Comparison of ELISA and GPAT in Diagnosis of Bovine Fasciolosis Using Excretory- Secretory Antigen

J Trop Med Parasitol 2001;24:1-7. ORIGINAL ARTICLE Comparison of ELISA and GPAT in Diagnosis of Bovine Fasciolosis Using Excretory- Secretory Antigen Montakan Vongpakorn 1, 2, Jitra Waikagul 1, Paron Dekumyoy 1, Tasanee Chompoochan 2, Wichit Rojekittikhun 1, Emsri Pongponratn 3 1 Department of Helminthology, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand 2 Parasitology Section, National Institute of Animal Health, Department of Livestock Development, Bangkok 10900, Thailand 3 Department of Pathology, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand T Abstract his study aimed to develop a sensitive and specific immunodiagnostic method for Fasciola gigantica infection using excretory-secretory (ES) antigen, from culturing living worms collected from the livers of naturally infected cattle at local slaughterhouses around Bangkok. The antigen was reacted with 269 cattle sera composed of 15 experimental fasciolosis sera, 15 calf sera, 119 heterologous sera, and 120 field-stool negative sera by indirect enzyme-linked immunosorbent assay (indirect ELISA) and gelatin particle indirect agglutination test (GPAT). From ELISA, the sensitivity, specificity, positive predictive value, negative predictive value, and efficacy of the test were 100%, 23.4%, 23.4%, 100%, and 37%, respectively. When the antigen was applied with GPAT, sensitivity, specificity, positive predictive value, negative predictive value, and efficacy of the test were 100%, 11.1%, 25%, 100%, and 31.4%, respectively. ELISA values and GPAT titers of cattle sera using ES antigen were moderately correlated, with a correlation coefficient of 0.57. From this study, both ELISA and GPAT were highly sensitive but gave low specificity. It showed that these methods, using ES antigen, might not be suitable for the diagnosis of fasciolosis as they showed cross-reaction with many parasitic infections. Keywords: Fasciola gigantica, ES antigen, ELISA, GPAT Introduction Fasciolosis gigantica causes considerable economic loss to the meat industry [1]. Diagnosis of bovine fasciolosis is by demonstration of parasitic eggs in the feces which can be found eight weeks post-infection. Clinical signs and symptoms appear three weeks post-infection, but antibody detection occurs earlier, about two weeks post-infection. This is an advantage of immunodiagnosis [2]. A number of immunodiagnoses have been developed for human and animal fasciolosis. In cattle, several pieces of research have been performed on Fasciola hepatica infection, while a few studies have been carried out on F. gigantica infection [3]. At present, the common immunodiagnostic test for F. gigantica infection in cattle is enzyme-linked immunosorbent assay (ELISA). Most reports have claimed that ELISA gave excellent sensitivity, but that crossreactivity with other helminthic infections occurred. In some Thailand bovine fasciolosis cases, Thammasart et al [4] found 47.6% false positive in the ELISA result for F. gigantica from Vol 24 (No. 1) June 2001 THE JOURNAL OF TROPICAL MEDICINE AND PARASITOLOGY 1

233 cattle sera using crude somatic antigen. Nevertheless, ELISA showed high sensitivity; the disadvantage is that expensive and specialized equipment is needed so that it cannot be performed in the field. The gelatin particle indirect agglutination test (GPAT) has the same principle as the indirect hemagglutination test (IHAT) [5], in which gelatin particles are used as antigen carriers instead of red blood cells (RBC). The advantages of gelatin particles over RBC are their inert antigenicity, that they can be lyophilized for keeping for a long period, the color helps convenient reading, the test is simple and rapid to perform without specialized equipment, and the method is helpful for mass screening. In 1990, Sato and Ryumon [6] used GPAT to diagnose human strongyloidosis, caused by Strongyloides stercoralis. They found a close relation to IHAT and ELISA. Kobayashi et al [7] used GPAT for mass screening of schistosomosis in humans in Brazil, compared with ELISA. They found a good correlation between GPAT and ELISA. In Thailand, Watthanakulpanich et al [8] used GPAT with crude somatic antigen for Opisthorchis viverrini diagnosis. They concluded that this method could be used for the serodiagnosis of human liver fluke infection. But there is no report using GPAT to diagnose helminthic infection in animals. In this study, we analyse the GPAT technique for the diagnosis of bovine fasciolosis using ES antigen, by comparing it with the ELISA method. Materials and methods F. gigantica adult worms were obtained from the livers of naturally infected cattle at the local slaughterhouses in Nonthaburi and Pathum Thani Provinces, Thailand. The worms were cultured by the method of Rivera Marrero et al [9], with some modifications. The worms were washed intensively 3-4 times in sterile NSS, then maintained at 37 C for 3 h (1 worm/5 ml) in sterile 0.01 M PBS at ph 7.4 supplemented with 0.8 mm phenyl methyl sulfonylfluoride (PMSF), 100 units/ml penicillin, 100 µg/ml streptomycin, changing the media every hour. The collected media was centrifuged at 10,000 g for 30 min at 4 C, the supernatant was dialyzed in DW by ultrafiltration using Amicon (MW cut-off 10,000). The ES antigens were filtered with a syringe plugged with glass wool. The antigens were stored at -70 C. The protein content of the antigen was measured by the method of Lowry et al [10]. Cattle sera Serum samples were divided into 4 groups, and depended on finding eggs of helminths by stool examination using the Beads and flotation techniques. Group A consisted of 15 sera of experimental F. gigantica infected cattle from Phra Nakhon Si Ayutthaya and Nakhon Ratchasima Provinces; group B consisted of 15 sera of calves that were free of any helminthic infection; group C consisted of 119 sera of cattle naturally infected with other helminthic infections, as shown in Table 1, and group D consisted of 120 cattle sera in which no helminthic eggs were detected in the stool samples. Serum samples in groups B, C and D were obtained from the National Institute of Animal Health (NIAH), Thailand. Table 1 Group C (heterologous cattle sera) used in this study. Infection Number of cases Schistosoma spindale (Sc) 23 Rumen fluke (RF) 35 Gastro-intestinal nematodes (GI) 18 Moniezia sp (Mo) 4 Trichuris sp (Tr) 1 Mixed infection 38 Total 119 Indirect method of enzyme-linked immunosorbent assay (Indirect ELISA) ELISA performance was done as described earlier by Voller et al [11], with some modifications. Briefly, wells of polystyrene microtiter plate (Nunc, Denmark) were filled with 100 µl of 0.5 µg/ml antigen with coating 2 THE JOURNAL OF TROPICAL MEDICINE AND PARASITOLOGY Vol 24 (No. 1) June 2001

buffer and incubated in a humidified chamber at 37 C for 1 h and 4 C overnight. The excess antigens were washed out by microshaker 3 times for 1 min each, with 150 µl of PBS-Tween; blocked with 150 µl of 1% skim milk at 37 C for 1 h; reacted with 100 µl of 1:400 cattle sera for 1 h; incubated with 1:1,000 rabbit anti-bovine horseradish peroxidase-conjugate IgG (DAKO, Denmark). The plate was washed 3 times with PBS-Tween between every change of solution. Finally, 100 µl of freshly prepared substrate solution containing paraphenylenediamine dihydrochloride (PPD) was added. The reaction was allowed to occur in the dark at room temperature for 30 min. The reaction was stopped by adding 50 µl of 1 N NaOH. Optical density (OD) at 492 nm was measured with an ELISA reader. The limit for discriminating negative from positive results was determined by the mean value of the negative controls plus 2 standard deviations. Gelatin particle indirect agglutination test (GPAT) The test was modified from that described by Watthanakulpanich et al [8]. Briefly, 10% of gelatin particles (Fujirebio Inc, Japan) were washed with 0.15 M NSS and adjusted to 5% suspension with PBS (0.15 M, ph 7). An equal volume of 5 mg/ml tannic acid was added to the suspension and incubated at 37 C for 15 min, washed 3 times with NSS. Two hundred and fifty ml of ES antigen (concentration 37 µg/ ml) were added into a tube containing 500 µl of 5% gelatin particles, incubated at 37 C for 1 h; washed 3 times with NSS and made into a 1% suspension with inactivated normal rabbit serum (NRS). The test was performed in a U-shaped bottom microtiter plate (Sartedt, USA). Twentyfive µl of 1% suspension in NRS was placed into each well. The test sera were dispensed into the wells starting at 1:4 to 1:8,192 by 2-fold dilution. Finally, 25 µl of antigen-coated particles were added to each of the wells. The plates were thoroughly shaken for 1 min, and allowed to settle for 3 h at room temperature. A positive reaction was shown by the diffusion of gel particles and a negative reaction was shown as smooth settlement at the bottom of the well. Agglutination titers were determined using the highest serum dilution that gave a positive reaction. In total, 45 cattle sera were used for GPAT, due to a limited supply of antigen for sensitizing the gelatin particles. The sera were from high, medium and low levels of ELISA results. Group A consisted of 8 experimental fasciolosis sera, group B consisted of 5 calf sera, group C consisted of 23 cattle sera infected with 3 Schistosoma spindale, 3 rumen fluke, 3 gastrointestinal nematodes, 3 Moniezia sp, 1 Trichuris sp, 6 mixed infection among rumen fluke and gastro-intestinal nematodes, 2 rumen fluke and gastro-intestinal nematodes and Moniezia sp, 1 rumen fluke and Moniezia sp, 1 gastro-intestinal nematode and Moniezia sp, group D consisted of 9 field-stool negative sera. Analysis and comparison of indirect ELISA and GPAT The Student t-test was used to evaluate the statistical significance of the data from cattle sera. Sensitivity, specificity, positive predictive value, negative predictive value, and efficacy of the test of each assay were worked out using the method of Galen [12] and the two techniques were compared by linear correlation and regression. Results F. gigantica ES antigens used in this study were taken from the supernatant of the culture medium after 950 worms were cultured for 3 h. The protein concentration of antigens was 3.57 mg (37.6 mg/worm) in total. Indirect ELISA The ELISA values are shown in Fig 1. The mean values of group A, group B, group C and group D were 0.593, 0.201, 0.43, and 0.514, respectively. A cut-off value was considered from the negative controls (group B) at X + 2 SD, 0.465. Nearly all sera of field bovines (groups C and D) had ELISA values higher than the cut-off value. By using this cut-off value, sensitivity, specificity, Vol 24 (No. 1) June 2001 THE JOURNAL OF TROPICAL MEDICINE AND PARASITOLOGY 3

positive predictive value, negative predictive value, and efficacy of the test, were found to be 100%, 23.4%, 23.4%, 100%, and 37%, respectively. Cross-reaction occured to a certain extent with cattle sera with single infection of S. spindale, Moniezia sp, Trichuris sp, mixed infection among gastro-intestinal nematodes and rumen fluke, gastro-intestinal nematodes and Moniezia sp, and mixed infection among gastro-intestinal nematodes, rumen fluke and Moniezia sp. GPAT When the serum dilution of 1:8 was used as the cut-off titer, it was found that the sensitivity, specificity, positive predictive value, negative predictive value, and efficacy of the test were 100%, 11.1%, 25%, 100%, and 31.4%, respectively. The GPAT results (log 2 ) are shown in Fig 2. Cross-reactions occurred with sera from most of the heterologous cattle sera and fieldstool negative sera. Correlation between ELISA and GPAT The associations were analyzed using GPAT titers (log 2 ) and OD values from the ELISA of the 48 cattle sera. The linear regression of ELISA and GPAT is shown in Fig 3. The correlation coefficient between the two tests was 0.57. Discussion In the present study, ELISA values of calf sera and field-stool negative cattle sera were both obtained and the titer of calf sera and field-stool negative cattle sera were very much different. The mean absorbance value was low in calf sera but high in field-stool negative cattle sera. There was a possibility that the cattle were previously exposed to F. gigantica infection and antibody to the infection already existed. They were not suitable for use as control sera. However, eventhough calf sera were used as control, our ELISA result showed cross-reaction with all schistosome sera and many sera of other parasites. Viyanant et al [13] reported that the 0.9 0.8 Optical density at 492 nm 0.7 0.6 0.5 0.4 0.3 0.2 X + 2 SD = 0.465 0.1 0 Sc RF Mo GI Tr RF+GI RF+Mo GI+Mo RF+GI+Mo Group A Group B Group C Group D Fig 1 A pattern of ELISA absorbance values of cattle sera against ES antigens. Group A : Experimental fasciolosis sera (15 samples) Group B : Calf sera (15 samples), X + 2 SD = 0.465 Group C : Field-heterologous sera (119 samples) Group D : Field-stool negative sera (120 samples) 4 THE JOURNAL OF TROPICAL MEDICINE AND PARASITOLOGY Vol 24 (No. 1) June 2001

9 Reciprocal in GPAT titers (log 2 ) 8 7 6 5 4 3 2 1 0 Group A Group B Group C Group D Fig 2 Distribution of antibody titers in cattle sera ( ) against ES antigens in GPAT. Group A : Experimental fasciolosis sera (8 samples) Group B : Calf sera (5 samples) Group C : Field-heterologous sera (23 samples) Group D : Field-stool negative sera (9 samples) 0.9 0.8 ELISA values (OD 492 nm) 0.7 0.6 0.5 0.4 0.3 0.2 y = 0.0498x + 0.1723 r = 0.57, n = 45 0.1 0 0 1 2 3 4 5 6 7 8 9 10 Titers of GPAT (log 2 ) Fig 3 Scatter plots of ELISA values and GPAT titers (log 2 ) of the same bovine sera reacted with ES antigen of F. gigantica. Vol 24 (No. 1) June 2001 THE JOURNAL OF TROPICAL MEDICINE AND PARASITOLOGY 5

sensitivity, when serum samples from uninfected calves was used as control cut-off value was 86.6%, but when the cut-off value was calculated from fetal calf sera and the trematode-free baby calf sera, the sensitivity improved to 100%. However, field cattle sera were not used in their study. Anderson et al [14] reported that positive and negative predictive values for ELISA using ES antigens of F. gigantica with 72 sera of stool positive cattle and 20 sera of stool negative cattle were 91.2% and 58.3%. But this present study correlated with Luong et al [15], who used ES antigens of Fasciola sp to detect antibody in Vietnamese cattle. They reported that the positive results in ELISA and fecal examination were 86% and 59%. These data also showed high positive predictive value. Fagbemi and Guobadia [16] reported that the negative predictive value was 45.5%. They concluded that there was no correlation between the fecal egg and the absorbance values in ELISA. In this present study, ELISA absorbance values of field cattle sera were high for cattle that were both stool positive with other helminths, and also with stool negative cattle. Our study aimed to develop a technique to be used in the field, so we used a large number of field sera for analysis. It can be concluded that crude ES antigen cannot be used to diagnose fasciolosis of field cattle by ELISA. The ELISA value in this present study showed that almost all S. spindale sera were crossreactive with F. gigantica ES antigens. Yagi et al [17] also found that primary infection with F. gigantica could cause cattle resistance to infection with S. bovis. They showed that cross-reaction occurred among different species of trematodes. GPAT showed a similar result to ELISA. It cannot be used for diagnosis of fasciolosis in field cattle. GPAT had been developed for 10 years, mostly for detection of human parasitic infections. The present study applied GPAT to the diagnosis of bovine fasciolosis. It was found that the correlation coefficient between GPAT and ELISA was moderate. In contrast to the study of Sato and Ryumon [6] who diagnosed human strongyloidosis, and reported a good correlation between GPAT and ELISA. Yang et al [18] reported the correlation between ELISA and GPAT was 0.742 in humans with S. japonicum infection. Kobayashi et al [6] also reported a good correlation between these two tests in human schistosomosis mansoni. To evaluate antigens, further investigation should be performed by a detection of subclass immunoglobins or by preparation of antigen with different molecular weight cut-off technique. Acknowledgement The authors would like to express their gratitude to Dr Suwannee Nithiuthai, Parasitology Unit, Faculty of Veterinary Medicine, Chulalongkorn University, for her useful suggestions for this study. Thanks to the SEAMEO-TROPMED for financial support. Thanks also to Mr Dorn Wattanakulpanich, Dr Suree Thammasart, Dr Wantanee Neramitmansook, Capt Dr Manahnun Prasittirat, Dr Reka Kanitpun, staff of the Department of Helminthology, Faculty of Tropical Medicine and staff of the Parasitology Section, NIAH, for their technical support. References 1. Sukapesna V, Tuntasuvan D, Sarataphan N, Imsub K. Economic impact of fascioliasis in buffalo production. J Thai Vet Med Assoc 1994;45:45-52. 2. Santiago N, Hillyer GV. Antibody profiles by EITB and ELISA of cattle and sheep infected with Fasciola hepatica. J Parasitol 1988;74:810-8. 3. Fagbemi BO, Obarisiagbon IO. Comparative evaluation of the enzyme linked immunosorbent assay in the diagnosis of natural Fasciola gigantica infection in cattle. Vet Q 1990;12:35-8. 4. Thammasart S, Chompoochan T, Prasittiratana P. Development of the ELISA technique for detection of bovine fasciolosis. J Nat Res Council Thailand 1995;27:47-55. 5. Boyden SU. The absorption of protein on erythrocytes treated with tannic acid and subsequent hemagglutination by 6 THE JOURNAL OF TROPICAL MEDICINE AND PARASITOLOGY Vol 24 (No. 1) June 2001

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