein using cattle Igs. The variation in isolation of different group of proteins using

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anti-F.-gigantica-infection-induced Ig ligands proved the expression of different antigens of the parasite in different host systems and thus further complicated the issue of developing a diagnostic test suitable for cattle, buffalo, sheep, and goat systems. Recently, Yokananth et al. (2005) isolated two polypeptides of 34 and 28kDa in dimer form using rabbit Igs and both the proteins were found

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immunodominant when probed with the sera of cattle, buffaloes, and sheep infected with F. gigantica and thus provided a significant clue of developing immunodiagnostic assays suitable for cattle, buffalo, and sheep systems by exploiting these two proteins. They also reported nonreactivity of these two proteins in goat sera experimentally infected with P. epiclitum and Schistosoma spindale. In the present investigation, the antigen was found 100% sensitive in detecting experimental F. gigantica infection (200 mc) in buffaloes on 3weeks PI and also highly sensitive in detecting noninfected animals. Besides the high level of sensitivity of F2 antigen, it did not show any cross-reaction when probed with G.-crumenifer- and G.-explanatuminfected field and P.-epiclitum-infected (1,000 mc; 6weeks PI) experimental buffalo sera. The cumulative effect of the dominant epitopes of the antigen at the coating level of 2.5µg/ml was found sensitive in detecting infection. Initially, indirect ELISA was standardized with the 3week postinfected buffalo sera and found that the antigen was 100% sensitive in detecting infection. Previously, F. gigantica cathepsin-L cysteine proteinase was isolated and found 100% sensitive in detecting experimental F. gigantica infection in buffaloes and cattle (Dixit et al. 2002, 2004; Yadav et al. 2005).To test the diagnostic potentiality of the isolated antigen in field cases, indirect ELISA and dot ELISA were standardized. Indirect ELISA has been found to be very suitable for the diagnosis of fasciolosis owing to its high sensitivity and possibility of processing many sera samples simultaneously. However, a drawback for this test is the need of an absorbance reader, which is expensive and, therefore, difficult to have at laboratories with limited resources. For this reason, dot ELISA was further standardized, so that it can be conducted by less trained persons. To test efficacy of the antigen and tests, a large number of sera were collected and grouped on the basis of individual expertization in PM. It was observed that in most of the cases the antigen could detect F. gigantica infection even when the infection was at the moderate level (six to eight parasites). This very high level of sensitivity of the test was previously reported by Fagbemi and Guobadia (1995) who have tested field animals using 25–28-kDa cysteine protease in Falcon assay screening test. It was quite interesting to note that in the present study in group C animals a strong antibody response was detected, even when only a single mature liver fluke was detected at PM (serum number 7), while on the other hand serum having moderate parasitic load (serum number 8) gave OD value which was just above the cutoff lines (Fig. 2) determined for F2. Among the eight sera of group C, the fifth serum collected from animal with heavy parasitic load gave highest OD values i.e., 1.285, while the serum of very heavy parasitic load stood at second position (Fig. 2; serum

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number 3). There appeared to be no correlation between serological responses, as measured by ELISA, and the number of flukes present in the liver at slaughter. Likewise, Reichel (2002) also failed to draw any correlation between these two variables. Both the tests gave 100% sensitivity and specificity in experimental negative (group A) and experimental monoinfection (F. gigantica) positive (group B) with no crossreaction with P.-epiclitum-infected serum. To test the specificity of the antigen, the sera of the groups (F–I) were tested and it was observed that the antigen was highly specific against G. crumenifer and G. explanatum infection (Figs. 2 and 3). The level of sensitivity observed in the present study using field buffalo sera was 81%, while specificity against G. crumenifer was 97%, G. crumenifer 95%, and in mixed infection of G. explanatum and G. crumenifer 94.2%. The decrease in level of sensitivity and specificity in field conditions were noted by many workers. For example, Cornelissen et al. (2001) using recombinant cathepsin-L-like protease in ELISA reported 100% sensitivity and specificity of 98.5% (cattle) and 96.5% (sheep) with experimentally infected sera but the sensitivity was as low as 90.2% and specificity of 75.3% were recorded when the test was conducted using sera collected from naturally infected cattle. The value of any diagnostic methodology can be judged only by their field applicability. In the cases of diagnosis of parasitic infection in ruminants, it should comply with the recommendation issued by the Office International des Epizooties, in the sense that it is necessary to develop diagnostic methods for parasitic diseases which should be cheap, simple, and useful under field conditions. Several immunodiagnostic assays were attempted for prepatent diagnosis of fasciolosis in field conditions viz., dot ELISA (Pappas et al. 1983), agar gel diffusion (Linh et al. 2003), DID (Ouchterlony 1964), dot immunoperoxidase (Maisonnave 1999), skin hypersensitivity reactions (Doyle 1973), etc., with their different acceptability level. Among these, dot ELISA is the most widely accepted because the test is easy to conduct and result can be ascertained rapidly. Realizing the immense potentiality of the test, Zimmerman et al. (1985) used E/S antigen of F. hepatica in dot ELISA format and antiparasite antibodies were detected by 2weeks PI in animals experimentally infected with 500 mc and 4weeks PI in sheep infected with 250 mc; further, in rabbits the antiarasite antibodies were detected even at initial dose of infection with 50 mc (Yadav and Gupta 1993). In the present study, at a coating level of 450 ng, sera dilution level of 1:50, and conjugate dilution level of 1:1,000, 8/ 8 known negative showed as negative and 4/4 known positive gave positive reaction and 8/8 F.-gigantica-only PM-positive group (group C) showed as positive, thus the test gave 100% sensitivity in detecting infection. To work

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out the sensitivity and specificity of a dot enzyme assay for the diagnosis of F. hepatica infection in cattle, Maisonnave (1999) standardized the assay using the bovine sera from seven different groups. The groups were F.-hepatica-only infected group (as group C of the present study), F. hepatica, Echinococcus-granulosus- and Paramphistomum-spp.-free group (as group J of the present study), E.-granulosus-and F.-hepatica-infected but Paramphistomum-free group (as group E of the present study), Paramphistomum-spp.-infected but F.-hepatica- and E.-granulosus-free group (as group G of the present study), and F.-hepatica- and Paramphistomum-spp.-infected group (as group D of the present study) and a sensitivity of 82% and specificity of 90% were recorded. Similarly, in the present study, the F2 antigen gave a sensitivity of 86.5% and specificity of 90% against G. crumenifer and G. explanatum infection, considered as significant in the detection of field infection. One limitation in this study is the use of slaughterhouse sera to represent a natural monospecific infection with a trematode. This is because a buffalo which is shown as coprologically negative for a particular trematode infection may harbor a prepatent infection or only very few adult worms that may be missed during PM inspection. However, sera from slaughterhouse animals are more representative under field situations than the sera from experimentally infected animals. Acknowledgement Sincere thanks are due to Indian Council of Agricultural Research, New Delhi, India for providing Research Fellowship to the senior author. Authors are grateful to the Director of the institute for providing necessary facilities.

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