Materials and methods
Results
Discussion
Conclusion
Bibliography
Short communications/brèves communications/ communicaciones breves
J.G. Bell, M. El Hakim El Alaoui and T. Jaouzi
The address of Drs Bell and Jaouzi is: Institut Agronomique et Vétérinaire Hassan 11, B.P. 6202, Rabat Instituts, Morocco. The address of Dr El Hakim El Alaoui is: Faculté des Sclences, Université Cadi Ayyad, Marrakech, Morocco. This work was supported by a technical cooperation project with the Animal Production and Health Section of the Joint FAO/IAEA Division, Vienna. The authors wish to thank Mohammed Sabate for his help throughout this work.
The Animal Production and Health Section of the Joint FAO/IAEA Division has developed an enzyme-linked immunosorbent assay (ELISA) kit for the screening of poultry sera for Newcastle disease antibodies. Based on the design of a kit for bovine antibodies, the simple indirect method uses a single serum dilution, peroxidase conjugate and orthophenylenediamine as substrate. The reagents are stable and incubation steps are carried out at 37°C, so the assay is easy to transport and suitable for widely fluctuating temperatures. ELISA titres were higher than haemagglutination inhibition titres and showed a high degree of correlation with them.
Newcastle disease is a fatal and highly contagious disease of poultry (Alexander, 1989). It is enzootic in most countries in Africa, Asia and South America, where it continues to cause serious losses despite the vaccination of industrialized poultry. The availability of a standard sensitive serological test adapted to the conditions in these countries would facilitate diagnosis and the accurate monitoring of vaccination programmes. The haemagglutination inhibition (HI) test, while simple to perform, is difficult to standardize among laboratories (Beard and Wilkes, 1985).
A number of methods have been published for the detection of antibodies against Newcastle disease virus (NDV) by means of enzyme-linked immunosorbent assays (ELISAs) (Snyder et al., 1983; Miers, Bankowski and Zee, 1983; Wilson et al., 1984; Mishra, Rai and Jaiswal, 1985; Adair et al., 1989) and commercial kits have also been produced.
The Joint FAO/IAEA Division (1989) produced a standard kit for the detection of bovine antibodies to particular antigens. It uses a simple, indirect ELISA and peroxidase conjugase, with ortho-phenylenediamine and hydrogen peroxide as substrates.
Unlike the methods cited above, the kit is designed to be easily transportable throughout the world and the reagents are sufficiently stable to withstand transport delays at ambient temperature. The incubation steps take place at 37°C in order to produce uniform results in widely fluctuating ambient temperatures. It was therefore considered worthwhile to adapt this kit for the detection of avian antibodies against NDV. This article describes such an adaptation and the standardization of the assay.
Concentrated NDV, prepared according to the method of Snyder et al. (1983) and supplied by the same authors, was used as the antigen. Plates were coated by way of overnight incubation at 4°C, following addition of 50 m l of antigen diluted in sodium bicarbonate coating buffer containing 0.2 g per litre of thiomersal. Plates were washed three times between each step in washing solution (0.014 g per litre of disodium phosphate and 0.01 percent Tween 20). Plates were incubated for 30 minutes at 37 °C following the addition of 50 m l per well of serum diluted in phosphatebuffered saline (PBS) containing 500 m l per litre of Tween 20. Peroxidase labelled affinity purified antibody to chicken IgG(H+L) (goat) was used as the conjugate.
Following washing, 50 m l per well of conjugate diluted in the same buffer as the sera - was added and plates were incubated for a further 30 minutes at 37°C. A substrate was prepared by adding one 30 mg tablet of ortho-phenylenediamine to 75 ml of distilled water followed by 40 m l of hydrogen peroxidase per 10 ml just before use. After washing, 50 m l of substrate was added to each well and incubated for ten minutes at 37°C. The reaction was stopped by adding 25 m l of 0.25 M citric acid and plates were read at 450 nm in an air-blanked ELISA reader. Subsequently, 1 M sulphuric acid was used as a stopper and plates were read at 492 nm.
The antigen dilution was fixed at 1:200 and the conjugate dilution at 1:1 600, since these dilutions gave the highest ratios of absorbance with positive anti-NDV serum divided by absorbance with negative serum.
In order to establish a reference graph relating absorbance at a fixed dilution directly to titre, 30 field serum samples and eight negative serum samples from specific pathogen-free (SPF) chickens were titrated at 12 doubling dilutions, starting at 1:400. The field serum samples, with HI titres varying from o to 7, were obtained from village chickens in the Haouz region of Morocco. A "positive-negative threshold" baseline was established, following the method of Snyder et al. (1983): the mean absorbance of all the negative samples at each dilution was taken and three standard deviations added.
On the basis of the titrations of positive serum samples, 1:104 was chosen as the fixed serum dilution since this dilution gave the maximum separation of absorbance values for different serum samples. All subsequent samples were tested in duplicate at this dilution, and positive and negative serum samples were included in every plate as controls. When the mean absorbance for the negative control exceeded 0.2, the results were rejected. Titres were calculated by first subtracting the mean absorbance for the negative control and then applying the equation calculated by relating the titre to absorbance at a dilution of 1:104.
1. Titration of sera - Titrage des sérums - Titulación de sueros
2. Correlation between absorbance at serum dilution 1:104 and titre - Corrélation entre l'absorbance à la dilution de sérum 1:104 et le titre - Correlación entre la absorbancia a la dilución de suero 1:104 y el título
In order to establish a curve showing expected ELISA titres for both vaccinated chickens and vaccinated chickens receiving a virulent challenge, the following experiment was carried out. According to the following schedule, 234 layer hens were vaccinated: day 7, Hitchner B 1 in drinking water; week 4, LaSota by eye-drop; week 8, clone 30 in drinking water; week 12, clone 30 in drinking water; week 19, combined inactivated vaccine for Newcastle disease and infectious bronchitis.
At 29 weeks, 69 birds were moved to different premises and challenged intraocularly with virulent velogenic NDV (109.5 EID50 per bird). Thirty blood samples were taken at the age of four weeks and, subsequently, every two weeks until 28 weeks of age. After challenging, 16 samples were taken from both challenged and unchallenged birds every week until the age of 33 weeks.
The samples from the experiment described above were also titred by the HI method (Allan and Gough, 1974) in order to establish whether a correlation existed between these titres and the ELISA titres.
Titration of serum samples. Fig. 1 shows an example of the titration of serum samples. The positive-negative threshold, calculated from the negative samples, is shown. The titre of each sample was determined as the inverse of the dilution at which the line relating absorbance to dilution intersected the positive-negative threshold line. The log 10 of the absorbance for each sample at a dilution of 10-4 was then plotted against this titre to provide a graph relating absorbance at a fixed dilution to titre (see Fig. 2). The equation relating absorbance and titre was calculated as log 10 absorbance = [0.28(log10 titre) - 1.786] with a coefficient of correlation r = 0.92.
Titres for vaccinated and challenged birds. Fig. 3 shows the mean ELISA titres together with the mean HI titres for a normally vaccinated flock of layer hens from the age of four weeks until 33 weeks as well as the titres reached after challenging with the virulent virus. The titres were calculated from the equation above. Absorbance values were between 0.1 and 1 for all samples.
Correlation with the Hl test. Fig. 4 shows the correlation of the ELISA titres with the HI titres, using the same serum samples as were used to provide the data for Fig. 3. A positive correlation was found to exist between the HI titres and ELISA titres (r = 0.61).
Titration of serum samples. All the samples titrated showed a more or less linear relationship between the log of the absorbance and the log of the reciprocal of the dilution over a certain range (see Fig. 1). The fixed dilution chosen (1:104) was such that it occured in this between the log of the absorbance and the log of the titre (r = 0.92) is similar to that obtained by Snyder et al. (1983) (r= 0.927).
3. ELISA and Hl titres in a vaccinated and challenged layer flock. The arrows show the ages at the time of vaccination and challenge with he virulent virus - Titres ELISA et IH selon l'âge dans une bande de pondeuses vaccinées et éprouvées. Les flèches indiquent l'âge aux différentes vaccinations et lors de l'épreuve avec le virus virulent - Títulos del ELISA y el HI para distintas edades en una población de gallinas ponedoras, vacunadas y expuestas a la infección. Las flechas indican las edades en las distintas vacunaciones y en la infección con virus virulento
4. Correlation between ELISA and Hl titres - Corrélation entre les titres en ELISA et en IH - Relación entre los títulos del ELISA y el Hl
Titres for vaccinated and challenged birds. From Fig. 2 it can be seen that the titres expected with this assay for a well-vaccinated flock are in the order of log2 (15), and that when challenged with virulent virus the titres can reach log2 (20) (titres are expressed as log2 to permit comparison with HI titres).
Correlation with the HI test. It is clear from Fig. 3 that there is a high degree of correlation between mean ELISA and mean HI titres. The coefficient of correlation between individual ELISA and HI titres (r = 0.61) is similar to that obtained by Adair et al. (1989) (r = 0.57). The ELISA titres, however, are considerably higher than HI titres, as found by other researchers (Snyder et al., 1983; Mishra, Rai and Jaiswal, 1985).
Once established, this assay is easy and rapid to perform and shows a good correlation with the existing serological method. It could be distributed to and used in different laboratories in widely varying climatic conditions, since this has already been achieved for the anti-bovine kit on which it is based. The assay could be expended for use with other avian pathogens using a uniform method.
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Alexander, D.J., ed. 1989. Newcastle disease. Norwell, MA, Kluwer Academic Publishers.
Allan, W.H. & Gough, R.E. 1974. A standard haemagglutination inhibition test for Newcastle disease. 1. A comparison of macro and micro methods. Vet. Rec., 95: 120-123.
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FAO/IAEA. 1989. Anti-bovine ELISA kit for animal disease diagnosis. Vienna, FAO/IAEA.
Miers, L.A., Bankowski, R.A. & Zee, Y.C. 1983. Optimising the enzyme-linked immunosorbent assay for evaluating immunity of chickens to Newcastle disease. Avian Dis., 27: 1112-1125.
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