Laboratory testing – serological methods

A laboratory was established at the Ethiopian Institute of Agricultural Research (EIAR) in Debre Zeit, which lies in the central highlands of Ethiopia, approximately 45km southeast of the capital, Addis Ababa. All serological and coprological examinations were carried out in this laboratory. Methanol-fixed blood smears and ectoparasites in 70% ethanol were exported under license to the University of Liverpool for microscopic examinations, and FTA cards were exported under license to the University of Nottingham, where they formed the basis of the genetics studies. The laboratory manuals used during the project which give full details of the Enzyme-linked immunosorbent assay (ELISA) and haemagglutination inhibition (HAI) test procedures, including preparation of reagents, are shown in General Appendix B.

2.5.1 Infectious Bursal Disease Virus (IBDV) ELISA

Serum samples were screened for antibodies to IBDV using a Flockscreen antibody ELISA kit (x-OvO, Dunfermline, UK). Samples were diluted 1 in 500 with the sample diluents provided, and added in triplicate to the antigen-coated wells. The positive and negative controls were provided with the kit and added in the last two positions on each plate. After a 30-minute incubation, the plates were manually washed with the wash buffer, and an enzyme conjugate was added. The plates were incubated for a further 30 minutes, then washed a second time before adding the enzyme substrate reagent. These were incubated

for 15 minutes and a stop solution was added. For the first two rounds of testing, the stop solution had to be replaced with 0.5M sodium hydroxide (NaOH), due to difficulties in transporting the kit’s stop solution from the UK on a commercial airline. Plates were read after 5 minutes on the spectrophotometer at an absorbance of 562, as the recommended 550 wavelength was not available. Where the test was not valid after 5 minutes, plates were read again at intervals, as the colour continued to develop. All plates gave a valid reading within 15 minutes.

2.5.2 Pasteurella multocida, Salmonella O9 serotype and Marek’s Disease virus ELISAs

2.5.2(a) Preparation of Antigen

Antigens for the bacterial serology tests were prepared according to the method described by Beal (2004). A nalidixic acid-resistant isolate of Salmonella enterica serovar Gallinarum was grown in Luria Bertani broth and an isolate of Pasteurella multocida grown in brain- heart infusion broth. Both were incubated overnight at 37° C in an orbital incubator (150 rpm). Bacterial cells were pelleted by centrifugation at 4100 g for 40 min and washed twice with phosphate buffered saline (PBS). Cultures were re-suspended in 40ml PBS, then heat inactivated at 65° C for 4 hours. They then underwent three freeze–thaw cycles at -70° C before sonication (10 x 20 second bursts) on ice. The suspensions were clarified by centrifugation at 4100 g for 40 min followed by centrifugation at 30,000 g for 20 min to remove insoluble fractions. Protein concentrations of the soluble antigen preparations were measured using the Fluka protein quantification kit rapid (Sigma-Aldrich, Gillingham, UK) and aliquots stored at -20 C.

Antigen for the Marek’s Disease virus (MDV) ELISAs was kindly provided by the Institute of Animal Health (Compton, UK), and consisted of a suspension of chicken kidney cells infected with a CV1988 strain of MDV, which had been lysed with 4 freeze-thaw cycles. In addition, the Institute provided non-infected chicken kidney cell lysate (CKCL) as a control.

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2.5.2(b) Antibody detection

Flat-bottomed polystyrene ELISA plates (Greiner Bio-one, Stonehouse, UK) were coated with antigen in 100 μl/well of carbonate buffer (pH 9.6) overnight at 4 °C. Dilutions of the different antigens are given in Table 2.1. Following removal of the antigen, plates were blocked with PBS Tween-20 (0.05%) (PBS-T) supplemented with 3% skimmed milk powder (diluent) for 1 hour at 37 °C, and washed once with PBS-T. Serum samples were diluted 1:100 in the diluent and plates were incubated at 37 °C for 1 hour and washed three times with PBS-T. Bound antibodies were detected by incubating with 100 µl rabbit anti-chicken IgY (Sigma-Aldrich, Gillingham, UK) diluted 1:1000 in diluent for 1 hour at 37°C. After three washes in PBS-T, bound antibodies were detected following the addition of 4-Nitrophenyl phosphate disodium salt hexahydrate tablets (Sigma-Aldrich, Gillingham, UK) dissolved in glycine buffer (pH 10.4) with 1mM zinc chloride and 1mM magnesium chloride. Plates were

incubated at 37 °C in the dark for 15 min, after which the reaction was stopped with 3M sodium hydroxide and absorbance read at 405 nm using an ELx808 Absorbance microplate reader (BioTek, Potton, UK).

In order to test for antibodies to MDV, samples were required to be tested against both infected and uninfected chicken kidney cells, to rule out cross-reactivity of the sera under evaluation to cellular antigens. To try and minimise the influence of external conditions such as atmospheric temperature, or timing differences on the test results, sera were tested against infected and uninfected cells on two plates run simultaneously.

Table 2.1 Dilution of antigen used in the laboratory-developed ELISA tests

Antigen Dilution in carbonate buffer

S. gallinarum antigen (6.4μg/ml) 1/80

P. multocida antigen (11.1 µg/ml) 1/80

MDV-infected chicken kidney cell lysate 1/20 Non-infected chicken kidney cell lysate 1/20

2.5.3 Controls and ELISA validation

The commercial kit for the IBD ELISA included positive and negative control sera. For the laboratory-developed assays, negative controls were sera collected from specific-pathogen free birds in the UK. Positive controls for each of the laboratory tests were produced as described below. The performance of the controls for the four assays is shown in the Appendix to Chapter 2.

2.5.3(a) Pasteurella

Positive controls were generated by vaccinating four adult white Leghorn birds at the research station in EIAR with an ovine Pasteurella multocida vaccine. Birds were given two doses of 300µl subcutaneously 19 days apart, and sera were collected 3 weeks after the second vaccination.

In order to validate the laboratory-developed Pasteurella ELISA, it was tested against a commercial kit (Flockchek, Idexx Laboratories, The Netherlands). Twenty field samples at a 1:50 dilution and both the laboratory and the kit positive and negative controls were tested in triplicate on both the commercial and laboratory ELISAs, run simultaneously.

Both the kit and the laboratory negative controls tested negative on both plates. The laboratory positives generated by vaccination were detected as only weakly positive or negative by the commercial kit. Nineteen of the twenty field samples were detected as positive, and the last one gave an equivocal result. All of the positive controls and all twenty field samples gave positive results on the laboratory ELISA (i.e. they had an optical density (OD405) at least three times greater than the negative control). However, the kit

positive was detected as being only weakly positive on the laboratory ELISA.

Apart from one equivocal test, both ELISAs showed good agreement in detecting antibody- positive sera. The laboratory test was likely to be less specific than the commercial kit, although this was not possible to demonstrate, as there was a lack of negative field

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samples available for testing. However, the laboratory test gave higher optical density readings for samples at the same dilution, and was likely to be more sensitive than the kit, which is particularly developed to evaluate vaccine efficacy. The most positive of the sera from the vaccinated birds was chosen to use as a control for testing the field samples (Positive 1); in addition a second positive control for the laboratory test was made by pooling sera from 4 positive field samples (Positive 2).

2.5.3(b) Salmonella O9 serotypes

An initial positive control was made from pooled sera collected from specific pathogen free chickens infected experimentally with S. Enteritidis in the UK. Pilot samples showed that many field samples had much higher titres than this; thus a new positive serum was selected from among the pilot samples for the testing of the study samples (Positive 1). As this was used up, a second positive was made by pooling positive field sera, and tested alongside the original control sera on 7 plates before being used alone.

2.5.3(c) Marek’s Disease Virus

An initial positive control was provided by the Institute of Animal Health, consisting of antiserum against the HPR516 strain of MDV. A positive serum from among the pilot samples was identified and validated as the positive control for the testing of field samples (Positive 1.) Pooled positive field samples were used to make up a replacement positive as the first one ran low (Positive 2) and was run in parallel with Positive 1 on 14 plates.

2.5.4 Plate and sample exclusion

The commercial IBD kit provided guidelines to assess the validity of the plates. These required that:

a) Mean Negative control absorbance must be <0.2

b) Mean Positive control absorbance must be at least 2 times the optical density of the negative control absorbance with a minimum difference of 0.2 OD.

Two IBDV plates were excluded because no positive control was added, and samples were tested again on different plates.

Out of 39 plates accepted, all satisfied the criterion that the positive should be at least 2 times the optical density of the negative, and all satisfied at least one of the other two criteria. One plate had a negative control value which was too high (Plate B9: negative control OD562 = 0.3025). Eighteen plates failed to meet the criterion of a minimum

difference of 0.2 OD562. This has occurred across all seasons of testing, so appears to be

unrelated to substituting NaOH for the stop solution in the first two seasons. There appeared to be a marked difference in colour development between different bottles of substrate, noticeable when two different bottles of substrate solution were used on the same plate. Therefore it may be an issue related to the transport / storage of the substrate reagent. Since it was not possible to retest all these plates due to lack of materials, we have accepted these plates, even though they do not meet the recommended criteria. However, we have adjusted for differences between plates in the analysis, and lowered the manufacturer’s recommended cut-off slightly, to account for the relatively low OD562

values.

No prior criteria were set for acceptance of the laboratory-developed ELISA tests, with the exception of the MDV ELISA, where positive controls were required not only to have a high OD405 against the infected cell lysate, but also to have a low OD405 against the uninfected

CKCL (Zelnik et al. 2004). We set a minimum difference of 0.1 for the positive control against infected and uninfected cells. Otherwise, for all assays, plates were evaluated against all previous plates as the assays were carried out, and a subjective assessment made as to whether they were valid, based on the normal range of values for their controls (Figure 2.2).

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Figure 2.2 Example of the daily log of positive and negative control values for the Salmonella ELISA plates over different batches of testing. The log was used to help determine invalid plates by comparing the control OD values to their normal range. Labels indicate plates which were rejected, because of (a) a high negative control OD and (b) the positive control OD was less than twice that of the negative control.

Four Pasteurella ELISA plates were rejected, because the mean positive control absorbance was less than twice that of the mean negative control absorbance, either because of unusually high negative or unusually low positive readings. Two Salmonella plates were rejected; one because the negative control OD405 was too high, and one because the

positive control was less than twice the negative control absorbance. One MDV test plate was rejected, as the OD405 of the positive and negative controls was too high against the

uninfected cells.

Samples were tested in triplicate where possible, or in duplicate when reagents began to run low. This affected all ELISA tests except Pasteurella, and samples from all regions and seasons of collection, with the general exception of those collected in the third season. The co-efficient of variation (CV) was calculated for all samples and all those with a CV >25%

0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 5 10 15 20 25 30 Season A negatives Season A positives Season B positives Season B negatives Season C positives Season C negatives a b

were retested. Where samples were tested in triplicate, one replicate may be excluded if it had an absorbance noticeably different from the other two readings, if this would bring the CV below 20%.

The test for MDV antibodies required that samples be tested against lysates of both virus- infected and uninfected chicken kidney cells; the OD405 against the uninfected cell lysate

was then subtracted from the OD405 against the infected lysate to give a net value which

was used in further calculations. Samples with a net value greater than 3x the standard deviation of the net values (after accounting for the effect of the different test plates) were deemed to be cross-reacting to the cells in which the virus was grown and were discarded from further analyses.

2.5.5 Newcastle Disease Haemagglutination Inhibition (HAI) assays

2.5.5(a) Test procedure

HAI assays were carried out according to the procedure described in the OIE Terrestrial manual (2009). Briefly, 250µl of PBS was dispensed into each well of a V-bottomed microtitre plate, 250 µl of test sera were dispensed into the first well of each row, and two- fold dilutions were made across the plate. Dilutions were made up to 1/64, in order to be able to test more samples per plate. Four haemagglutinating units (HAU) of virus in 250µl of PBS were then added to each well, and plates were incubated for 30 minutes at room temperature. A volume of 250µl of a 1% solution of chicken red blood cells (RBCs) was then added to each well, gently mixed and allowed to settle for 45 minutes at room temperature.

Agglutination was read by gently tilting the plates, and the haemagglutination (HI) titre was the highest dilution of serum which caused complete inhibition of 4 HAU of virus. A line of 6 red blood cell control wells, containing 250µl of a 1% chicken RBC solution and 500µl of

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PBS, were included on each plate, and only those wells in which red blood cells streamed at the same rate as the control wells were considered to show inhibition.

A virus control was also included on each plate. This consisted of twofold dilutions of the virus suspension in volumes of 250µl, plus 250µl of PBS and 250µl of chicken RBCs. This was included to confirm the virus solution had been correctly titrated and was able to agglutinate chicken RBCs. Positive and negative control sera were also included on each plate. Plates were rejected if the negative control gave a titre > 1/4, if the positive control was not within one dilution of the known titre, if the virus was not within one dilution of the expected titre, or if the red blood cell control did not stream. Samples were considered as positive if they inhibited agglutination at a dilution of 1/16 or above.

2.5.5(b) Validation

Inter-reader variation was assessed for the two main readers who carried out the HAI assays. This consisted of both readers being asked to read four plates independently of each other and provide the titre for each of the 12 samples on each plate. I made a comparison of the two scores, and where the two scores differed, I and one other observer also read the plates and allocated a third score.

The four readings of the positive control agreed either exactly or to within one dilution. However, Reader 1 scored the negative control two- to fourfold higher than Reader 2, and a similar pattern was seen for the readings of the test samples. Where Reader 1 had classed a sample as negative, there was good agreement between the two scores. However, of the 10 samples which Reader 1 classed as positive, none were scored as positive by Reader 2, or by the third arbitrator. Where there was disagreement between the two readers, the third score invariably agreed with Reader 2’s assessment of the titre. In addition, we were able to test 17 samples with an NDV ELISA test (Biocheck, Hounslow, UK). This confirmed as negative 12 samples which both readers had scored as negative.

One sample which both readers scored as positive was not classed as positive by the ELISA. One sample which Reader 2 had classed as positive, and Reader 1 had classed as equivocal was classed as positive by the ELISA, and one sample which Reader 1 had classed as positive and Reader 2 as negative was classed as negative by ELISA. Two samples tested by ELISA were read by only one reader, of which the ELISA agreed with the negative reading by Reader 2, but disagreed with the positive reading by Reader 1.

From this, it was evident that there was considerable subjectivity in the reading of the test, and Reader 1’s assessment appeared to be much less specific than Reader 2’s interpretation. In order to try and establish a consistent measurement of samples, it was therefore decided to accept only the samples scored as negative by Reader 1, and for Reader 2 to retest all samples which Reader 1 had given a titre 4 or greater, providing there was sufficient serum left to do so.