LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. XLIII (1), 2010 TIMISOARA IMMUNOLOGICAL DIAGNOSIS OF FASCIOLOSIS BY ELISA METHOD IN SHEEP AND CATTLE FROM ARAD COUNTY I. OPRESCU¹, GH. DARABU޹, S. MORARIU¹, NARCISA MEDERLE¹, M.S. ILIE¹, MIRELA IMRE¹, K. IMRE¹, P. ONITA² ¹Faculty of Veterinary Medicine Timisoara, 300645, Calea Aradului, No. 119, Timisoara, Romania ²DSVSA ARAD E-mail: [email protected] Summary The aim of the study " Immunological diagnosis of fasciolosis by Elisa method in sheep and cattle from Arad County " was to make a serological screening in sheep and cattle from Arad County in 2008. Results obtained in sheep show a medium infestation of animals from six localities and a lower infestation in other 13 CSV units. ELISA test in bovines painted out negative results in four units, in three units the infestation was medium and in the other 12 units infestation was low. Based on the obtained results the extensivity of faciolosis was determined, and a mapping of this helminthosis in Arad County was made. Key words: immunological, diagnosis, fasciolosis Fasciolosis is one of the most important helmintosis affecting the liver, of ruminants and produces significant losses in livestock populations (7). Early diagnosis of fasciolosis creates the opportunity of a prompt intervention with narrow-spectrum products, but with a high degree of efficiency, so that economic losses are minimal (3, 8, 9, 10, 11, 15, 16, 17). The surveillance program implemented by A.N.S.V.S.A. is trying to perform the serological monitoring of sheep and cattle for Fasciola hepatica infestations. ELISA immunoassay method is a paraclinic sensitive and accurate test, being used in most Western laboratories to monitorize the infections in sheep and cattle in endemic areas (1, 4, 6, 13, 14). These imunodiagnosis methods are widespread used also in human medicine (2, 5, 12). Materials and methods Economical impact of fasciolosis is in a continuous increase. This disease with populational extinction can be quickly diagnosed by serological tests and can easily be treated with specific anthelmintic drugs if it is diagnosed. For domestic and pet animals, the antigen hemaglutination "f2" method, proved to be more sensitive than immunoelectrophoresis. Recently, ELISA method using purified extract of Fasciola hepatica was improved (45). 21 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. XLIII (1), 2010, TIMISOARA Antigen "f2" is very immunogenic and has a high specificity for F. hepatica . This kit was standardized according to the haemaglutination method developed by Dr. Levieux team. The kit allows determining the concentration of antibodies to F. hepatica . It was validated for serum of sheep cattle and cow lactoser. Since in most cases the antibody concentration is correlated with the infestation, the analysis can be done on a single serum sample, but also a mixture of 5-10 sera, or a mixture of serum from all animals in the herd can be evaluated. Also, the test can be used on the milk from cooling tank. Procedure: dilute control samples 1/20, put 190 µl of “dilution buffer 2”, add 10 µl of undiluted negative control solution into A1 and A2, and 10 µl positive control solution in B1, B2 and C1, C2. Serum samples are diluted 1/20, put 190µl dilution buffer, adding 10µl of each serum in a coated tube and 10µl of each serum in uncoated tubes. Milk samples are processed as follows: put 200 µl of each sample of undiluted milk in a coated tube and the same amount of undiluted milk in an uncoated tube. The tube content is well homogenized by slightly shaking the tray on which the samples are arranged. The tray is then covered with an aluminum or plastic foil and incubated for one hour (± 5 min) at 37° C (± 3 º C). Samples distribution in the titration plates is shown below: N N 6 6 P P .. .. P P .. .. 1 1 2 2 3 3 4 4 5 5 N- negative control sample; P-positive control sample; 1 - sample No. 1; 2 - sample No. 2……; The next operation is washing. A bottle of "concentrated washing solution" is diluted in 1900 ml of distilled water. Mixture is called "washing solution", tray is emptied of content (can be made by automatic method) after that all the tubes on the tray will be filled with washing solution, then emptied (it takes repeat of 2 - 3 times). Conjugate storage: conjugate is diluted 1/100 with "dilution buffer1" and placed 100 ml/tube, cover the pan with aluminum foil and incubated 30 min (± 3min) at 37° C (± 3°C). After this stage another washing is performed (operation is repeated three times). 22 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. XLIII (1), 2010 TIMISOARA Revelation is made by putting a quantity of 100 µl of "Revealing solution 3” in each tube, followed by incubation at 21°C for 20 minutes (protect from light) after incubation 100 µl of "Stop Solution" are added to each tube and shake the pan gently until the solution is homogeneous. Carefully wipe the tray. The plate is read at an optical density of 450 nm (OD 450). Photometry must be proved first in goals (air) and calculate its correction OD 450 nm for each serum sample. It transcribes the value of OD 450 obtained from an uncoated tube and the value obtained from a sample. The result can be considered valid if: value uncorrected OD 450 nm of positive control is higher than 0.350 and if the ratio of value corrected OD 450 of positive control and correction value OD 450 of negative control is greater than or equal to 3.5. Interpretation of results: for each sample the ratio of S / P is calculated: S/P= 100 x corrected value OD 450 for analysed sample / mean corrected value OD 450 of positive control sample S/P % from Correlation between The correlation between test results and the sample the result of test and proportion of herd infestation the level of infestation (individual serum sample) %E/P ≥150% + + + Severe infestation (>50%) + + + 80< %E/P< 150% + + Medium infestation (20% and 50%) + + 30 <%E/P≤ 80 + Low infestation (<20%) + % E/P≤ 30% 0 Very low infestation 0 Note: Values described in the above table are only indicative. Indeed, if serum or milk is analyzed, results depend on the antibody concentration of each sample. A positive serological test reveals the presence of infected animals and contaminated land. Even after propriate treatment, with an effective drug, the animal will remain a carrier of antibodies up to 12 weeks (during this time, the concentration of antibodies may fluctuate). An animal treated in autumn can not be distinguished from one untreated 12 weeks ago. Furthermore, a serological examination (early in spring) will not be able to estimate the effectiveness of treatment. Blood samples were collected under sterile conditions, in special vacuum tubes without anticoagulant from sheep and cattle from 19 localities of Arad County. 3% of samples (Table 1) were analyzed for fasciolosis by the surveillance program, from the total herds of sheep and cattle. Corrected optimal density of 450 nm for samples examined in sheep have varied between 0.350 and 0.450 and values for cattle ranged from 0.365 to 0.450. Mean corrected density value 450 nm of positive control sample was 3.500 for sheep and 3.550 for cattle. 23 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. XLIII (1), 2010, TIMISOARA Table 1 Situation of examined serum samples from sheep and cattle in Arad County in 2008 Crt. C.S.V.A. Effectives Total of no. Sheep Cattle analyzed samples 1. Petris 720 500 22/15 2. Savarsin 500 600 15/18 3. Bata 820 540 25/16 4. Varadia de Mures 1600 500 48/15 5. Barzava 3339 969 100/28 6. Conop 5780 280 171/29 7. Odvos 556 230 17/7 8. Ususau 5800 180 172/5 9. Bocsig 13400 410 402/12 10. Cermei 2568 327 76/10 11. Apateu 5990 660 180/20 12. Chisineu-Cris 11850 395 357/12 13. Simand 5500 400 165/12 14. Sintea Mare 3200 221 96/7 15. Sepreus 6504 371 195/11 16. Archis 710 414 21/13 17. Carand 500 330 15/10 18. Santana 6932 793 208/24 19. Siria 5800 350 174/11 Results and discussions All serum samples from sheep and cattle were processed according to the instructions for ELISA method developed by the Institute Pourquier, France and have been described in the previous chapter. The reading of the samples was performed at 450 nm optical density. The degree of infestation has been established after a correlation between test results and the proportion of infestation in the herd. The results obtained from processed samples are presented in Table 2. Table 2 Results of positive tests samples for Fasciola hepatica in sheep and bovine serum by ELISA, in 2008 Crt. C.S.V.A. Total of analyzed samples Total positive samples nr. Sheep Cattle Sheep Cattle 1. Petris 22 15 2 1 2. Savarsin 15 18 1 1 3. Bata 25 16 3 1 4. Varadia de Mures 48 15 3 2 24 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. XLIII (1), 2010 TIMISOARA 5. Barzava 100 28 6 2 6. Conop 171 29 7 3 7. Odvos 17 7 1 1 8. Ususau 172 5 5 0 9. Bocsig 402 12 12 1 10. Cermei 76 10 3 1 11. Apateu 180 20 14 1 12. Chisineu-Cris 357 12 17 1 13. Simand 165 12 6 1 14. Sintea Mare 96 7 4 0 15. Sepreus 195 11 5 1 16. Archis 21 13 1 1 17. Carand 15 10 1 0 18. Santana 208 24 5 1 19.