Veterinary Research Communications, 16 (1992) 403-406 Copyright C) Kluwcr Academic Publishers by - Printed in the Netherlands
Short Communication A MODIFIED FILTER PAPER TECHNIQUE FOR SEROSURVEILLANCE OF NEWCASTLE DISEASE P. ROY, K. NACHIMUTHU AND A.T. VENUGOPALAN Centre for Animal Health Studies, Tamil Nadu Veterinary and Animal Sciences University, Madhavaram Milk Colony, Madras 600 051, India
Roy, P., Nachimuthu, K. and Venugopalan, A.T., 1992. A modified filter paper technique for serosurveillance of Newcastle disease. VeterinaryResearch Communications, 16 (5), 403-406
Keywords: blood, haemagglutination-inhibition, Newcastle disease, serology, virus
INTRODUCTION Newcastle disease (ND) poses a continuous and unresolved threat to poultry farmers. In tropical countries, with a dense population of poultry in open houses near farms, eradication of ND is difficult. Hence, vaccination is necessary to control the disease. Assessment of the post-vaccinal immune response is essential to monitor the efficacy of a vaccination schedule and of the vaccines used. This can be done by the haemagglutination-inhibition (HI) test, which is easy to perform and rapid, but large-scale serosurveillance presents some problems. These include collection of the sera, their storage and transportation to a laboratory. To overcome some of these difficulties, the use of filter paper to collect blood samples for serosurveillance has been recommended (Beard and Brugh, 1977; Brugh and Beard, 1980; Giambrone, 1981; Rivetz et al., 1985). An attempt was made to reduce the test time by modifying the fdter paper technique using Whatman No. I filter paper for collection of blood samples, Brij-35 solution for quick elution of antibody, and a micro-HI procedure to evaluate the antibody content.
MATERIALS AND METHODS Filter paper: Whatman No. 1 f'dter paper was cut into strips of approximately 1.3 x 10 cm. Three strips were overlapped in the middle and stapled together. Each duster of three strips could be used to collect six samples, blood being collected on both ends of each strip. Sample collection: Blood samples were collected from a wing vein of birds with a known history of vaccination against ND. The blood was allowed to diffuse into the filter paper strip. About 1 cm at the end of each strip was saturated with blood, as indicated by equal staining on both sides. At the same time blood was collected
404
aseptically into a sterile test tube for serum. Filter papers carrying blood were folded to give a concave shape to facilitate easy drying and allowed to dry. After drying, half the f'dter papers were kept at room temperature and the other half at +4°C in polypropylene bags. Sera collected for comparative studies were stored at -200C until used.
Antigen: Newcastle disease virus (NDV-K) obtained from the Institute of Veterinary Preventive Medicine, Ranipet, was used for conducting the HI test. Chicken erythrocytes: Chicken red blood cells (CRBC) collected into Alsever's solution from 6-8-week-old birds were washed three times with normal szllne solution and a 0.5% suspension was used as the indicator in the HI test. Phosphate-buffered saline: Phosphate-buffered saline was used to elute the antibody from filter paper discs. Elution" Discs of 5 mm diameter were cut from the blood-stained sample area with a hand punch and placed in 0.1% (v/v) polyoxyethylene-23,1auryl-ether (Brij-35) in normal saline solution in a fiat-bottomed microtitre plate. Complete elution was indicated by uniform light coloration of the discs after 2 h at room temperature. Microhaemagglutination inhibition test: V-bottomed microplates were charged with 25 /zl of normal saline solution, except for the first well in each row. To the first well of each row, 50/zl of the elute obtained from 2 discs in 100/zl of Brij-35 solution for each sample was transferred using a micropipette (HLT, Germany) and doubling dilutions were made across the plate. The micro-HI test was conducted by the method of Allan and Gough (1974). For comparison, f'tlter paper eluate and whole serum samples were used in parallel tests. The reciprocal of the highest dilution of serum showing complete button formation by the CRBC was taken as the HI titre of the serum. RESULTS The results of micro-HI tests carried out at weekly intervals for 4 weeks on eluates from filter paper samples stored at room temperature or at refrigerator temperature (+ 4°C) and on comparable whole serum samples are presented in Tables I and II. When the filter paper eluate from one disc in 200/zl, two discs in 200/zl and two discs in 100/zl of Brij-35 solution was compared with the respective whole serum samples in preliminary studies, differences in the titre of 25, 24 and 23 were observed, respectively. Hence, to reduce the difference in the HI titres between whole serum and filter paper eluate, two discs in 100/zl of Brij-35 solution was used routinely. This eluate is equivalent to a 1:16 dilution of the serum samples. Two hours' treatment with 0.1% Brij-35 solution was found to be sufficient for complete elution as overnight elution did not improve the recovery of antibody. A 0.2% Brij-35 solution did not otherwise alter the elution process or HI results. Phosphate-buffered saline and normal saline were unsuitable for rapid elution.
405 TABLE I Comparison of micro-HI tests on serum samples stored at -200C and f'dter papers stored at room temperature
Mean HI titre (log2) a
Test interval (weeks) Whole serum
1 2 3 4
7.3 6.3 6.3 6.7
--- 1.03 - 0.93 - 0.63 -+ 0.88
Filter paper eluate
4.7 4.0 3.6 3.5
__. 0.81 +_ 0.71 - 0.42 - 0.60
aMeans are the average of 10 samples
T A B L E II Comparison of micro-HI tests on serum samples stored at -200C and f'dter papers stored at refrigerator temperature (+ 4°C)
Test interval
Mean HI titre (log2)a
(weeks) Whole serum
1 2 3 4
5.9 5.1 6.6 6.1
-+ 0.66 __. 0.79 _+ 0.43 _+ 0.54
Filter paper eluate
3.7 2.4 3.7 3.0
- 0.63 +__0.33 - 0.30 _+ 0.51
aMeans are the average of 10 samples except at week 3 when 23 samples were tested
DISCUSSION The HI titres of filter papers stored at room temperature or at refrigerator temperature (+ 4°C) did not vary significantly. Filter paper samples can be stored at room temperatures but should be screened within 3 weeks as there was a reduction in HI titres by the fourth week. Several of the main constraints in a serosurveillance programme for ND, such as trained personnel, collection of blood under sterile conditions, packing and despatch
406
and cold storage of serum samples are avoided by use of this simple technique. Filter papers are readily available and inexpensive. Rivetz et al. (1985) used Whatman No. 1 f'dter paper in an immuno-comb study. In earlier reports (Beard and Brugh, 1977; Brugh and Beard, 1980; Giambrone, 1981) PBS was used as an eluting agent and overnight soaking was needed for complete elution, but this problem was avoided by using the Brij-35 solution as described by Rivetz et al. (1985). Brij-35 solution hastens the elution process and HI results can be read within 4 h. Since the eluate from two discs in 100 /zl Brij-35 solution was found to be equivalent to a 1:16 dilution of whole serum, HI values below 16 cannot be estimated by this technique. This does not greatly affect serological monitoring programmes since HI values of less than 64 are considered non-specific (Giambrone, 1981) and only HI values of 80 and above were found to be protective (Saifuddin et al., 1990). Hence, this technique could be used for serosurveillance of ND in commercial flocks under field conditions. REFERENCES Allan, W.H. and Gough, ILE., 1974. A standard haemagglutination inhibition test for Newcastle disease. 1. A comparison of macro and micro methods. VeterinaryRecord, 95, 120-123 Beard, C.W. and Brugh, M., 1977. Use of the Nobuto blood sampling paper strip for Newcastle disease serology. Avian Disease, 21, 630-636 Brugh, M. and Beard, C.W., 1980. Collection and processing of blood samples dried on paper for micro assay of Newcastle disease virus and avian influenza virus antibodies. American Journal of Veterinary Research, 41, 1495-1498 Giambrone, JJ., 1981. Antibody levels - how they arc determined, what is their significance. Poultry Digest, 40, 624-627 Rivvtz, B., Wcisman, Y., Ritterband, M., Fish, F. and Herzherg, M., 1985. Evaluation of a Novel Rapid Kit for the visual detection of Newcastle disease virus antibodies. Avian Disease, 29, 929-941 Saifuddin, M.D., Chaudhury, T.I.M.F.IL, Sanker, AJ. and Arun, M.M., 1990. Protection confirmed by vaccination with Blacksburg and Komarov strains of Newcastle disease virus against Newcastle disease in Bangladesh. TropicalAnimal Health and Production, 22, 263-272 (Accepted 25 August 1992)
Short Communication A MODIFIED FILTER PAPER TECHNIQUE FOR SEROSURVEILLANCE OF NEWCASTLE DISEASE P. ROY, K. NACHIMUTHU AND A.T. VENUGOPALAN Centre for Animal Health Studies, Tamil Nadu Veterinary and Animal Sciences University, Madhavaram Milk Colony, Madras 600 051, India
Roy, P., Nachimuthu, K. and Venugopalan, A.T., 1992. A modified filter paper technique for serosurveillance of Newcastle disease. VeterinaryResearch Communications, 16 (5), 403-406
Keywords: blood, haemagglutination-inhibition, Newcastle disease, serology, virus
INTRODUCTION Newcastle disease (ND) poses a continuous and unresolved threat to poultry farmers. In tropical countries, with a dense population of poultry in open houses near farms, eradication of ND is difficult. Hence, vaccination is necessary to control the disease. Assessment of the post-vaccinal immune response is essential to monitor the efficacy of a vaccination schedule and of the vaccines used. This can be done by the haemagglutination-inhibition (HI) test, which is easy to perform and rapid, but large-scale serosurveillance presents some problems. These include collection of the sera, their storage and transportation to a laboratory. To overcome some of these difficulties, the use of filter paper to collect blood samples for serosurveillance has been recommended (Beard and Brugh, 1977; Brugh and Beard, 1980; Giambrone, 1981; Rivetz et al., 1985). An attempt was made to reduce the test time by modifying the fdter paper technique using Whatman No. I filter paper for collection of blood samples, Brij-35 solution for quick elution of antibody, and a micro-HI procedure to evaluate the antibody content.
MATERIALS AND METHODS Filter paper: Whatman No. 1 f'dter paper was cut into strips of approximately 1.3 x 10 cm. Three strips were overlapped in the middle and stapled together. Each duster of three strips could be used to collect six samples, blood being collected on both ends of each strip. Sample collection: Blood samples were collected from a wing vein of birds with a known history of vaccination against ND. The blood was allowed to diffuse into the filter paper strip. About 1 cm at the end of each strip was saturated with blood, as indicated by equal staining on both sides. At the same time blood was collected
404
aseptically into a sterile test tube for serum. Filter papers carrying blood were folded to give a concave shape to facilitate easy drying and allowed to dry. After drying, half the f'dter papers were kept at room temperature and the other half at +4°C in polypropylene bags. Sera collected for comparative studies were stored at -200C until used.
Antigen: Newcastle disease virus (NDV-K) obtained from the Institute of Veterinary Preventive Medicine, Ranipet, was used for conducting the HI test. Chicken erythrocytes: Chicken red blood cells (CRBC) collected into Alsever's solution from 6-8-week-old birds were washed three times with normal szllne solution and a 0.5% suspension was used as the indicator in the HI test. Phosphate-buffered saline: Phosphate-buffered saline was used to elute the antibody from filter paper discs. Elution" Discs of 5 mm diameter were cut from the blood-stained sample area with a hand punch and placed in 0.1% (v/v) polyoxyethylene-23,1auryl-ether (Brij-35) in normal saline solution in a fiat-bottomed microtitre plate. Complete elution was indicated by uniform light coloration of the discs after 2 h at room temperature. Microhaemagglutination inhibition test: V-bottomed microplates were charged with 25 /zl of normal saline solution, except for the first well in each row. To the first well of each row, 50/zl of the elute obtained from 2 discs in 100/zl of Brij-35 solution for each sample was transferred using a micropipette (HLT, Germany) and doubling dilutions were made across the plate. The micro-HI test was conducted by the method of Allan and Gough (1974). For comparison, f'tlter paper eluate and whole serum samples were used in parallel tests. The reciprocal of the highest dilution of serum showing complete button formation by the CRBC was taken as the HI titre of the serum. RESULTS The results of micro-HI tests carried out at weekly intervals for 4 weeks on eluates from filter paper samples stored at room temperature or at refrigerator temperature (+ 4°C) and on comparable whole serum samples are presented in Tables I and II. When the filter paper eluate from one disc in 200/zl, two discs in 200/zl and two discs in 100/zl of Brij-35 solution was compared with the respective whole serum samples in preliminary studies, differences in the titre of 25, 24 and 23 were observed, respectively. Hence, to reduce the difference in the HI titres between whole serum and filter paper eluate, two discs in 100/zl of Brij-35 solution was used routinely. This eluate is equivalent to a 1:16 dilution of the serum samples. Two hours' treatment with 0.1% Brij-35 solution was found to be sufficient for complete elution as overnight elution did not improve the recovery of antibody. A 0.2% Brij-35 solution did not otherwise alter the elution process or HI results. Phosphate-buffered saline and normal saline were unsuitable for rapid elution.
405 TABLE I Comparison of micro-HI tests on serum samples stored at -200C and f'dter papers stored at room temperature
Mean HI titre (log2) a
Test interval (weeks) Whole serum
1 2 3 4
7.3 6.3 6.3 6.7
--- 1.03 - 0.93 - 0.63 -+ 0.88
Filter paper eluate
4.7 4.0 3.6 3.5
__. 0.81 +_ 0.71 - 0.42 - 0.60
aMeans are the average of 10 samples
T A B L E II Comparison of micro-HI tests on serum samples stored at -200C and f'dter papers stored at refrigerator temperature (+ 4°C)
Test interval
Mean HI titre (log2)a
(weeks) Whole serum
1 2 3 4
5.9 5.1 6.6 6.1
-+ 0.66 __. 0.79 _+ 0.43 _+ 0.54
Filter paper eluate
3.7 2.4 3.7 3.0
- 0.63 +__0.33 - 0.30 _+ 0.51
aMeans are the average of 10 samples except at week 3 when 23 samples were tested
DISCUSSION The HI titres of filter papers stored at room temperature or at refrigerator temperature (+ 4°C) did not vary significantly. Filter paper samples can be stored at room temperatures but should be screened within 3 weeks as there was a reduction in HI titres by the fourth week. Several of the main constraints in a serosurveillance programme for ND, such as trained personnel, collection of blood under sterile conditions, packing and despatch
406
and cold storage of serum samples are avoided by use of this simple technique. Filter papers are readily available and inexpensive. Rivetz et al. (1985) used Whatman No. 1 f'dter paper in an immuno-comb study. In earlier reports (Beard and Brugh, 1977; Brugh and Beard, 1980; Giambrone, 1981) PBS was used as an eluting agent and overnight soaking was needed for complete elution, but this problem was avoided by using the Brij-35 solution as described by Rivetz et al. (1985). Brij-35 solution hastens the elution process and HI results can be read within 4 h. Since the eluate from two discs in 100 /zl Brij-35 solution was found to be equivalent to a 1:16 dilution of whole serum, HI values below 16 cannot be estimated by this technique. This does not greatly affect serological monitoring programmes since HI values of less than 64 are considered non-specific (Giambrone, 1981) and only HI values of 80 and above were found to be protective (Saifuddin et al., 1990). Hence, this technique could be used for serosurveillance of ND in commercial flocks under field conditions. REFERENCES Allan, W.H. and Gough, ILE., 1974. A standard haemagglutination inhibition test for Newcastle disease. 1. A comparison of macro and micro methods. VeterinaryRecord, 95, 120-123 Beard, C.W. and Brugh, M., 1977. Use of the Nobuto blood sampling paper strip for Newcastle disease serology. Avian Disease, 21, 630-636 Brugh, M. and Beard, C.W., 1980. Collection and processing of blood samples dried on paper for micro assay of Newcastle disease virus and avian influenza virus antibodies. American Journal of Veterinary Research, 41, 1495-1498 Giambrone, JJ., 1981. Antibody levels - how they arc determined, what is their significance. Poultry Digest, 40, 624-627 Rivvtz, B., Wcisman, Y., Ritterband, M., Fish, F. and Herzherg, M., 1985. Evaluation of a Novel Rapid Kit for the visual detection of Newcastle disease virus antibodies. Avian Disease, 29, 929-941 Saifuddin, M.D., Chaudhury, T.I.M.F.IL, Sanker, AJ. and Arun, M.M., 1990. Protection confirmed by vaccination with Blacksburg and Komarov strains of Newcastle disease virus against Newcastle disease in Bangladesh. TropicalAnimal Health and Production, 22, 263-272 (Accepted 25 August 1992)
