Veterinary Parasitology Dicrocoelium1[1]

veterinary parasitology ELSEVIER

Veterinary Parasitology 61 (1996) 151-156

Short Communication

Comparative evaluation of agar gel precipitation, counterimmunoelectrophoresis and passive haemagglutination tests for the diagnosis of Dicrocoelium dendriticum infection in sheep and goats K.P. Jithendran *, J. Vaid *, L. Krishna Disease InvestigationLaboratory, Indian VeterinaryResearch Institute, Regional Station, Palampur, H.P., India

Received 18 October 1994; accepted 9 February 1995

Abstract The sensitivity and specificity of the agar gel precipitation test (AGPT), counter immunoelectrophoresis (CIEP) and passive haemagglutination test (PHT) were evaluated for the diagnosis of Dicrocoelium dendriticum infection in naturally infected sheep and goats. Two hundred and forty five sheep and goat sera samples were tested using phosphate buffered saline, pH 7.2 extracted adult fluke antigen. CIEP detected 69.8% of the infected animals and was found to be the most sensitive, followed by PHT which detected 50.0% of the infected sheep and goats. AGPT was found to be the least sensitive, detecting only 23.8% of the infected animals. The specificity of AGPT, CIEP and PHT were 93.3%, 84.0% and 93.3%, respectively. CIEP was found to be the most sensitive, specific and rapid test for the seroepidemiological survey of dicrocoeliosis in sheep and goats. Keywords: Sheep-Trematoda; Goat; Dicrocoelium dendriticum; Diagnosis-Trematoda

1. Introduction Dicrocoeliosis caused by Dicrocoelium dendriticum is a trematode infection that occurs in sheep, goats, cattle and buffalo (Soulsby, 1982). Animals suffering from it

* Corresponding author. t Deceased. 0304-4017/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0304-4017(95)00808-X

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show clinical symptoms consisting of anaemia, oedema, emaciation and, in advanced cases, extensive cirrhosis, scarring of the liver surface and marked distension of bile ducts. Though the disease is less severe than fascioliosis, economic losses as a result of liver condemnation are high in the North Temperate Himalayan Region of the Indian subcontinent. The failure to detect prepatent infections by faecal examination is one of the contributing factors which hinders the chemotherapeutic control of this disease among livestock. Corba et al. (1987) and Sanz et al. (1987) have reported that even at low doses, luxabendazole (10 mg kg 1 body weight) and netobimin (15-20 mg kg ] body weight) respectively, are highly efficacious (98-99%) against this parasite. The diagnostic value of serological tests for detection of dicrocoeliosis in sheep and goats has not been critically addressed. The present study was therefore conducted to evaluate the suitability of some serological tests for the diagnosis of dicrocoeliosis in migratory sheep and goats and the possibility of its control in these flocks using such drugs.

2. Materials and methods 2.1. Antigen Adult flukes of Dicrocoelium dendriticum were collected from the bile ducts of naturally infected sheep and goats. After removal from the bile ducts, the flukes were washed in several changes of chilled phosphate buffered saline (PBS, pH 7.2), homogenised in a glass homogeniser and kept at 4°C. The homogenate was centrifuged at 10 000 × g for 30 min at 4°C. The supernatant fluid was collected and used as adult fluke antigen (ALVA). The protein concentration of the antigen was determined by the method of Lowry et al. (195l). 2.2. Sera Blood samples were collected from 2 - 6 year old sheep and goats before slaughter at the local abattoir and their livers were subsequently examined for D. dendriticum infection. Sera samples from 70 sheep and 56 goats, which were confirmed to be positive for this infection, were selected for the study. Sera samples from 47 sheep and 54 goats which were free from D. dendriticum infection were used as control sera. Additionally, 18 Fasciola gigantica infected reference sera from sheep and goats were employed for testing apparent cross reactivity. Sera samples, containing 0.2% sodium azide (1: 100) were stored at - 2 0 ° C until further use. Prior to their use in serological tests these sera samples were inactivated at 56°C for 30 min. 2.3. lmmunodiagnostic tests The agar gel precipitation test (AGPT) was performed as described by Edwards (1979). One per cent Noble agar (Difco Laboratories, Detriot, MI, USA) in PBS, pH 7.2 was used in the preparation of gel. The diluted antigen (protein content 4 - 6 mg m l - ] ) was used against the undiluted sera and diffusion was allowed to take place in a moist

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chamber at 37°C for 48 h. The precipitation lines were examined after washing in normal saline and the slides were stained with amido black. Counterimmunoelectrophoresis (CIEP) was carried out according to the method of Edwards (1979) using 1% agarose (Loba Chemie, Vienna) in 0.02 M barbital buffer, pH 8.6. The electrophoresis was conducted at room temperature at 10 mA per slide for 90 min. Known positive and negative control sera were also run with each test. The slides were washed and stained as for AGPT. Passive haemagglutination tests (PHT) were carried out according to the method described by Sundick and Rose (1980). The tests were standardised using the antigen containing 1 mg ml-~ protein and diluted ten times in PBS. In brief, sheep red blood cells (SRBC) were collected aseptically in Alsever's solution (1:1, v / v ) and washed three times with 0.15 M PBS, pH 7.2 by sedimentation (800 X g). The washed SRBC were incubated for 15 min at 37°C with an equal volume of PBS containing 0.005% tannic acid. The tanned cells were concentrated by centrifugation at 800 × g for 20 min, washed three times and divided into two volumes. Dicrocoelium dendriticum A F A diluted in PBS (1 mg m l - ~ protein conc.) was added to one aliquot of the cells and the mixture was incubated for 30 min at 37°C. The sensitised cells were washed three times and resuspended to 1% suspension in PBS containing 1% inactivated normal rabbit serum. The other aliquots of cells served as unsensitised controls. Two-fold dilutions of test serum in 0.25 ml PBS were made in a perspex plate and 0.25 ml of sensitised SRBC were added to each dilution. In addition, each test sample was tested against unsensitised cells for heterophilic antibodies. To compare the test results, 0.25 ml of sensitised SRBC were also added to 0.25 ml PBS. The plates were incubated at 37°C and read after 2 h. The agglutination pattern of sensitized SRBC in test dilutions was compared with appropriate controls and the absence of a button formation was taken as positive end point. The sensitivity and specificity of these tests were calculated as follows True P o s i t i v e s - False Negatives Sensitivity (%) =

True Positives

X 100

True N e g a t i v e s - False Positives Specificity (%) =

True Negatives

× 100

3. Results The results of the serological tests for the diagnosis of D. dendriticum infection in sheep and goats by AGPT, CIE and PHT are presented in Tables 1 and 2, respectively. In AGPT, sharp precipitation lines were evident in 16 out of 70 known positive sera samples from sheep and 14 out of 56 such samples from goats. The sensitivity of AGPT was found to be lower than that of the other tests as it gave 22.9% for sheep and 25.0% for goats. Using the resolving power of CIEP, it was possible to detect 71.4% infection in sheep and 67.9% in goats with a reasonably high degree of specificity of 85.5% and 82.8%

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Table 1 Comparative sensitivity and specificity of different immunodiagnostic tests using D. dendriticum adult fluke antigen in sheep Serum samples

Total sera tested

D. dendriticum infected

70

Non-infected

47

F. gigantica infected

Total non-specific reaction

8 55

Specificity (%)

No. positive (%) AGPT

CIEP

PHT

16 (22.9) 2 (4.3) 2 (25.0) 4 (7.3) 92.7

50 (71.41 4 (8.5) 4 (50.111 8 (14.5) 85.5

35 (50.0) 3 (6.4) 4 (50.0) 7 (12.71 87.3

respectively. The test, h o w e v e r , g a v e 8.5% and 13.0% false reaction in non infected sheep and goat sera, respectively. In PHT, the control (uninfected) sera samples g a v e titres ranging from 1:2 to 1:32, while it ranged from 1:2 to 1:1024 in infected sheep and goats. Since 96.1% of the uninfected sera showed titres ranging from 1:2 to 1:16, titres of o v e r 1:32 were considered as significant test titre for P H T in the present study.

Table 2 Comparative sensitivity and specificity of different immunodiagnostic tests using D. dendriticum adult fluke antigen in goats Serum samples

Total sera tested

D. dendriticum infected

56

Non-infected

54

F. gigantica infected

111

Total non-specific reaction

64

Specificity ( %)

No. positive (%) AGPT

CIEP

PHT

14 (25.0) 0 (0.0) 4 (40.0) 4 (6.3) 93.8

38 (67.9) 7 (13.111 4 (40.0) I1 (17.2) 82.8

28 (50.0) 1 (1.9) 0 (0.0) 1 (1.6) 98.4

Table 3 Overall sensitivity and specificity of different immunodiagnostic tests using D. dendriticum adult fluke antigen in sheep and goats Immunodiagnostic tests

Sensitivity (%)

Specificity (%)

AGPT CIEP PHT

23.8 69.8 50.0

93.3 84.0 93.3

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The comparative sensitivity and specificity of different immunodiagnostic tests used in the present study are presented in Table 3. The overall sensitivity of AGPT, CIEP and PHT were 23.8%, 69.8% and 50.0%, respectively. However, the specificity of the tests was variable with 93.3%, 84.0%, and 93.3% in AGPT, CIEP and PHT, respectively. The cross reactivity of these tests with F. gigantica infected sheep and goat reference sera indicated 25-40%, 40-50% and 0-50% positive reaction in AGPT, CIEP and PHT, respectively.

4. Discussion Immunodiagnostic tests have an important role to play in the seroepidemiology of helminthosis and serve as a valuable adjunct to existing diagnostic methods. The specificity and sensitivity of the three serodiagnostic tests were evaluated in the present study. The AGPT showed the lowest sensitivity of the three tests as it gave a rate of 22.8% for sheep sera and 25.0% for goat sera. The low sensitivity of this test was previously reported by Pathak and Gaur (1986) in porcine cysticercosis and by Swamp et al. (1987) in bovine fascioliosis. In contrast, CIEP has been reported to be highly sensitive in the serodiagnosis of various parasitic infections including F. gigantica in sheep (Pathak et al., 1986) and buffaloes (Swarup et al., 1987), and cysticercosis in pigs (Kumar and Gaur, 1989). PHT showed a higher sensitivity than AGPT but was less specific than CIEP. However, the specificity was found to be reasonably high in PHT followed by AGPT and least in CIEP. In the present study, 1:32 was considered the minimum diagnostic titre for PHT. Recently, Somvanshi et al. (1993) also reported the use of PHT and CIEP for the diagnosis of D. dendriticum in goats and indicated that CIEP was more sensitive than PHT, which is in agreement with the present observations. The background titre at the lower dilutions of the control sera probably reflects the apparent lack of specificity a n d / o r is due to overt cross reactivity with other parasites. In the present study, CIEP was found to be more sensitive, quite specific and a rapid test for the diagnosis of dicrocoeliosis in sheep and goats. However, purification of fluke antigens may qualitatively improve the diagnostic value of the tests, which needs further elucidation.

Acknowledgement The authors are thankful to the Director, Indian Veterinary Research Institute, Izatnagar, (U.P.) for providing facilities.

References Corba, J., Hovorka, J., Spaldonova, R., Stoffa, P., Legeny, J. and Andrasko, H., 1987. Efficacy of luxabendazole (Hoe 216 V) susp. 5% in sheep naturally infected with the most important helminths. Helminthologia, 24: 227-235.

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Edwards, E.A., 1979. Counterimmunoelectrophoresis and double immunodiffusion. In: W. Ryfel (Editor), Rapid Diagnosis in Infectious Disease. CRC Press, Boca Raton, FL, pp. 19-37. Kumar, D. and Gaur, S.N.S., 1989. Comparative evaluation of various immunodiagnostic tests for thc diagnosis of T. solium cysticercosis in pigs using fractionated antigens. J. Helminthol., 63: 13-17. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., 1951. Protein measurement with the Folin-phenol reagent. J. Biol. Chem., 193: 265-275. Pathak, K.M.L. and Gaur, S.N.S., 1986. Evaluation of some serodiagnostic tests in porcine cysticercosis. Indian J. Anim. Sci., 56: 1059-1061. Pathak, K.M.L., Kumar, D. and Gaur, S.N.S., 1986. lmmunodiagnosis of fascioliasis by counterimmunoelectrophoresis in sheep. Indian J. Vet. Med., 6:38 40. Sanz, F., Tarazona, J.M., Jurado, R., Frias, J.. Tarazona, J.V. and Duncan, J.L., 1987. An evaluation of the efficacy of netobimin against Dicrocoelium dendriticum in sheep. Vet. Rec., 120: 57-58. Somvanshi, R., Vaid, J., Biswas, J.C. and Jithendran, K.P., 1993. Clinicopathological observations on dicrocoeliasis in goats. Indian J. Vet. Pathol., 16:112-114. Soulsby, E.J.L., 1982. Helminths, Arthropods and Protozoa of Domesticated Animals, 7th edn. The English Language Book Society/Bailliere Tindall, London, 809 pp. Sundick, R.S. and Rose, N.R., 1981). Agglutination reactions. In: N.R. Rose and P.E. Bigazzi (Editors), Methods in Immunodiagnosis, 2nd edn. John Wiley, New York, pp. 109-127. Swarup, D., Pachauri, S.P., Sharma, B. and Bandhopadhyay, S.K., 1987. Serodiagnosis of Fasciola gigantica infection in buffaloes. Vet. Parasitol., 24: 67-74.