Antibody detection-based differential ELISA for NDV-infected or vaccinated chickens versus NDV HN-subunit vaccinated chickens

Antibody detection-based differential ELISA for

NDV-infected or vaccinated chickens versus

NDV HN-subunit vaccinated chickens

Andrea M. Makkaya, Peter J. Krellb, EÂ va Nagya,*

aDepartment of Pathobiology, Ontario Veterinary College, University of Guelph,

Guelph Ont., Canada N1G 2W1bDepartment of Microbiology, University of Guelph, Guelph Ont., Canada N1G 2W1

Received 27 May 1998; accepted 3 February 1999

Abstract

With the advent of subunit vaccines for microbial diseases it is becoming increasingly important

to be able to differentiate naturally infected animals from those vaccinated with the corresponding

subunit vaccine. For avian viruses such as Newcastle disease virus (NDV), a whole virus-based

ELISA cannot make such a differential diagnosis since in both cases the antisera would react with

the whole virus. The nucleocapsid protein (NP) gene of the NDV Hitchner B1 strain was cloned,

sequenced and expressed to develop a differential ELISA. The B1 NP had 95.7 and 96.1% amino

acid identities with the NP of the d26 and Ulster 2C strains, respectively. The B1 NP expressed in a

baculovirus expression vector (recNP) was the expected size and reacted with NDV-specific

antibodies (Ab) in Western blots and by radioimmunoprecipitation. The ELISA using recNP-coated

wells, tested on serum samples from flocks pretested with a commercial NDV kit gave results

corresponding to those of the kit. Furthermore, use of both the recNP-based ELISA and a whole

virus ELISA allowed the differentiation of birds vaccinated with a NDV haemagglutinin±

neuraminidase (HN) expressing fowlpox virus from birds infected with NDV. This provides the

basis for establishing an ELISA that discriminates between the antibody response to a recombinant

fowlpox vaccine (expressing NDV HN protein) and that to live and inactivated NDV. # 1999

Elsevier Science B.V. All rights reserved.

Keywords: Chicken±Viruses; Newcastle disease virus; Differential ELISA; Baculovirus expression; NDV

nucleocapsid protein

Veterinary Microbiology 66 (1999) 209±222

* Corresponding author. Tel.: +1-519-824-4120 x 4783; fax: +1-519-767-0809; e-mail:[email protected]

0378-1135/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved.

PII: S 0 3 7 8 - 1 1 3 5 ( 9 9 ) 0 0 0 1 6 - 4

1. Introduction

Newcastle disease virus (NDV) is a widespread and economically important poultry

pathogen. Although vaccines have long been available and administered to control

Newcastle disease, the virus remains an ongoing threat to commercial flocks. The forms

of the disease vary and are dependent upon several factors, but mainly on the strain of the

virus (Alexander, 1997).

NDV, a member of the family Paramyxoviridae (Rima et al., 1995), is an enveloped

virus with helical nucleocapsid symmetry and a single-stranded, negative sense RNA

genome, about 15 kb in length. In the 30±50 orientation, the genome encodes six major

proteins: nucleocapsid (NP), phosphoprotein (P), matrix (M), fusion (F), haemagglutinin±

neuraminidase (HN) and RNA-dependent RNA polymerase (L) (Chambers et al., 1986;

Wilde et al., 1986). The HN and F glycoproteins are important for virus infectivity and

virulence, and either of these two proteins can induce protective immunity (Meulemans

et al., 1986; Nagy et al., 1991).

NDV-specific antibodies (Ab) have traditionally been monitored by the haemagglu-

tination inhibition test (Alexander, 1997). ELISA procedures based on whole virus as

coating antigen have been described (Miers et al., 1983; Snyder et al., 1983; Wilson et al.,

1984; Rivetz et al., 1985), and, more recently, commercial kits (e.g. IDEXX Laboratories,

Westbrook, ME) are widely being used in diagnostic laboratories. Errington et al. (1995)

recently reported on an NDV NP-based ELISA using NP expressed in a baculovirus

system and compared their NP ELISA-based scores with HI and the commercial IDEXX

ELISA scores. They did not observe any false positive readings for samples scored

positive by HI. However, they did report that among sera expected to be negative were

some that scored positive by IDEXX ELISA and negative by their NP-based system.

Numerous, live and inactivated virus vaccines exist against NDV. There has recently

been growing interest in using subunit vaccines against poultry diseases including ND

(Meulemans et al., 1988; Nishino et al., 1991; Morgan et al., 1993; Nagy et al., 1993).

Consequently, there is a need to be able to differentiate between birds naturally infected

with NDV and vaccinated with such a recombinant subunit in the surveillance of NDV.

In this study, we sought to develop an ELISA test for Ab detection that would

discriminate between the antibody response to a subunit vaccine (HN expressed by a

recombinant fowlpox virus) and the response to live and inactivated NDV. We cloned the

NP gene of the Hitchner B1 NDV strain and expressed the NP in a baculovirus vector for

use as a coating antigen in the ELISA. Serum samples were analysed from chickens

vaccinated with a recombinant fowlpox virus expressing the NDV±HN and subsequently

infected with NDV.

2. Materials and methods

2.1. Viruses and cells

NDV strains Hitchner B1 and LaSota were used (Nagy et al., 1990). Virus was propagated

in embryonated eggs and in chicken embryo lung (CELu) cells by standard procedures.

210 A.M. Makkay et al. / Veterinary Microbiology 66 (1999) 209±222

Autographa californica nucleopolyhedrovirus (AcMNPV) and Spodoptera frugiperda

(Sf) 9, Sf21 and Trichoplusia ni High Five cells were from Invitrogen. Baculovirus and

insect cell techniques were performed as described by Summers and Smith (1987) and

O'Reilly et al. (1992).

Fig. 1. Construction of NP plasmid vector and baculovirus transfer vector.

A.M. Makkay et al. / Veterinary Microbiology 66 (1999) 209±222 211

2.2. Generation of recombinant baculovirus

The strategy for recombinant plasmid construction is outlined in Fig. 1. All molecular

biology techniques were based on Sambrook et al. (1989). Complementary DNA was

made (cDNA Synthesis System; Gibco-BRL, Burlington, ON) from genomic NDV RNA

using a primer (50-GCCTTCTGCCAAAATGTC-30; the initiation codon is in bold)

derived from the NP genes of d26 (Ishida et al., 1986) and Ulster 2C (GenBank accession

#Z300084). Blunt-ended cDNA was ligated into the SmaI site of pGEM-7Zf(�) to

generate pNP103, with a 2.5 kb insert containing the NDV NP gene. A PCR product

containing just the NP gene was generated using the same primer used for cDNA

synthesis, and primer B (50-TGTTGTTGGTCAGTACCC-30) with the complement of the

termination codon (in bold) and based on the NP gene of the Ulster 2C strain. The PCR

product was blunt-end ligated into the SmaI site of pGEM-7Zf(�) to generate

pNP103.22. The 1.5 kb EcoRI/Csp45I fragment from pNP103.22 was blunt-ended with

Klenow enzyme and NheI linkers (New England Biolabs, Beverly, MA) were added for

cloning into the NheI site of the baculovirus pETL vector (Richardson et al., 1992) to

generate pEN159 which contained the NP gene under the control of the polyhedrin (polh)

promoter. Recombinant baculovirus was generated as described by Summers and Smith

(1987) and purified by three rounds of plaque purification to generate one recombinant

referred to as rBNP.

2.3. Sequencing

The NP gene in pNP103 was sequenced at the Guelph Molecular Supercentre

(Laboratory Services Division, University of Guelph) and sequence data were analysed

with `Align' (version 1.02, #1989 Scientific and Education Software). Sequences were

deposited into GenBank, accession number AF060483.

2.4. Polyacrylamide gel electrophoresis and Western blot analysis

Sf9 cells infected with rBNP at a multiplicity of infection (m.o.i.) of 10 were analysed

by sodium dodecyl sulfate 10% polyacrylamide gel electrophoresis (SDS-PAGE;

Laemmli, 1970), and Western blotting. Infected cells, harvested at different times post-

infection (p.i.), were lysed in an electrophoretic sample buffer (ESB; 62.5 mM Tris [pH

6.8], 10% glycerol, 2% SDS, 5% b-mercaptoethanol, 0.002% bromophenol blue).

Western blotting onto nitrocellulose membrane with a 0.45 mm pore size (Schleicher and

Schuell, Keene, NH) was as described by Towbin et al. (1979). Blots were blocked in 5%

skim milk powder (SMP; Difco, Detroit, MI) in PBS for 3 h at room temperature. NDV-

specific rabbit polyclonal serum at a dilution of 1/2000 in 2% SMP in PBS±Tween

(0.05% Tween-20 in PBS) was added to the blots and incubated at room temperature for

2 h. Blots were then incubated with goat anti-rabbit alkaline phosphatase-conjugated Ab

(Bio-Rad, Mississauga, ON) at a dilution of 1/1000 in 2% SMP in PBS and developed in

a 1 : 1 : 200 (by volume) solution of BCIP (Bio-Rad; 15 mg/ml stock in N,N-

dimethylformamide), NBT (Bio-Rad; 30 mg/ml stock in 70% N,N-dimethylformamide)

in 0.1 M Tris (pH 9.5), 0.5 mM MgCl2.


2.5. Radioimmunoprecipitation

Sf9 cells infected with rBNP were incubated at 48 h p.i. in methionine-free Grace's

medium with [35S] methionine at 100 mCi/ml (ICN; specific activity 1175 Ci/mmol) for

4 h at 288C. Cells were lysed in RIPA buffer (150 mM NaCl, 10 mM Tris [pH 7.2], 1%

sodium deoxycholate, 1% Triton X-100, 0.1% SDS). NDV-infected chicken CELu were

similarly labelled in methionine-free EMEM at 8 h p.i. for 2 h at 378C. Immunopreci-

pitation with NDV-specific rabbit serum and analysis was as described by Nagy et al.

(1990).

2.6. Antigen preparation and conditions for ELISA

The rBNP-infected Sf21 cell lysates were used as antigens for ELISA and the

appropriate conditions were determined by a series of checkerboard plates as described

by Carpenter (1992). Cells infected at an m.o.i. of 5 and harvested at 40 h p.i. were

washed twice, resuspended in PBS, and sonicated over a 1.5 min period on ice.

Concentrations of soluble protein were determined with a Bio-Rad protein assay kit.

ELISA plates (Becton Dickinson Laboratories, Lincoln Park, NJ) were coated at 2.5 mg

total protein/well in 0.5 M carbonate±bicarbonate buffer (pH 9.6), and blocked with 5%

BSA (Fraktion V, Boehringer Mannheim) for 1 h at 378C. Plates were incubated at 378Cfor 1.5 h with primary Ab diluted 1/500 in PBS±Tween. Plates were next incubated at

378C for 1.5 h with goat anti-chicken IgG alkaline phosphatase conjugate (Kirkegaard

and Perry, Gaithersburg, MD) diluted 1/1000 in PBS±Tween. Plates were developed with

Nitrophenyl±Phosphate (Sigma, p-NPP tablets; Sigma, Oakville, ON) and OD405 was

read in a Bio-Tek microplate autoreader.

Samples considered positive for NDV were from the Animal Health Laboratory

(University of Guelph) as a collection of serum samples from individual chickens from

flocks which tested positive for NDV Ab. For flock testing, random samples of five birds

per flock were tested with the commercial IDEXX kit. The individual samples were

divided into 48 pooled serum samples (five individuals per pooled sample) for testing

with our NP ELISA. Samples considered negative for NDV were from 58 birds reared in

isolation and confirmed to be negative by IDEXX ELISA.

Specificity and sensitivity of the ELISA were calculated according to Smith (1995).

Sensitivity was the number of true positive samples divided by the sum of the true

positive and false negative samples, and the specificity was the number of true negative

samples divided by the sum of the true negative and false positive samples. The true

positive and negative samples were those scored as positive or negative, respectively, by

both IDEXX and NP ELISA. False positives were those that scored negative by IDEXX

but positive by NP ELISA while false negatives were those that scored positive by

IDEXX but negative by NP ELISA. An OD405 cut-off value for the NP ELISA to

differentiate positive from negative samples was set at two standard deviations above the

average OD405 from IDEXX-negative samples. Values above this cutoff were scored as

positive.


2.7. Vaccination experiment

Three-week-old Leghorn chickens (Arkell Research Station, University of Guelph)

were separated into three groups of five birds each. Group 1 and Group 2 chickens were

vaccinated with a recombinant fowlpox virus expressing the HN of NDV with the

vaccinia virus promoter P7.5 (rFPV7.5) or P11 (rFPV11), respectively, at Weeks 0 and 2.

Group 3 chickens were mock-vaccinated. At Week 4 all birds were infected with 0.1 ml

of NDV (LaSota strain) via the intranasal route. Weekly blood samples were collected

beginning with a pre-immune bleed at Week 0, and ending with post-infection samples at

Week 5. Serum samples were tested using recNP ELISA and whole NDV ELISA. Plates

for whole NDV ELISA were coated with a hundred-fold dilution of NDV-infected

allantoic fluid (initial HA 1 : 256) at 100 ml/well.

3. Results

3.1. Sequence analysis of the NP gene

The open reading frame of the NP gene of the Hitchner B1 NDV strain was 1,467

nucleotides (nt) long and coded for a 489 aa protein with a predicted molecular mass (Mr)

of 53.03 kDa. The number of amino acids for the B1 strain NP gene was identical to that

from d26 and Ulster 2C. The nucleotide homology of the NP gene was 90.03 and 89.45%

with that of d26 (Ishida et al., 1986) and Ulster 2C (GenBank accession #Z30084),

respectively. Amino acid identities were 95.71 and 96.11%, respectively. The NP gene

sequences of the d26 and Ulster 2C strains showed nucleotide homology and amino acid

identity of 96.4 and 98.6%, respectively.

3.2. Expression of the recombinant NP protein in insect cells

Sf9 cells infected with rBNP were labelled with [35S] methionine and cell lysates were

immunoprecipitated (Fig. 2). A very prominent band was detected at 53 kDa (Lane 3) for

rBNP and the position of this band was identical to that of the NP band in NDV-infected

CELu cells (Lane 2). An NDV-specific band was also detected at 43 kDa for rBNP but a

corresponding band was not detected for NDV-infected CELu cells and may represent

degradation of the 53 kDa NP. Similarly, a 39 kDa protein band was detected in NDV-

infected CELu cells but not in the rBNP lanes. This represents the NDV matrix protein

that would co-precipitate with the anti-NDV Ab. No proteins of the uninfected CELu

(Lane 1) or Sf9 (Lane 5) cells reacted with the NDV-specific Ab. A single band at

approximately 30 kDa which corresponds to the position of the polyhedrin protein was

detected in the AcMNPV-infected Sf9 cell lysates treated with either anti-NDV or pre-

immune sera (Lanes 4 and 7, respectively). Polyhedrin is present as a protein complex

which does not solubilize in cell lysis buffer and pellets with immunoprecipitated proteins

(O'Reilly et al., 1992).

In a time course of expression of the recNP in Sf9 cells infected with rBNP at an m.o.i.

of 10, a Coomassie blue stained band corresponding in size to recNP (53 kDa) was seen


by 36 h p.i. (arrow) and increased in intensity up to 48 h p.i., and remained constant

thereafter (Fig. 3, panel A). By Western blotting an NDV-specific protein band

corresponding to the unique 53 kDa band in the stained gel, was first detected at 24 h

p.i. (arrow) and increased in intensity over time (Fig. 3, panel B). Some bands of lower

molecular weight appeared at 36 h p.i. and increased in amount with time. The NP of

NDV grown in embryonated eggs, migrated at 55 kDa.

NP expression was compared in Sf9, Sf21 and High Five cell lines, at m.o.i. of 0, 1, 5

and 10 and harvested at 24, 36, 48 and at 60 h p.i. By Coomassie blue staining, recNP

expression in Sf9 and Sf21 cells was similar at all time points and m.o.i., but expression

in High Five cells was extremely low. Based on densitometric analysis of the gels, Sf9

and Sf21 cells infected with an m.o.i. of 5 and collected at 48 h p.i. contained

approximately 0.60 and 0.53%, respectively, of recNP relative to the total amount of

protein in the lane. RecNP expression was greater in Sf21 cells than in Sf9 cells by

Western blot analysis (data not shown). Since optimal production of recNP occurred in

Fig. 2. Radioimmunoprecipitation of rBNP-infected [35S] methionine-labelled Sf9 cell lysates. Lysates in Lanes

1 to 5 were precipitated with NDV-specific Ab, in Lanes 6 and 7 were precipitated with pre-immune serum.

Lane 1: uninfected CELu cells; Lane 2: NDV infected CELu cells; Lanes 3 and 6: rBNP infected cells; Lanes 4

and 7: AcMNPV-infected cells; and Lane 5: uninfected Sf9 cells. M: radiolabelled molecular weight marker.


Sf21 cells infected at an m.o.i. of 5 and harvested between 36 and 48 h p.i. these

conditions were used for production of recNP for subsequent ELISA studies.

3.3. Development of recombinant NP-based ELISA

Appropriate conditions of the recNP-based ELISA were determined through a series of

checkerboard ELISAs testing antigen concentrations, primary and secondary Ab

dilutions, and use of different blocking agents. The final conditions, as described in

Section 2, were then used to screen a number of known positive and negative samples.

The specificity and sensitivity of the ELISA were evaluated from a panel of 48 IDEXX-

positive and 58 IDEXX-negative serum samples (Fig. 4). Of the samples tested, the

recNP ELISA gave a total of 48 true positive, 55 true negative, three false positive, and no

false negative readings (i.e. 48 of the 48 positive sera were scored as positive and 55 of

the 58 negative sera were scored as negative using the recNP ELISA). The OD405 values

for the three false positives were just above the cut-off OD405 value. The specificity and

sensitivity of the recNP ELISA were 94.8 and 100%, respectively. Raising the OD405 cut-

off value to three standard deviations above the average for the negative values (0.251)

would result in 48 true positive samples and 58 true negative samples for 100%

specificity and 100% sensitivity.

Fig. 3. Time course analysis of recNP expression by SDS-PAGE and Western blotting. A: Coomassie blue

stained gel, and B: Western blot analysis with NDV-specific Ab. The numbers above the lanes indicate rBNP-

infected cells harvested at 12±72 h p.i. AcMNPV-infected (wt) and uninfected (un) cell lysates, and purified

NDV (NDV) were also run. M: molecular weight markers, in kDa. Arrows show recNP band.


3.4. Vaccination experiment

The ability of the recNP ELISA to differentiate between NDV-infected birds, and those

vaccinated with an HN subunit vaccine was tested. Chickens were vaccinated with two

recombinant fowlpox viruses expressing HN and differing only in the nature of the

promoters used, and at Week 4, all chickens were inoculated with the LaSota NDV strain.

Blood samples were collected at 1 week intervals and tested using both the recNP ELISA

and a whole NDV ELISA. OD405 values for each group were averaged and results for the

pre-immune, post-FPV vaccination (Week 4) and post-NDV infection (Week 5) bleeds are

presented in Fig. 5. Recombinant FPV-vaccinated birds, which were protected from

virulent NDV challenge (unpublished), were positive by whole NDV ELISA at Week 4,

Fig. 4. Graphs of absorbance values obtained in testing known positive and negative serum samples in recNP

ELISA to determine specificity and sensitivity values. Cut-off values were set at two standard deviations above

the average absorbance of negative standard sera (OD405 0.230).


but were negative by recNP ELISA. These recombinant FPV-vaccinated birds would

produce antibodies only to HN of NDV and consequently react with only this NDV

polypeptide in the wells. The resulting OD405 values would consequently be lower than

that for NDV-inoculated birds whose antibodies would react against all NDV

polypeptides. Even though the OD405 values for the vaccinated birds were close to the

cut-off values they were clearly above those of the pre-immune sera. Mock-vaccinated

birds remained negative throughout Weeks 1±4 in both ELISAs. Serum samples taken at

Week 5, 1 week after live NDV inoculation, were positive in both tests for all birds.

4. Discussion

The NP of NDV was expressed in a baculovirus expression vector system and was used

as a coating antigen for a diagnostic and differential ELISA.

Fig. 5. Histograms of average OD405 values in ELISAs of serum samples from chickens vaccinated with

recombinant fowlpox virus expressing the haemagglutinin±neuraminidase of NDV and subsequently infected

with NDV. Panel A shows the results of the whole NDV ELISA, and panel B shows the results of the same serum

samples in the recNP ELISA. Average values per group are shown for Week 0 (pre-immune), Week 4 (post-

vaccination) and Week 5 (post-infection).


The NP gene of the Hitchner B1 strain was sequenced and compared to those of strains

d26 and Ulster 2C (Ishida et al., 1986; GenBank accession #Z30084), the only complete

NP sequences of any NDV strains reported to date. The predicted molecular mass of the

B1 NP according to sequence information was 53,029 Da which is in excellent agreement

with the size of the virion NP and with the expressed NP determined by PAGE. The B1

NP amino acid sequence showed a high degree of identity to those of Ulster 2C and d26

(95.71 and 96.11%, respectively). Since there is a very high degree of amino acid

sequence conservation, the recNP ELISA based on the B1 strain should be effective

against all NDV strains. The recombinant NP expressed in insect cells was detected as a

single, Coomassie blue-stained band at approximately 53 kDa in size. The same protein

was shown to be NDV-specific by Western blotting and radioimmunoprecipitation with

rabbit polyclonal serum raised against purified NDV (Figs. 2 and 3, panel B). The

recombinant NP of 53 kDa migrated at a size comparable to that of NP (55 kDa) from

virus grown in embryonated eggs (Smith and Hightower, 1981; Ishida et al., 1986;

Samson, 1988). The slight discrepancy in size between the recNP (53 kDa) and the virion

NP (53 or 55 kDa) in Fig. 3 might reflect slightly different forms of the NP proteins from

NDV grown under different conditions as has previously been described (Smith and

Hightower, 1981). For radioimmunoprecipitation (Fig. 2), CELu cells infected with NDV

were used to compare with the rBNP-infected Sf9 cells. Both these samples showed NP

bands of the same mobility, estimated at 53 kDa. In addition to the major recNP band

several faster migrating bands were seen in Western blots and radioimmunoprecipitation

of rBNP-infected cell lysates. In time course experiments, these bands appeared only

after the major recNP band was seen, and they accumulated over time. These lower

molecular weight proteins may be due to proteolytic degradation of the recombinant NP,

since the NP of NDV is susceptible to such degradation (Mountcastle et al., 1970). This is

also common for other paramyxoviruses including simian virus 5 (Mountcastle et al.,

1974), measles virus (Rozenblatt et al., 1979) and canine distemper virus (Hall et al.,

1980). Lower molecular weight species were also obtained by Kamata et al. (1993) when

the rinderpest virus nucleocapsid protein was expressed using baculovirus. The 53 and

43 kDa bands present in the radioimmunoprecipitated rBNP-infected cell sample could

represent the undigested and digested forms of NP, respectively, as described by

Mountcastle et al. (1974).

Initial ELISA experiments showed no cross-reactivity of chicken sera with uninfected

and AcMNPV-infected Sf21 cell lysates. ELISA plates were therefore coated with cell

lysates from rBNP-infected cells.

Baculovirus-expressed nucleocapsid proteins have been used as antigens in ELISAs for

measles virus (Hummel et al., 1992), rinderpest virus (Kamata et al., 1993), vesicular

stomatitis virus (Ahmad et al., 1993) and NDV (Errington et al., 1995). In comparison to

commercially available ELISAs, there was a better correlation of the recombinant NP

ELISAs with neutralising antibody levels than with commercial ELISAs (Hummel et al.,

1992; Errington et al., 1995).

The recNP-based ELISA was developed and optimised, and positive and negative sera

were tested. Since pooled samples are commonly used to determine overall flock

immunity, use of pooled samples for positive sera in determining the specificity and

sensitivity of this test was considered appropriate. Specificity and sensitivity of the test


were 94.8 and 100%, respectively, and no false negative readings were observed (Fig. 4).

Since IDEXX itself might not be 100% accurate this might lead to some lack of

correlation with the recNP ELISA. The few false positive samples were very close to the

cut-off values determined, and thus may be an artifact of a cutoff set too low. The cut-off

value used in the recNP ELISA was two standard deviations above the average OD405 of

the negative standards. If the cutoff was three standard deviations above the average

negative value, the three false positives would be true negatives. Since the sensitivity and

specificity of this recNP ELISA are high we feel that it is a reliable test for determining

the presence of NDV-specific antibodies.

Errington et al. (1995) earlier described a baculovirus-expressed NP-based ELISA for

NDV Ab. They did not report any false positive NP ELISA readings (for samples positive

by HI) although they reported some false negatives relative to some IDEXX-positive

samples which they felt may have been due to PMV-2 or PMV-3 interference. In our

recNP-based ELISA we did not observe any false negative samples, even using the

IDEXX ELISA kit which is more sensitive than HI. One of the differences in the

protocols of the two NP-based ELISAs was the cut-off values set; in our study we did not

use an S/P ratio as described by Errington et al. (1995). An S/P ratio is the ratio of the

unknown value to that of a weak positive control, after the subtraction of the negative

control value from both. It is possible that incorporating weak positive standards and an

S/P ratio cutoff into the recNP ELISA described herein could eliminate the false positives

and thus increase the specificity of our test to 100%. Thus our test is potentially more

reliable than that of Errington et al. (1995) for the detection of NDV Ab. Furthermore, we

showed that the recNP described can be used to differentiate between birds exposed to

NDV in either a whole NDV vaccine or by natural infection, from those vaccinated with a

subunit vaccine not containing the NP.

Presently, there is a great deal of interest in the development of recombinant and

protein subunit vaccines. For NDV, protective antibodies are induced against either

the HN or F proteins. The recNP ELISA described here could be used to dif-

ferentiate between birds naturally infected with NDV from those immunised with

HN or F protein subunit vaccines since antibody against the NP would be present

only in birds exposed to whole virus. To evaluate this, sera from chickens which

were vaccinated with recombinant fowlpox viruses expressing the HN only of NDV,

were tested. Chickens immunised with either recombinant FPV construct were

weakly positive using whole NDV ELISA after two vaccinations, whereas using recNP

ELISA they were negative (Fig. 5). Unvaccinated chickens were negative in both

ELISAs. All chickens, after receiving an intranasal inoculation of live NDV, became

strong positives according to both ELISAs. The OD405 values of the whole NDV ELISA

after vaccination with the FPV-HN were considerably lower than after exposure to

NDV, but were still positive for all birds within a group and significantly higher than

that in pre-immune sera. These indicate that a positive result in a whole NDV ELISA

can be due to antibodies to HN alone and that the recNP ELISA can be used to

differentiate between subunit-vaccinated birds and those exposed to whole virus. Most

importantly, a positive result with a recNP-based ELISA would indicate immunisation

with a whole NDV vaccine or/and infection with NDV and excludes birds immunised

with only a subunit vaccine.


The feasibility of using baculovirus-expressed nucleocapsid protein as an antigen for

an ELISA to differentiate between vaccinated and infected animals was also explored by

Ahmad et al. (1993) for vesicular stomatitis virus, and Kamata et al. (1993) for rinderpest

virus. As in our study, both groups showed that a baculovirus-expressed recombinant NP-

based ELISA was capable of differentiating subunit-vaccinated animals from those

infected with whole virus.

Acknowledgements

This work was funded by the Natural Sciences and Engineering Research Council

(NSERC) of Canada. We thank Paul Huber for his photographic and technical expertise

and Sean Walter for his technical assistance. We are indebted to Dr. D. Key (Animal

Health Laboratory, University of Guelph) for providing the pre-tested chicken serum

samples.

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Antibody detection-based differential ELISA for NDV-infected or vaccinated chickens versus NDV HN-subunit vaccinated chickens