Linear hoof defects in sheep infected with foot-and-mouth disease.

During the epidemic of foot-and-mouth disease (FMD) in The Netherlands in 2001, a sheep farm was identified that had been subclinically infected with the disease. The FMD virus genome was detected in 12 of 16 probang samples collected from the sheep and the virus was isolated from four of these samples. Linear defects were observed, 1 to 3 cm from the coronary band, in the hooves of several of the sheep. The defects were thought to have been caused by the FMD infection. It was thought that the distance of the defects from the coronary band might be an indication of the time since the animals had been infected. To determine the growth rate of the claws of sheep, the growth of the hoof horn of uninfected lambs and ewes was measured; in the lambs the growth rate was 0.44 mm per day and in the ewes it was 0.29 mm per day.

Oral transmission of porcine reproductive and respiratory syndrome virus by muscle of experimentally infected pigs.

The current study was performed to determine if porcine reproductive and respiratory syndrome virus (PRRSV) could be transmitted to pigs by feeding muscle tissue obtained from recently infected pigs. Muscle obtained from pigs infected with either a European strain (EU donor pigs) or American strain (US donor pigs) of PRRSV was fed to PRRSV-free receiver pigs. The donor pigs were slaughtered 11 days post-infection (dpi). PRRSV was detected by conventional virus isolation in muscle at 11 dpi from 7 of 12 EU donor pigs and 5 of 12 US donor pigs. In contrast to conventional virus isolation, all muscle samples from infected pigs were positive for viral nucleic acid by PCR, except for muscle from one animal infected with the American strain of PRRSV. Five hundred grams of raw semimembranosus muscle from each of the donor pigs was fed over a 2 days period (250 g per day) to each of two receiver pigs (48 receiver pigs). The receiver pigs were housed separately in five groups. One of the five groups was fed muscle obtained from US donor pigs that was also spiked with the American strain of PRRSV. Sentinel pigs were placed in-contact with the group of receiver pigs fed spiked muscle. All receiver pigs became viraemic by 6 days post-feeding (dpf). There was evidence of horizontal transmission with sentinel pigs, in-contact with receiver pigs, becoming viraemic. The study demonstrates that PRRSV could be infectious through the oral route via the feeding of meat obtained from recently infected pigs.

Safety and protective efficacy of porcine reproductive and respiratory syndrome recombinant virus vaccines in young pigs.

Three porcine reproductive and respiratory syndrome virus (PRRSV) recombinants, generated by mutagenesis of an infectious cDNA clone of the Lelystad virus (LV) isolate, were tested for their safety and protective efficacy as potential PRRSV vaccines in pigs. Recombinant vABV688 contains two amino acid substitutions in the minor structural protein GP(2) resulting in improved growth on cell line CL2621; in recombinant vABV707 the region encoding the ectodomain of the major unglycosylated membrane protein M has been replaced by that of the murine lactate dehydrogenase-elevating arterivirus; recombinant vABV746 lacks the six C-terminal amino acids of the nucleocapsid protein N. First, we determined the safety of these recombinant viruses by monitoring the stability of the introduced mutations in 8-week-old pigs. We showed that the introduced genomic mutations were maintained throughout the viraemic period. Second, the protective efficacy of immunization with the recombinant viruses against challenge with a homologous and a heterologous PRRSV strain was determined in two pigs and compared with the efficacy of vABV437, a virus derived from the parental LV cDNA. The viraemia in pigs immunized with the recombinant viruses was reduced compared to pigs immunized with vABV437. In addition, the length of viraemia was reduced in the sentinel pigs that were introduced into the groups immunized with vABV746, vABV688, and vABV707, however, all of the sentinel pigs became infected. Pigs immunized with vABV707 and vABV437 were protected against challenge with homologous virus LV-Ter Huurne and transmission of the latter virus. None of the immunized pigs were protected against heterologous challenge with the virulent US isolate SDSU#73, but the vABV707- and vABV746-immunized pigs were protected against transmission of this virus from challenged pigs. In conclusion, the obtained viral recombinants are interesting candidates to be further explored for their use as vaccines against PRRSV.

Identification of porcine alveolar macrophage glycoproteins involved in infection of porcine respiratory and reproductive syndrome virus.

The aim of this study was to identify the receptor(s) for PRRSV on porcine alveolar macrophages (PAMs) by producing monoclonal antibodies (MAbs) against these cells. Hybridoma supernatants were selected for their ability to block PRRSV infection. Four MAbs, 1-8D2, 9.4C7, 9.9F2, and 3-3H2 inhibited infection and recognised cell surface, PAM-specific antigens as shown by immunofluorescence and immunoperoxidase monolayer assay. These MAbs were then used to identify cellular proteins involved in PRRSV infection by radioimmunoprecipitation assays (RIPAs). MAbs 1-8D2 and 9.9F2 each recognised a 150 kDa-polypeptide doublet, while MAbs 9.4C7 and 3-3H2 both recognised a 220 kDa-polypeptide. Glycosidase treatment demonstrated all these polypeptides to be N-glycosylated. Thus, multiple glycoproteins appear to be involved in infection of PAMs by PRRSV.

Development of organs and intestinal mucosa leukocytes in four broiler lines that differ in susceptibility to malabsorption syndrome.

Growth retardation in young broiler chicks due to poor nutrient metabolism, commonly known as malabsorption syndrome (MAS), is a widespread problem caused by enteric infections with a combination of pathogens mainly viruses. Genetic lines of broiler chickens differ in susceptibility to the syndrome. A difference in growth retardation was observed among four broiler lines (BL) after oral inoculation at 1 d of age with intestinal homogenates obtained from MAS-affected birds. Two of the lines that are more susceptible to MAS had severe weight gain depression. To uncover the factors that play a role in the susceptibility to MAS, we analyzed the growth rate of the body and vital organs and the quantity of leukocytes in the peripheral blood and intestinal mucosa. The development of the intestine, liver, bursa of Fabricius, and spleen was similar among the BL. The resistant BL had higher numbers of peripheral blood leukocytes, especially lymphocytes, at 1 d of age. A significant difference was noted in the numbers of CD4+ T cells and CD8+ T cells in the intestinal villi. At the ages of 3 and 8 d, the susceptible BL had more CD8+ T cells in the villi, whereas the ratios of CD4+:CD8+ T cells were higher in the resistant BL. This difference in the number of T-cell subpopulations in the intestinal mucosa might be an important factor in the difference in susceptibility to the enteric infections associated with MAS.

Experimental reproduction of malabsorption syndrome with different combinations of reovirus, Escherichia coli, and treated homogenates obtained from broilers.

Attempts to reproduce malabsorption syndrome (MAS) by oral inoculation with several different combinations including intestinal homogenate, reovirus, and hemolytic Escherichia coli obtained from MAS-affected chickens and intestinal homogenate from healthy chickens (healthy homogenate) were performed in 1-day-old specific-pathogen-free (SPF) broilers. The MAS homogenate, serving as a positive control, induced weight gain depression and intestinal lesions such as cystic crypts of Lieberkuhn, villus atrophy, and lymphoid and/or granulocytic infiltration. The healthy homogenate, the formalin-treated MAS homogenate, the formalin-treated healthy homogenate, and phosphate-buffered saline caused neither weight gain depression nor intestinal lesions. We were able to reproduce both weight gain depression and intestinal lesions by inoculation of reovirus either combined with the formalin-treated MAS homogenate or combined with healthy homogenate. Surprisingly, when hemolytic E. coli was added to the combination of reovirus with formalin-treated MAS homogenate, this did not cause weight gain depression although this combination caused the described intestinal lesions. Identical results were obtained with the combination of formalin-treated MAS homogenate with hemolytic E coli or the combination of reovirus with hemolytic E. coli. The intestinal lesions were more severe and developed faster by combinations including reovirus and formalin-treated MAS homogenate. This study indicates that a combination of enteropathogenic reovirus with other agents or substances that are present in an intestinal homogenate from MAS-affected and healthy chickens can induce MAS in SPF broilers. Escherichia coli is not essential for induction of weight gain depression but can play a role in development of intestinal lesions. Furthermore, intestinal lesions alone will not always result in weight gain depression.

Cellular immune response in the small intestine of two broiler chicken lines orally inoculated with malabsorption syndrome homogenates.

We studied the cellular immune response against malabsorption syndrome (MAS) in two broiler chicken lines, A and B. We determined the number of pan T-lymphocytes (CD3), helper T-lymphocytes (CD4), cytotoxic T-lymphocytes (CD8) and macrophages/monocytes in the small intestine in the first 2 weeks after oral inoculation of two MAS homogenates, MAS80 and MAS97-1. The immune cells were detected on cryostat tissue by immunohistochemistry and counted by villus area. In trial 1, we compared the two broiler lines for weight gain depression, intestinal lesion and number of CD3, CD4, CD8 cells and macrophages/monocytes after MAS80 inoculation. Although there was no significant difference in weight gain depression between the two broiler lines, line B had significantly higher numbers of CD8+ T-cells per villus area than had line A. To confirm part of the results of trial 1, trial 2 was done in which we compared different homogenates in broiler line B. Broiler line B was orally inoculated with either MAS97-1, intestinal homogenate obtained from healthy chickens (healthy homogenate), or phosphate buffered saline (PBS). In this trial, the MAS97-1 homogenate also induced weight gain depression and intestinal lesions, whereas the "healthy homogenate" and PBS did not induce weight gain depression or intestinal lesions. The broilers inoculated with MAS97-1 homogenate had significantly more CD8+ T-cells per villus area than had broilers inoculated with "healthy homogenate" or PBS. Increased CD8+ T-cells per villus area in the affected small intestines of broilers suggests an increase of cytotoxic T-cell activity.

A quantitative assessment of the effectiveness of PRRSV vaccination in pigs under experimental conditions.

This paper presents a quantitative approach to evaluate effectiveness of vaccination under experimental conditions. We used two consecutive experimental designs to investigate whether PRRSV transmission among vaccinated pigs was reduced compared to control pigs and to estimate the reproduction parameter R. Based upon data analysis and power calculations the series of small-scale vaccination-challenge experiments ended with multiple one-to-one experiments. This new experimental design has considerable power to detect the effect of vaccination on transmission if R is close to but still above one in vaccinated pigs. The last experiment showed that transmission was not significantly reduced and the R for vaccinated pigs was estimated to be larger than 4.9. This is remarkable because duration and level of viremia were significantly reduced by vaccination.

Deletion of the UL21 gene in Pseudorabies virus results in the formation of DNA-deprived capsids: an electron microscopy study.

We studied the morphogenesis of three pseudorabies virus mutants lacking parts of the gene homologous to the UL21 gene of the herpes simplex virus type 1. The mutants were examined in an SK-6 cell-line, in an SK-6 cell-line expressing the UL21 gene product, in porcine lung alveolar macrophages (PLAM) and in porcine nasal mucosa explants. Although on SK-6 cells and PLAM, the virus-assembly and egress of mutant virus M155, lacking almost the entire UL21 gene, was similar to that of the rescued PRV mutant, M155 producing virions containing little or no DNA (A-type particles). Virus mutants M133 and M134 (lacking 23 and 232 amino acids respectively) produced more C-type particles. In SK-6 cells stably expressing the UL21-encoded protein, all mutants produced C-type particles. All mutants produced C-type particles in nasal mucosa explants, indicating that the UL21-gene product is not essential for virus production in porcine tissue. These results support and extend previous work that indicated a role for the UL21 encoded protein in the packaging of newly replicated viral DNA.

Efficacy of inactivated infectious bursal disease (IBD) vaccines: comparison of serology with protection of progeny chickens against IBD virus strains of varying virulence.

The efficacy of inactivated infectious bursal disease vaccines was determined by measuring both the antibody response of vaccinated chickens and clinical protection of progeny chicks from vaccinated dams. Similar virus neutralizing (VN) antibody titres were obtained in 4-week-old chickens and mature hens after vaccination with one vaccine dose. VN titres below 10 log 2 increased considerably between the fourth and seventh week after vaccination in 4-week-old chickens as well as in mature chickens. All 2-week-old progeny chicks with serum VN antibody titres of at least 9 log 2 were clinically protected against the classical virulent 52/70 infectious bursal disease virus (IBDV) strain, as well as against the very virulent IBDV (vvIBDV) strain D6948. However, vaccination often did not prevent subclinical infection in these 2-week-old progeny chicks, which often resulted in severe lymphocyte depletion in the bursa of Fabricius. Even a serum VN titre of 11 log 2 was not always sufficient to prevent severe bursal damage. Although 52/70 IBDV and vvIBDV were equally pathogenic in 2-weekold specific pathogen free chickens, significant higher maternal antibody titres were required to prevent the adverse effects of vvIBDV in comparison with 52/70 IBDV. The relation between the serological response of chickens after application of inactivated IBD vaccines and the protection of progeny chicks of vaccinated dams depended on both the virulence of the IBDV challenge strain and the IBDV strain in the vaccine.

A comparative study of the pathogenesis of malabsorption syndrome in broilers.

Five malabsorption syndrome (MAS) homogenates from The Netherlands and Germany were used to reproduce MAS in broilers. We studied the histopathology after inoculation of 1-day-old broiler chicks and the agents that might be involved. Generally, the MAS homogenates induced signs that differed in severity and pathobiology. We could distinguish and classify the inoculated groups best by histopathology: proventriculitis, lesions in the small intestines in combination with proventriculitis, or lesions of the small intestines only. Lesions in the small intestine had more impact on weight gain depression than lesions in the proventriculus. In three out of five inoculated groups, microscopic lesions of the pancreas were found. Reovirus was detected in the inoculated groups by virus isolation and seroconversion, and reoviral antigen was detected by immunohistochemistry of the small intestine. Also, enteroviruslike particles were detected in three of the five inoculated groups, although not in the most affected group. Additionally, bacteriophages and bacteria (hemolytic Escherichia coli, Pasteurella hemolytica, and Enterococcus durans) were isolated from inoculated chicks. The role these agents play in pathogenesis of MAS is still unsolved.

Systemic and mucosal isotype-specific antibody responses in pigs to experimental influenza virus infection.

The immunoglobulin isotype-specific responses in serum and at the respiratory mucosa of pigs after a primary infection with influenza virus were studied. To do this, we developed an aerosol challenge model for influenza in specified pathogen-free (SPF) pigs and isotype-specific enzyme-linked immunosorbent assays (ELISAs). Ten-week-old pigs were inoculated without anesthesia in the nostrils with an aerosol of the field isolate influenza A/swine/Neth/St. Oedenrode/96 (H3N2). The infection caused acute respiratory disease that closely resembled the disease observed in some outbreaks of influenza among finishing pigs, which were not complicated by bacterial infections. Pigs showed clinical signs characterized by fever, dyspnea, and anorexia. At necropsy on postinfection days 1 and 2, an exudative endobronchitis was observed throughout the lung. Viral antigen was present in the epithelial cells of the bronchi and bronchioli and virus was isolated from bronchioalveolar and nasal lavage fluids and from pharyngeal swabs until 5 days after infection. With the isotype-specific ELISAs, viral nucleoprotein specific immunoglobulin (Ig) M, IgG1, and IgA antibody responses were measured in serum and bronchioalveolar and nasal lavage fluids. To determine whether the antibodies were produced and secreted at the respiratory mucosa or were serum-derived, the specific activity (ie, the ratio of antibody titer to Ig concentration) was calculated for each isotype. The IgA and interestingly also a substantial part of the IgG1 antibody response in pigs upon infection with influenza virus was shown to be a mucosal response. Local production of specific IgA in the nasal mucosa, and of specific IgA and IgG1 in the lung was demonstrated. These results indicate that protective efficacy of vaccination can be improved by an immunization procedure that preferentially stimulates a mucosal immune response. The aerosol challenge model in SPF pigs and the isotype-specific ELISAs that we developed can be useful for evaluating various strategies to improve efficacy of porcine influenza vaccines and to study the immune mechanisms underlying the observed protection.

Introduction, persistence and fade-out of porcine reproductive and respiratory syndrome virus in a Dutch breeding herd: a mathematical analysis.

The objective of this study was to investigate the dynamics of PRRSV infection and to quantify transmission within a breeding herd, and its impact on herd performance. For this purpose a longitudinal study was performed in a closed breeding herd of 115 sows. Statistical methods and Monte Carlo simulations based on stochastic SIR models were used to analyse the observational data. Moreover, a case-control study was performed to determine whether seroconversion of sows during gestation was associated with aberrant litters. The transmission parameter R was estimated to be 3.0 (95% confidence interval 1.5-6.0) for the model version based on the most plausible assumptions that the infectious period lasts 56 days and no lifelong immunity exists after infection. Based on simulations using a breeding herd of equal size the average time-to-extinction was estimated to be 6 years; using a herd of twice the size, it was 80 years. Furthermore, in contrast to the epidemic phase of the disease, the endemic phase was not detrimental to herd performance.

Changes of leukocyte phenotype and function in the broncho-alveolar lavage fluid of pigs infected with porcine reproductive and respiratory syndrome virus: a role for CD8(+) cells.

Porcine reproductive and respiratory virus (PRRSV) primarily infects and destroys alveolar macrophages of the pig. The aim of the present study was to characterize the changes of leukocyte populations in the broncho-alveolar lavage fluid (BALF) of PRRSV-infected pigs. Piglets were inoculated intranasally with PRRSV strain LV ter Huurne. On various days post-infection the piglets were sacrificed and the lungs removed, washed semi-quantitatively and analysed by flow cytometry. The total number of recovered BALF cells increased approximately 10 times between day 10 and day 21 of infection and decreased thereafter. The number of small low-autofluorescent cells (SLAC), i.e. lymphocytic and monocytic cells, increased very strongly from day 2 until day 21 of infection; in contrast, the number of large highly autofluorescent cells (LHAC), i.e. mostly macrophages, remained constant until day 14 of infection, increased slightly on day 21 and then decreased. On day 21 of infection in specific-pathogen-free piglets approximately 60% of the SLAC consisted of CD2(+)CD8(+)CD4(-)gammadeltaTCR(-) cells, which were partly CD8(+)CD6(+) and partly CD8(+)CD6(-). These phenotypes correspond to that of cytotoxic T-cells and natural killer cells respectively. From these results we can conclude that during a PRRSV infection the total number of BALF cells increases mainly due to an influx of lymphocytic cells with a cytolytic phenotype.

Detection of early infection of swine vesicular disease virus in porcine cells and skin sections. A comparison of immunohistochemistry and in-situ hybridization.

Sensitive methods are required to study the early pathogenesis of swine vesicular diseases (SVD). Therefore, two new methods, immunohistochemistry (IHC) and in-situ hybridization (ISH), were developed and tested for their specificity and sensitivity. With these methods the SVD virus (SVDV) infection in cytospins of primary porcine kidney cells and in frozen skin sections was investigated. Both IHC and the ISH showed a specific cytoplasmic staining, but the IHC detected more infected cells than the ISH. Furthermore, both IHC and ISH were able to detect SVDV in skin sections 4.5 h after infection. It is concluded that IHC is the most suitable and simplest method to identify cells and tissues that support the initial replication of swine vesicular disease virus. However, IHC can only be applied to frozen sections, whereas ISH can also be used in paraformaldehyde-fixed tissues.

Comparative morphogenesis of three PRRS virus strains.

The morphogenesis of a Dutch PRRS field strain virus (Lelystad virus) was studied and compared to that of a U.S. field strain VR2332 and its attenuated vaccine strain JJ1882. Porcine lung alveolar macrophages (PLAM) and CL2621 cells were infected with high doses of virus (MOI = 10). At 4, 6, 9, 12, 18, 24, and 48 h post infection (hpi) cells were fixed for electronmicroscopy or for detection of viral antigens by immunoperoxidase staining. From 6 hpi on, viral antigens were detected in the cytoplasm and from 9 hpi on completely assembled virus particles could be detected in infected cells. The three strains were similar in assembly of new virus particles, envelopment at the smooth endoplasmic reticulum, and egress from infected cells. However, distinct differences were seen in replication time of the three strains in various cell types. The Lelystad virus replicated very fast and efficiently in PLAM while VR2332 and JJ1882 replicated preferably in CL2621 cells. JJ1882 replicated faster in CL2621 cells than VR2332 did, probably because of increased adaptation to the cell-line. Although the U.S. and European strains differ at the level of the genome, morphogenesis is not visibly altered. There is however a distinct difference in preferred cell type between the European strain and the two U.S. strains.

Dual infections of PRRSV/influenza or PRRSV/Actinobacillus pleuropneumoniae in the respiratory tract.

To study the effect of a previous porcine respiratory and reproductive syndrome-infection (PRRS) of the respiratory tract on influenza virus and Actinobacillus pleuropneumoniae (App) infections, 3-week-old specific-pathogen-free (spf) piglets were intranasally infected with PRRS virus. One week later, when the lung alveolar macrophages of PRRSV infected pigs were lowest in number, a second infection was applied by intranasal aerosol of influenza virus H3N2 or by endobronchial instillation of a mildly virulent App. The first experiment consisted of two groups (only influenza infection or dual PRRSV/influenza infection). A second experiment consisted of 4 groups (only influenza infection, only PRRSV infection, dual PRRSV/influenza infection and uninfected controls). At day 2, 4, 14 and 21 after influenza infection, two pigs were killed and sampled for virological and histopathological examination. Influenza H3N2 virus caused only a mild inflammation of the smaller bronchioli. Previous PRRSV infection did not influence clinical signs during influenza infection. Next, we studied in two experiments the effect of dual PRRSV/App infection during the acute stage at two days after App infection. In a third experiment, the influence of PRRSV on more chronic stages of App infection was studied at two weeks after the App infection. At the end of the experiments, the pigs were killed. Lungs were ranked according to size and kind of the lesions. Lesions were cut and measured, samples were taken for virological and histopathological examination. Statistical analysis of the ranked lung-lesions in the first experiment showed a distinct but small effect of previous PRRSV infection on the development of App-lesions. In PRRSV infected pigs. App produced a more severe disease. The second and third experiment however failed to show any influence of the previous PRRSV infection on the App infection. We conclude that previous PRRSV infection of the respiratory tract of spf pigs does not necessarily enhance the severity of secondary infections of the respiratory tract.

Pseudorabies virus infections in pigs. Role of viral proteins in virulence, pathogenesis and transmission.

This paper reviews new findings on the biological functions of pseudorabies virus (PRV) proteins. It focuses on the role of PRV proteins in the pathogenicity, immunogenicity and transmission of PRV vaccine strains in pigs. Furthermore, it evaluates potential risks that are connected with the use of PRV vector strains. Special emphasis is placed upon the spread of genetically engineered vaccine strains within pigs or between pigs.

Experimental quantification of transmission of genetically engineered pseudorabies virus.

There is concern that live pseudorabies virus (PRV) vaccine or PRV vector vaccine strains may spread from vaccinated to unvaccinated pigs. Moreover, it is feared that recombining PRV vaccine strains with related vaccine or wild-type strains may lead to spread and survival of recombinant PRV. To learn more about to what extent different PRV vaccine strains could spread we used a previously described experimental model to study the transmission of intranasally inoculated PRV mutant strains under experimental conditions. We used PRV strains that lacked glycoprotein E (gE) or thymidine kinase (TK), and a PRV vector vaccine (gE-, TK-, gG-) that expresses the glycoprotein E1 (E1) of hog cholera virus. In addition, we investigated whether intranasally co-inoculated gE-negative and gE-positive PRV strains competed in transmission among pigs. The extent of transmission was estimated using the reproduction ratio R. This ratio has a threshold property; when R1, the infection can spread; when R < 1, the infection will disappear. We found that R for a gE-negative strain was 10.1, and R for a TK-negative strain was 5. Furthermore, the R for the vector vaccine (gE-, TK-, gG-) expressing E1 was 0.18, and did not differ significantly from the R for the control strain without E1. The R of gE-negative strain was significantly 1 (P = 0.0005). Co-inoculation with a gE-positive field strain did not prevent the transmission of a gE-negative strain. This study shows that a small-scale experiment can be used to estimate the transmission of genetically engineered organisms in their host species. The results of this study indicate that the deletion of gE alone or TK alone is not enough to prevent spread of PRV among susceptible pigs, and that transmission of gE-negative PRV is not firmly limited by co-presence of a gE-positive strain.

Nucleocapsid protein N of Lelystad virus: expression by recombinant baculovirus, immunological properties, and suitability for detection of serum antibodies.

The ORF7 gene, encoding the nucleocapsid protein N of Lelystad virus (LV), was inserted downstream of the P10 promoter into Autographa californica nuclear polyhedrosis virus (baculovirus). The resulting recombinant baculovirus, designated bac-ORF7, expressed a 15-kDa protein in insect cells. This protein was similar in size to the N protein expressed by LV in CL2621 cells when it was analyzed on sodium dodecyl sulfate-polyacrylamide gels. The N protein expressed by bac-ORF7 was immunoprecipitated with anti-ORF7 was immunoprecipitated with anti-ORF7 peptide serum, porcine convalescent-phase anti-LV serum, and N protein-specific monoclonal antibodies, indicating that this N protein had retained its native antigenic structure. The recombinant N protein was immunogenic in pigs, and the porcine antibodies raised against this protein recognized LV in an immunoperoxidase monolayer assay. However, pigs vaccinated twice with approximately 20 micrograms of N protein were not protected against a challenge with 10(5) 50% tissue culture infective doses of LV. Experimental and field sera directed against various European and North American isolates reacted with the N protein expressed by bac-ORF7 in a blocking enzyme-linked immunosorbent assay. Therefore, the recombinant N protein may be useful for developing diagnostic assays for the detection of serum antibodies directed against different isolates of LV.