Expression of capsid proteins and non-structural proteins of waterfowl parvoviruses in Escherichia coli and their use in serological assays

Abstract

While there are a number of methods available for detection of antibodies against waterfowl parvoviruses, none is able to differentiate responses against the capsid and non-structural proteins. To enable this, the capsid and non-structural proteins of goose parvovirus (GPV) and Muscovy duck parvovirus (MDPV) were expressed in Escherichia coli. These proteins were purified and used as antigens in western blotting assays of antibodies against GPV and MDPV. The results showed that 94.7% of the goose and 90.0% of the duck sera collected from the field contained antibodies against GPV or MDPV. Moreover, these sera could be classified into distinct groups based on differences in patterns of western blot reactivity. These different patterns might indicate different stages in infection. Western blotting assays of sera collected from experimentally infected ducks showed that antibodies against the non-structural protein appeared first after infection, followed by antibodies against the capsid protein. It was concluded that the recombinant capsid and non-structural proteins might serve as useful antigens for assays for antibodies against GPV and MDPV. Moreover, because these assays could discriminate between antibodies against the non-structural protein and those against the capsid protein, they may be useful in differentiating vaccinated from infected birds when recombinant capsid protein is used as the vaccine.

Expression des protéines de capside et des protéines non structurales des parvovirus des palmipèdes dans Escherichia coli et utilisation de ces protéines en sérologie

Alors qu'il existe un certain nombre de méthodes disponibles pour la détection des anticorps contre les parvovirus des palmipèdes, aucune n'est capable de différencier les réponses contre les protéines de capside et non structurales. Afin de le permettre, la protéine de capside et la protéine non structurale du parvovirus de l'oie (GPV) et du parvovirus du canard de barbarie (MDPV) ont été exprimées dans E. coli. Ces protéines ont été purifiées et utilisées comme antigène en Western blot pour la détection des anticorps anti GPV et anti MDPV. Les résultats ont montré que 94,7% des sérums d'oie et 90,0% des sérums de canard prélevés sur le terrain avaient des anticorps anti GPV ou anti MDVP. De plus, ces sérums pouvaient être classés en deux groupes distincts, basés sur les caractéristiques de la réaction du Western blot. Ces différentes caractéristiques peuvent indiquer différents stades de l'infection. Les résultats des Western blots réalisés à partir des sérums prélevés chez des canards infectés expérimentalement ont montré que les anticorps anti protéine non structurale apparaissent en premier, suivis des anticorps anti protéine de capside. Il a été conclu que les protéines recombinantes de capside et non structurale pourraient servir d'antigènes utiles pour la détection des anticorps anti GPV et anti MDPV. De plus, du fait que ces tests peuvent discriminer les anticorps anti protéine non structurale de ceux anti protéine de capside, ils peuvent être utiles pour différencier les animaux infectés de ceux vaccinés quand la protéine recombinante de capside a été utilisée comme vaccin.

Expression der Kapsid- und der Nicht-Strukturproteine der Parvoviren des Wassergeflügels in Escherichia coli und ihre Verwendung in serologischen Tests

Obwohl es eine Vielzahl von Methoden zum Nachweis von Antikörpern gegen die Parvoviren des Wassergeflügels gibt, ist keine davon in der Lage zwischen den Immunantworten gegen die Kapsid- und die nicht-strukturellen Proteine zu unterscheiden. Um dies zu ermöglichen, wurden die Kapsid- und die nicht-strukturellen Proteine des Gänseparvovirus (GPV) und des Flugentenparvovirus (MDPV) in E. coli exprimiert. Diese Proteine wurden gereinigt und als Antigene in Western blotting-Tests zur Untersuchung auf Antikörper gegen GPV und MDPV verwendet. Die Ergebnisse zeigten, dass 94,7 % der Gänse- und 90,0 % der Flugentenfeldseren Antikörper gegen GPV und MDPV aufwiesen. Darüber hinaus konnten die Seren basierend auf Unterschiede im Western blot-Reaktionsmuster in verschiedene Gruppen unterteilt werden. Die unterschiedlichen Reaktionsmuster können auf unterschiedliche Infektionsstadien hinweisen. Western blot- Tests von Seren, die von experimentell infizierten Flugenten stammten, zeigten, dass die Antikörper gegen die Nicht-Strukturproteine zuerst nach einer Infektion erschienen, gefolgt von den Antikörpern gegen das Kapsidprotein. Aus diesen Ergebnissen wurde geschlossen, dass die rekombinanten Kapsid- und Nicht-Strukturproteine geeignete Antigene für Antikörpernachweisverfahren gegen GPV und MDPV sein können. Da diese Tests zwischen den Antikörpern gegen die nicht strukturellen Proteine und solchen gegen das Kapsidprotein unterscheiden können, erscheint ihr Einsatz darüber hinaus sinnvoll bei der Differenzierung zwischen vakzinierten und infizierten Tieren, wenn rekombinantes Kapsidprotein als Vakzine verwendet wurde.

Expresión de la proteína de la cápside y de proteínas no estructurales de parvovirus de aves acuáticas en Escherichia coli y su uso en ensayos serológicos

Existen una serie de métodos disponibles para la detección de anticuerpos frente a parvovirus de aves acuáticas, pero ninguno de ellos es capaz de diferenciar si la respuesta es frente a la cápside o a las proteínas no estructurales. Con este objetivo, se expresaron en E.coli las proteínas de la cápside y no estructurales del parvovirus del ganso (GPV) y del parvovirus del pato de Muskovy (MDPV). Estas proteínas fueron purificadas y utilizadas como antígeno en ensayos de Western blotting con anticuerpos frente a GPV y MDPV. Los resultados demostraron que el 94.7% de los sueros de ganso y el 90.0% de los sueros de pato recogidos a partir de casos de campo contenían anticuerpos frente a GPV o MDPV. Además, estos sueros podrían ser clasificados en diferentes grupos en base a las diferencias en el patrón de reactividad en el Western blot. Estos diferentes patrones pueden indicar diferentes estados de infección. Los ensayos de Western blotting de los sueros de patos infectados experimentalmente demostraron que los anticuerpos frente a las proteínas no estrcuturales aparecían en primer lugar durante la infección, mientras que los anticuerpos frente a proteínas de la cápside aparecían más tarde. Se concluye que las proteínas de cápside y no estructurales recombinantes podrían ser útiles como antígenos para los ensayos para detectar anticuerpos frente a GPV y MDPV. Además, ya que estos ensayos pueden discriminar entre los anticuerpos frente a proteínas no estructurales y frente a proteínas de la cápside, pueden ser útiles para diferenciar aves vacunadas de infectadas cuando se ha utilizado una proteína recombinante de la cápside como vacuna.

Introduction

Waterfowl parvovirus can cause disease with high mortality and morbidity in geese and ducks (Barnes, 1997; Gough, 2003). Studies have shown that waterfowl parvovirus can be divided into the goose parvovirus (GPV)-related group and the Muscovy duck parvovirus (MDPV)-related group (Le Gall-Reculé & Jestin, 1994; Zádori et al., 1994 1995; Brown et al., 1995; Chang et al., 2000). The genomes of GPV and MDPV contain two major open reading frames: the first encoding the non-structural protein (NS) involved in viral replication and regulatory functions, and the second the three capsid proteins VP1, VP2 and VP3. The proteins VP1, VP2 and VP3 are derived from the same gene, and the entire amino acid sequences of VP2 and VP3 are contained within the carboxyl terminal portion of VP1 (Zádori et al., 1995). VP3 appears to be the most abundant of the three capsid proteins in purified virions (Le Gall-Reculé & Jestin, 1994). Sequence comparisons have shown that VP1 of GPV and MDPV have 87.4% amino acid sequence identity and the NS proteins have 90.6% identity (Zádori et al., 1995; Chu et al., 2001).

Serological assays are important for evaluation of the efficacy of vaccination and diagnosis of recent outbreaks of GPV and MDPV (Gough, 2003). A number of serological methods have been developed for detection of antibodies against GPV and MDPV, including agar gel precipitation and virus neutralization tests (Gough, 1984), enzyme-linked immunosorbent assays (Jestin et al., 1991; Kardi & Szegletes, 1996), the sperm agglutination-inhibition test (Malkinson et al., 1974), a plaque neutralization assay (Takehara et al., 1994), and an indirect fluorescent antibody test (Takehara et al., 1999). While these methods are useful for serological assays to detect waterfowl parvovirus infection, none is able to differentiate NS-specific antibodies from capsid-specific antibodies. This differentiation may enable discrimination of vaccinated birds from infected birds when the recombinant capsid protein is used as the vaccine (Le Gall-Reculé et al., 1996). It might also enhance understanding of how the titres of NS-specific and capsid-specific antibodies vary at different stages of infection. In this study, NS and capsid proteins of GPV and MDPV were expressed as recombinant proteins in Escherichia coli the recombinant proteins purified and then used as antigens in western blotting assays of NS-specific and capsid-specific antibodies against GPV and MDPV.

Materials and Methods

Polymerase chain reaction amplification and gene cloning

A GPV-related virus (strain 92-217) and an MDPV-related virus (strain 90-215) (Chang et al., 2000) were used as the sources for isolation of viral DNA. Procedures used for the DNA isolation and polymerase chain reaction (PCR) amplification were as described previously (Chang et al., 2000). Sequences of primers used for PCR amplification were based on published sequences of the genes for NS and capsid proteins of GPV and MDPV (Zádori et al., 1995). A total of seven primers were synthesized (Table 1). The primers included restriction endonuclease (BamHI or XhoI) cleavage sites at their 5′ ends (underlined bases). Primers NS(+) and NS(–) amplified a 1893 base pair (bp) fragment encoding the full-length NS protein (amino acids 1 to 627) of GPV and MDPV. Primers VP1N-GPV(+) and VP1N(–) amplified a 606 bp fragment encoding the amino terminal portion (residues 1 to 198) of VP1 of GPV. This truncated form of VP1 was designated VP1N and contained sequences present in VP1 and VP2, but not in VP3 (Figure 1). Primers VP1N-MDPV(+) and VP1N(–) amplified the same region of the VP1 gene in MDPV. Primers VP3(+) and VP3(–) amplified a 1614 bp fragment encoding the full-length VP3 (amino acids 1 to 534) of GPV and MDPV. The full-length VP3 contained residues 199 to 732 of VP1 (Zádori et al., 1995).

Figure 1.  Genomic structure of waterfowl parvovirus and locations of recombinant proteins. The genome is shown by a horizontal open bar and the coding regions are shaded. The NS, VP1, VP2 and VP3 proteins are indicated with horizontal arrows above the bar. The recombinant proteins expressed in this study are shown by horizontal dashed lines below the bar.

Table 1.  Sequences of primers used in this study

Primer	Position^a	Sequence^b
NS(+)	Nucleotides 537 to 557	5′-GGATCCATGGCA(C/T)TTTCTAGGCCTCTT-3'
NS(–)	Nucleotides 2397 to 2417	5′-CTCGAGTTGTTCATT(C/T)TC(A/C)(G/A)(C/T)ATCATC-3′
VP1N-GPV(+)	Nucleotides 2439 to 2460	5′-GGATCCATGTCTACTTTTTTAGATTCTT-3'
VP1N-MDPV(+)	Nucleotides 2450 to 2471	5′-GGATCCATGTCTACTTTTTTAGAGAAAT-3′
VP1N(–)	Nucleotides 3011 to 3032	5′-CTCGAG(T/G)T(C/T)AG(A/G)TGCTGCCACAGGTTCG-3′
VP3(+)	Nucleotides 3033 to 3054	5′-GGATCCATGGCAGAGGGAGGA(G/A)GCGGAG-3′
VP3(–)	Nucleotides 4612 to 4634	5′-CTCGAGCAGATT(T/C)TGAGT(T/C)A(G/A)ATA(T/C)CTGG-3′
^aPositions based on the sequences of GPV and MDPV reported by Zádori et al.(1995).
^bSequences of restriction endonuclease (BamHI or XhoI) cleavage sites are underlined.

The PCR products were cloned in the pCR-TOPO vector (Invitrogen, Carlsbad, California, USA) and the recombinant plasmid was purified using the Plasmid Miniprep kit (Qiagen GmbH, Hilden, Germany). The recombinant plasmid was cleaved with BamHI and XhoI. The inserted fragment was purified using a Geneclean III kit (BIO101, Vista, California, USA) and then cloned into the expression vector pET32a (Novagen, Inc., Madison, Wisconsin, USA). The identity of the insert in pET32a was verified by DNA sequencing. The recombinant protein produced by the pET32a vector was a fusion protein containing a tag at its amino terminus. This tag itself had a size of 179 amino acids, comprising three domains: a thioredoxin domain, a polyhistidine domain and an S-tag domain. The thioredoxin domain can enhance the solubility of recombinant proteins, the polyhistidine tag was used in purification of the recombinant proteins by nickel chromatography, and the S-tag was used in detection of recombinant proteins in western blots probed with alkaline phosphatase conjugated S protein (Novagen, Inc.).

Expression and purification of recombinant proteins

The recombinant proteins were expressed in E. coli strain BL21 (DE3) and purified by nickel chromatography (Novagen, Inc.) or by elution from sodium dodecyl sulphate (SDS) polyacrylamide gels as described previously (Shien et al., 2000; Chang et al., 2002). The yield was about 4 mg purified recombinant proteins from 1 l E. coli culture. The concentration of the proteins was determined using a Protein Assay kit (Bio-Rad, Hercules, California, USA).

Field sera

Sera were collected from 58 flocks (38 goose flocks and 20 duck flocks) in 2002 to 2003. The ducks were Muscovy (Cairina moschata), Tsaiya (Anas platyrhynchos) or mule ducks (Cairina moschata x Anas platyrhynchos), but the numbers of each type were not recorded. Each of the flocks was from a different farm and none of these flocks was vaccinated against GPV or MDPV. Most flocks were over 5 weeks of age and sera were collected in each flock from 10 randomly selected birds that appeared healthy and then pooled. Data about the disease history of these flocks were not available.

Experimental sera

Twelve Muscovy ducks from breeders that had not been vaccinated against GPV or MDPV were divided into two groups. The two groups were housed in separate rooms. At 1 week of age, ducks in the first group were infected by intraocular and intranasal inoculation of a total of 0.2 ml allantoic fluid containing 10⁵ median embryo infective dose/ml strain 92-217, while ducks in the second group were not infected. Strain 92-217 had been passed 60 times in goose fibroblast cells and 21 times in Muscovy duck (C. moschata) embryos prior to this, and appeared to be apathogenic in Muscovy ducks. Inoculation was repeated weekly until the ducks were 9 weeks old. Sera were collected from ducks every week for 8 weeks. The sera collected from each group were pooled each week and regarded as single samples for western blot analysis.

Western blotting

The purified recombinant NS and capsid proteins (2.5 µg per lane) were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using the Mini-Protean 3 electrophoresis system (Bio-Rad) and then transferred onto nitrocellulose membranes using the Semi-Dry Transfer system (Bio-Rad). The membrane was blocked with 3% skim milk in TBST buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20) for 1 h and then washed three times, each time for 5 min, using TBST buffer. The membrane was probed with goose or duck sera at a dilution of 1:100 in TBST buffer containing 3% skim milk for 1 h and then washed three times, each time for 5 min, using TBST buffer. The membrane was then incubated with alkaline phosphatase-conjugated goat anti-duck IgG (Kirkegaard and Perry Laboratories, Gaithersburg, Maryland, USA) at a dilution of 1:1000 in TBST buffer containing 3% skim milk for 1 h or with alkaline phosphatase-conjugated S protein (Novagen, Inc.) at a dilution of 1:5000 in TBST buffer for 15 min. Following three 5-min washes in TBST buffer, each membrane was incubated in 10 ml of AP buffer (100 mM Tris–HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl₂) containing 33 µl 5-bromo-4-chloro-3-indolyl phosphate (50 mg/ml in 100% dimethylformamide) and 66 µl of nitro blue tetrazolium (50 mg/ml in 70% dimethylformamide). The membranes were incubated for 20 min and then washed in distilled water to stop the reaction.

Multiscreen western blotting

Multiscreen western blotting (also known as slot blotting) allows the screening of up to 40 different sera simultaneously without having to cut the blot into individual strips. Recombinant NS of GPV (10 µg) was subjected to SDS-PAGE in a gel cast using a full-width comb in the Mini-Protean 3 system. After electrophoresis, the protein was transferred onto a nitrocellulose membrane and blocked with 3% skim milk in TBST buffer. Following three 5-min washes in TBST buffer, the membrane was held between the two plates of the multiscreen apparatus (Bio-Rad) and 500 µl each serum (1:100 dilution in TBST buffer containing 3% skim milk) were loaded individually into slots to react with the antigen. The immune complex was detected with alkaline phosphatase-conjugated goat anti-duck IgG (1:1000 dilution in TBST buffer containing 3% skim milk) as already described.

Results

Cloning and expression of NS, VP3 and VP1N proteins of GPV and MDPV

A total of six recombinant proteins, three from GPV and three from MDPV, were expressed in E. coli (Figure 1). The calculated molecular weights of recombinant NS, VP3 and VP1N of GPV, including the fusion tag, were 91.7 kDa, 78.7 kDa and 41.2 kDa, respectively. The recombinant proteins of MDPV had similar molecular weights.

Figure 2.  Bacterial expression and purification of recombinant proteins. (2a) Coomassie blue-stained SDS-PAGE of total cell lysates of E. coli expressing the tag protein (lane 1) and NS, VP3 and VP1N of GPV and MDPV (lanes 2 to 7). Lane M, molecular weight marker. (2b) Coomassie blue-stained SDS-PAGE of purified recombinant proteins. (2c) Western blots of purified recombinant proteins probed with the S protein. (2d) Western blots of sera from Muscovy ducks infected with GPV. (2e) Western blots of sera from uninfected Muscovy ducks.

SDS-PAGE of the total cell lysate of E. coli expressing the recombinant proteins showed that all these proteins were highly expressed in E. coli (Figure 2a). The majority of the recombinant NS and VP3 was insoluble, whereas the majority of the recombinant VP1N was soluble (data not shown). Therefore, recombinant NS and VP3 were purified by elution from gels, while VP1N was purified by nickel chromatography (Figure 2b). Western blotting showed that the purified recombinant NS, VP3 and VP1N were all recognized by S protein (Figure 2c), indicating that these recombinant proteins were expressed as fusions with the tag protein. Some minor bands with molecular weights lower than that of the full-length proteins were also detected (Figure 2c). These bands appeared to be degraded products of the full-length proteins.

Western blotting assays showed that purified recombinant NS, VP3 and VP1N from both GPV and MDPV were recognized by the sera collected from Muscovy ducks experimentally infected with GPV (Figure 2d), but not by sera collected from uninfected ducks (Figure 2e).

Western blotting assays of field sera

The purified tag protein and recombinant NS, VP3 and VP1N were used as antigens in western blotting assays of sera collected from the field. The sera from the 58 flocks could be classified into four groups based on their reactivity in the western blots. Group I had antibodies against only NS, group II had antibodies against NS and VP3, group III had antibodies against NS, VP3 and VP1N, and group IV had no detectable antibody (Figure 3). The numbers of flocks falling into each group are summarized in Table 2. If flocks in groups I, II and III were considered positive for having GPV-specific or MDPV-specific antibodies, 94.7% (36/38) of the goose flocks and 90.0% (18/20) of the duck flocks were positive.

Figure 3.  Western blotting assays of field sera. (3a) Coomassie blue-stained SDS-PAGE of purified recombinant proteins (lanes 1 to 7). Lane M, molecular weight marker. (3b to 3e) Western blots of purified recombinant proteins probed with sera yielding different patterns of reactivity.

Table 2.  Western blotting assays of field sera

Western blot reactivity	Number of flocks^a
	Geese	Ducks^b
NS	0	1
NS and VP3	8	0
NS, VP3 and VP1N	28	17
None	2	2
Total	38	20
^aNone of these flocks was vaccinated with GPV or MDPV and most flocks were over 5 weeks of age.
^bMuscovy, Tsaiya or mule ducks, but the numbers of each type were not recorded.

Western blotting assays of sera from experimentally infected ducks

Western blotting assays showed that Muscovy ducks experimentally infected with GPV had antibodies against NS at 1 week after infection, with a faint VP3 band detected (Figure 4a). This pattern was similar to that seen with field sera in group I. At 2 weeks after infection, antibodies against both NS and VP3 were clearly detected (Figure 4b), a pattern similar to that seen with field sera in group II. At 6 or 8 weeks after infection, antibodies against NS, VP3 and VP1N were all clearly detected (Figure 4c,d), a pattern similar to that seen with field sera in group III. Western blotting assays of sera collected from uninfected ducks detected no antibodies against NS, VP3 or VP1N (data not shown).

Figure 4.  Western blotting assays of sera from experimentally infected ducks. The blots were prepared using purified recombinant proteins. (4a to 4d) Western blots of purified recombinant proteins probed with duck sera collected at 1, 2, 6, or 8 weeks after infection.

Multiscreen western blotting assay

Sera collected from 58 flocks (38 goose and 20 duck flocks) were screened using a multiscreen western blotting assay. The results showed that sera from six flocks (one goose flock and five duck flocks) reacted with NS at high intensity, those from 19 flocks (18 goose flocks and one duck flock) at medium intensity, those from 29 flocks (17 goose and 12 duck flocks) at low intensity, and those from four flocks (two goose and two duck flocks) yielded no visible band. Representative results are shown in Figure 5. If sera yielding a visible NS band were scored positive, the assay found that 54 of 58 flocks had antibodies against GPV or MDPV, a result consistent with those obtained with conventional western blotting assays.

Figure 5.  Multiscreen western blotting assay of field sera. Recombinant NS of GPV was used as the antigen. Lane M, molecular weight marker. Lanes 1 and 2, sera showing intense reactivity; lanes 3 and 4, sera showing a medium level of reactivity; lanes 5 and 6, sera yielding a low level of reactivity; lanes 7 and 8, sera with no reactivity. Sera in lanes 1, 3, 5 and 7 were collected from geese, and those in lanes 2, 4, 6 and 8 were collected from ducks.

Discussion

We have shown that recombinant NS, VP3 and VP1N expressed in E. coli can serve as antigens in serological assays for GPV and MDPV infection. The advantage of using E. coli-expressed proteins is that the cost is relatively low and the recombinant protein is easy to purify by either chromatography or elution. Many reports have demonstrated that E. coli-expressed proteins are useful antigens for detecting antibodies against a variety of viral diseases (Crabb & Studdert, 1995; Ro et al., 1995; Boshoff et al., 1997; De Diego et al., 1997; Ndifuna et al., 1998; Anderson et al., 1999; Johne et al., 2004). For detection of antibodies against GPV and MDPV, an added advantage of using recombinant NS as the antigen is that it might be able to discriminate between infected and vaccinated birds when recombinant capsid protein (VP2 and VP3) is used as the vaccine (Le Gall-Reculé et al., 1996). This might allow the elimination of infected birds and thereby control the spread of this disease, and might eventually lead to the eradication of GPV and MDPV. A similar strategy has been suggested for the control of porcine parvovirus (Madsen et al., 1997).

Western blotting assays showed that purified recombinant NS, VP3 and VP1N from both GPV and MDPV were recognized by the sera collected from Muscovy ducks experimentally infected with GPV, and thus there appeared to be partial cross-reactivity between antibodies against GPV and MDPV. Partial cross-reactivity was probable because the protein sequences of NS, VP3 and VP1N of GPV and MDPV exhibited 90.6%, 90.4% and 79.8% sequence identity, respectively (Zádori et al., 1995).

Western blots probed with sera collected from experimentally infected ducks showed that antibodies against NS appeared first after infection, followed by antibodies against VP3 and then by those against VP1N. This is reminiscent of findings about the immune response to infection with the human B19 parvovirus, which have shown that the appearance of antibodies against NS can confirm a recent infection (Ennis et al., 2001; Heegaard et al., 2002) and the appearance of antibodies against the amino terminal end of VP1 (amino acids 1 to 227) may indicate a later stage of infection (Kurtzman et al., 1989; Schwarz et al., 1988).

Western blotting assays showed that 54 of 58 goose and duck flocks had antibodies against GPV or MDPV. These antibodies were unlikely to result from vaccination because only a limited number of geese and ducks are vaccinated in Taiwan. They were unlikely to be derived from maternal transfer of antibodies because most of the sera were collected from birds over 5 weeks of age. Thus most of these antibodies can be assumed to have resulted from natural infection. It was therefore concluded that the great majority of geese and ducks in Taiwan have been infected with GPV or MDPV. This conclusion is consistent with the high morbidity seen in outbreaks of disease due to GPV and MDPV in the field. Although infection with GPV can result in 100% mortality in goslings under 10 days of age (Gough, 2003), such high mortality has not been seen in Taiwan, possibly because of protection provided by transfer of maternal antibodies from breeders that have been either naturally infected or vaccinated.

The multiscreen western blotting assay using recombinant NS of GPV yielded results consistent with those from conventional western blotting assays, indicating that it may be suitable for large-scale screening for the presence of GPV-specific or MDPV-specific antibodies in goose or duck sera. The differences in intensity of the NS band may result from collection of sera from flocks at different stages of infection. It is also possible that flocks repeatedly infected with GPV and MDPV might produce a more potent immune response and thus an NS band of higher intensity.

Translations of the abstract in French, Germany and Spanish are available on the Avian Pathology website.

The authors thank Dr D.F. Lin in National Animal Research Institute, Taipei, Taiwan for providing parvovirus strains. This investigation was supported by grant NSC-93-2313-B-005-069 from the National Science Council, Taiwan.