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Lactobacillus casei strain 393 was selected as an antigen delivery vehicle for the development of oral vaccine to express recombinant classical swine fever virus (CSFV) T cell epitope E290 peptide and porcine parvovirus (PPV) VP2 protein. The recombinant genes encoding CSFV T cell epitope E290 peptide and PPV VP2 protein, respectively, were cloned into the secretion expression vector pPG, and then the pPG-VP2-E290 was electrotransformed into L. casei 393 giving rise to recombinant strain pPG-VP2-E290/L. casei 393. The recombinant L. casei 393 was induced by 2% lactose in MRS and about 70kDa protein was detected with SDS-PAGE in induced recombinant strain and culture supernatants. The result of Western blot indicated that the expressed protein possessed the antigenic specificity same as the native virus protein. The indirect ELISA test also indicated that the interest protein was expressed and secreted from the recombinant strain. Specific anti-PPV VP2 secret immunoglobulin A (sIgA) antibody was detected by indirect ELISA in the feces, anti-PPV VP2 and anti-CSFV E290 peptide immunoglobulin G (IgG) antibody was detected by indirect ELISA in the serum of immunized mice after intragastric administration. The results indicated that the mice immunized with recombinant strain pPG-VP2-E290/L. casei 393 could produce clear antibody level, which establish important material basement for the development of lactic acid bacteria oral vaccine of recombinant CSFV and PPV.

The effect of different Parvovirus+Erysipelas vaccination schemes in PCV2-infected sows on PMWS outcome in the offspring was investigated under experimental conditions. Six PCV2-free sows were first infected oro-nasally with PCV2 two months before insemination (D0; "Day 0") and then by the intra-uterine route at insemination (D62). On D21 and D42, vaccinated sows received either the two commercial monovalent vaccines, A1(PPV) and A2(Erysipelas), or the bivalent vaccine B (PPV+Erysipelas). In addition, three SPF sows (foster-sows) were synchronized for farrowing dates to enable them to foster piglets born to infected sows and removed at birth before colostrum intake. A significantly higher proportion of mummified fetuses was obtained from PCV2-infected non-vaccinated sows than from vaccinated sows. Acute myocarditis lesions were found in their piglets, together with a high PCV2 genome load. The latter was significantly higher than in those born to PCV2-infected vaccinated sows. Sentinel PCV2-negative piglets, born to SPF foster-sows, seroconverted at almost the same time as piglets without PCV2 passive immunity and born to infected sows. Sixteen of the 84 liveborn piglets born to infected sows and foster-sows were affected by a syndrome possibly related to PMWS, as judged by clinical signs and histological lesions. Most were born to PCV2-infected non-vaccinated sows and 12/16 did not receive PCV2 passive immunity. The probability of PCV2 infection and the number of PCV2 genome copies per gram of tissue were significantly increased in piglets that did not receive PCV2 passive immunity.

The pathogenicity of two recent German field isolates of Porcine parvovirus (PPV-27a and PPV-143a) and two vaccine viruses [PPV-NADL-2 and PPV-IDT (MSV)], which are used for the production of inactivated vaccines, was investigated by inoculation of pregnant sows at day 40 of gestation. Post-infection sera of these sows as well as antisera prepared in rabbits by immunization with the four above-mentioned PPV isolates and with the virulent strain PPV-Challenge (Engl.) were tested for their homologous and heterologous neutralization activities. All antisera had high neutralization activity against the vaccine viruses, the PPV-Challenge (Engl.) virus and PPV-143a, but much lower activity against PPV-27a. These results suggest that PPV-27a represents a new antigenic variant or type of PPV and vaccines based on the established vaccine viruses may not be fully protective against this field isolate. PPV-27a has been characterized based on the amino acid sequences of the capsid protein as a member of a new and distinct PPV cluster (Zimmermann et al., 2006). Interestingly, the homologous neutralizing antibody titres of the sera of all three pigs and both rabbits inoculated or immunized with PPV-27a were 100- to 1000-fold lower than the heterologous titres against any of the other viruses. The low homologous neutralizing antibody titres suggest a possible, yet undefined, immune escape mechanism of this PPV isolate.

A novel vaccine against porcine parvovirus (PPV), composed of recombinant virus-like particles (PPV-VLPs) produced with the baculovirus expression vector system (BEVS) at industrial scale, was tested for its immunogenicity and protective potency. A formulation of submicrogram amounts of PPV-VLPs in a water-in-mineral oil adjuvant evoked high serum antibody titres in both guinea pigs, used as reference model, and target species, pigs. A single immunisation with 0.7microg of this antigen yielded complete foetal protection against PPV infection after challenge with a virulent strain of this virus. Furthermore, also in the presence of mild adjuvants the protective action of these PPV-VLPs is excellent. This recombinant subunit vaccine overcomes some of the drawbacks of classical PPV vaccines.

Background: Nucleic acid amplification allows the detection of single infectious agents. Protein-based assays, although they provide information on ongoing infections, have substantially less detection sensitivity., Methods: We used proximity ligation reactions to detect proteins on bacteria and virus particles via nucleic acid amplification. Antibodies recognizing viral or bacterial surface proteins were equipped with DNA strands that could be joined by ligation when several antibodies were bound in proximity to surface proteins of individual infectious agents., Results: Detection sensitivities similar to those of nucleic acid-based detection reactions were achieved directly in infected samples for a parvovirus and an intracellular bacterium., Conclusions: This method enables detection of ligated DNA strands with good sensitivity by real-time PCR and could be of value for early diagnosis of infectious disease and in biodefense.

To construct gene vaccine of PPV and to investigate the effects of interleukin 2 (IL-2) as an adjuvant on immune responses in mouse, the recombinant expression plasmid of pCIneo-IL2-VP2 was constructed and transfected into PK-15 cells by lipofectamine, the expressed product was detected by immunofluore assay. To study the immune effects of DNA vaccine in vitro and in vivo, mice were used as the animal model. The recombinant plasmid pCIneo-IL2-VP2, the control plasmid pCI-neo and the PPV live vaccine were immunized by intramuscular injection. Anti-PPV antibodies were measured by ELISA, lymphocyte proliferation activity was detected using MTT method, and the specific killing activities of CTL were assayed too. The results show that the immunized mice produced PPV antibody after one week, and reached to highest after four weeks. Compared with the control group, the pCIneo-IL2-VP2 immunized group produced significant differences in the antibody titers, the lymphocyte proliferation activity and the specific killing activities of CTL. The pCIneo-IL2-VP2 induced humoral and cellular immunity responses similarly to that the live vaccine induced. These results manifested that the PPV DNA vaccine successfully induced humoral and cellular immunity response in mice with the IL-2 gene as an adjuvant.

We studied the seroprevalence of six reproductive pathogens in Spanish hunter-harvested wild boar females. The sample was representative of the hunting harvest in the studied hunting estates. Mean antibody prevalences were: 60.6+/-0.06% for Aujeszky's disease virus (ADV), 56.6+/-0.09% for porcine parvovirus (PPV), 51.8+/-0.06% for porcine circovirus type 2 (PCV2), 29.7+/-0.09% for Brucella spp. and 36.3+/-0.1% for Toxoplasma gondii. We did not detect antibodies against porcine reproductive and respiratory syndrome virus (PRRSv). ADV seroprevalence was associated with PPV and PCV2 seroprevalence in Spanish wild boar females. Ovulation rate in the studied wild boar females was 4.41+/-0.16 (n=120), mean litter size was 3.91+/-0.16 (n=82) and the partial resorption index 0.92+/-0.17 (n=66). Ovulation rate and litter size were statistically associated with age. T. gondii seroprevalence was negatively related to ovulation rate and partial resorption index. Wild boars from managed fenced estates had antibodies against more pathogens than wild boars from open estates. Potential relations between management of wild boar populations and exposure of individuals to different reproductive pathogens are discussed.

A slide latex agglutination test (LAT) was developed and evaluated to detect serum antibodies against porcine parvovirus. Porcine parvovirus antigen was obtained by 10% PEG-6000 and 0.5 mol/l sodium chloride precipitation, and inactivated by 0.1% methanal. Two per cent suspensions of latex particles (0.5-0.8 microm) were coated by adding an equal volume of porcine parvovirus antigen at 0.34 microg/ml. Repeatability of latex agglutination test was evaluated with a panel of 100 sera using the same and different antigen lots. A good agreement between LAT and haemagglutination inhibit assay was observed. Because of convenience and speed of performance, this method would be used widely in clinic examination.

This study was conducted to determine the antibody response for porcine parvovirus (PPV) of 39 gilts in field conditions after vaccination. Gilts from four herds endemically infected with PPV were injected twice with a commercial vaccine of inactivated PPV and Erysipelothrix rhusiopathiae. The PPV antibodies were analysed both with haemagglutination inhibition (HI) and enzyme-linked immunosorbent assay (ELISA) in order to study the agreement between these methods. The possible association between high-antibody titres and reproductive failure (repeat breeding, culling for infertility, < or = 6 piglets born alive) was also investigated. In these study herds, endemically infected by PPV, most gilts (84.6%) had not seroconverted by the age of 6 months. On-field vaccination resulted in a consistent increase of humoral immunity not exceeding the antibody level of 1 : 512 in the majority of gilts in all herds examined. The agreement between ELISA and HI tests was moderate (Spearman's rho = 0.87, kappa = 0.63). The seroconversion over the level >1:512 by mid-pregnancy was not associated with reproductive failure.

To differentiate pigs infected with porcine parvovirus (PPV) from those vaccinated with inactivated whole-virus vaccine, an enzyme-linked immunosorbent assay (ELISA) based on detection of a nonstructural polyprotein 1 (NS1) was developed. A threshold of 0.23 optical density units was established and the assay had high specificity (100), sensitivity (88), accuracy (90) and positive predictive value (100) using haemagglutination inhibition as the standard method. A reproducibility test revealed that the coefficients of variation of sera within-plates and between-run were less than 10%. The assay showed no cross-reactivity with antibodies to porcine reproductive respiratory syndrome virus, pseudorabies virus, foot and mouth disease virus, Actinobacillus pleuropneumoniae, Toxoplasma or Chlamydia. Sera obtained from pigs infected with PPV reacted with recombinant NS1 protein in the ELISA. Sera from pigs vaccinated with inactivated whole virus did not recognize this protein in the ELISA. In contrast, antibodies against PPV whole virus were present in both PPV-infected and vaccinated animals. Serum conversion against NS1 was first detected 10 days after infection and NS1-specific antibodies were detectable up to half a year post infection. In conclusion, the PPV-NS1 ELISA can differentiate PPV-infected versus inactivated PPV-vaccinated pigs and could be applied in disease diagnosis and surveillance.

Porcine parvovirus (PPV) vaccines containing different adjuvants were evaluated for inducing Th1 or Th2 type of immunity in mice. Isotypes of antigen specific antibodies and levels of cytokines in serum and in lymphocyte culture supernatants measured by ELISA and the Gyrolab Bioaffy were used to determine the polarisation of the immune response. Enumeration of cytokine secreting cells was carried out by ELISPOT assays. Vaccines containing the ginseng-fraction Rb1 induced serum-detectable amounts of IL-4 and IL-10 as early as 24h after primary injection that was confirmed in sera collected at 24 and 72 h post re-vaccination. Five weeks after booster, immune lymphocytes were still producing large amounts of cytokines including IFN-gamma, IL-2, IL-4, IL-10 and TNF-alpha and the antibody titres were still similar to those titres recorded 1 week post booster. The Rb1 adjuvanted vaccines stimulated similar titres of antigen specific IgG1, IgG(2a) and IgG(2b). Thus, the cytokine and the serological data indicated that the Rb1 fraction of ginseng elicits a balanced Th1 and Th2 immune response.

Serologic evidence of exposure to porcine parvovirus (PPV) in the wild boar (Sus scrofa) in Croatia was investigated. Serum samples from 219 wild boars captured during 2003 from 12 different locations in the Republic of Croatia were tested by using a commercial enzyme-linked immunoassay (ELISA) and a hemagglutination inhibition (HI) test. Antibodies to PPV were detected in 91 (41.6%) of tested samples and positive results were detected in wild boar from all sample locations. Adults had a significantly higher prevalence (70%) than juveniles (31%; P < 0.01). Our results indicate that wild boar populations throughout the Republic of Croatia are exposed to PPV.

The aim of this study was to investigate the tissue distribution of two different field isolates and two vaccine strains of porcine parvoviruses (PPV) in infected piglets after transplacental infection. The viral load in 10 different foetal organs was determined by real-time polymerase chain reaction assays with SYBR Green targeting the viral VP2 gene and the genomic c-myc gene in 12 foetuses. The viral load in foetal tissues differed greatly among the different parvoviruses. Between one virulent field isolate compared with the other field isolate and the vaccine strains, the detected viral copy number differed in an order of magnitude of 10(9). The virulent isolate contained PPV in all 10 organs with viral loads varying between 10(11) and 10(15) per 10(6) cells. Concerning the other field isolate and the two vaccine strains, if PPV was detected, in most of the cases the highest viral load was found in foetal kidneys with a maximum viral load of 10(3) per 10(6) cells. Additionally, PPV was found in the heart of one foetus, in the liver and duodenum of one foetus and in the thymus of one foetus with viral loads varying between 10(2.1) and 10(3.5) per 10(6) cells. In completely mummified foetuses with no discriminable organs of foetuses infected with the vaccine strains and the less virulent isolate, PPV was present in very low amounts or even below the detection limit.

Three experiments were conducted to evaluate viral inactivation by the spray-drying process used in the production of spray-dried animal plasma (SDAP). In Exp. 1, bovine plasma was inoculated with pseudorabies virus (PRV) grown in PK 15 cells. Three 4-L batches were spray-dried in the same manner and conditions of industrial SDAP production but with laboratory spray-drying equipment. Presence of infectivity was determined before and after spray-drying by microtiter assay in PK 15 cell cultures. Before spray-drying, all three samples contained 10(5.3) tissue culture infectious dose50 (TCID50)/mL of PRV. After four consecutive passages, no viable virus was detected in samples of spray-dried bovine plasma. In Exp. 2, bovine plasma was inoculated with porcine respiratory and reproductive syndrome (PRRS) virus propagated previously in MARC cell culture to provide approximately 10(6.3) TCID50/mL. Three 4-L batches were spray-dried in the same manner as Exp. 1. Before spray-drying, samples contained TCID50 of 10(4.0), 10(3.5), and 10(3.5)/mL, respectively. After four consecutive passages in MARC cell cultures, no viable virus was detected in spray-dried bovine plasma. In Exp. 3, 36 weaned piglets (28 d of age) were fed a common diet for 14 d and were determined to be negative for PRV, PRRS, and porcine parvovirus titer. Afterwards, pigs were allotted to six pens with six pigs per pen and fed diets containing either 0 or 8% SDAP (as-fed basis) for 63 d. The SDAP used in the feed contained antibody (titer 1:400) against porcine parvovirus. Blood samples were collected from pigs on d 0 and 63 to determine whether feeding SDAP caused seroconversion and development of antibodies against parvovirus, PRRS, or PRV. Inclusion of SDAP in the diet improved growth of pigs without seroconversion. Spray-drying conditions used in this study were effective in eliminating viable pseudorabies and PRRS viruses from bovine plasma. In this study, feeding SDAP that contained functional antibodies did not promote seroconversion in naïve animals.

This report describes an experimental infection with porcine circovirus type 2 (PCV2) in combination with porcine parvovirus (PPV) in 3-week-old conventional colostrum-fed pigs with maternal antibodies to both viruses. Two groups of four pigs each were inoculated with PCV2 and PPV. One of the groups received also a commercial inactivated vaccine against porcine pleuropneumonia to evaluate possible effects of the stimulation of the immune system of pigs on the infection. Another group of four pigs was kept as uninfected control. Clinical signs, rectal temperatures and body weights were recorded. Serum antibody titers to PCV2 and PPV were determined at weekly intervals. Pigs were killed 42 days after inoculation and tissue samples were examined for the presence of gross and microscopic lesions. Tissues were also analyzed for the presence of PCV2 and PPV DNA by PCR, and for the presence of PCV2 antigen by immunohistochemistry (IHC). All the pigs had serum antibodies to PCV2 and PPV at the beginning of the trial. None of them developed clinical symptoms or pathological lesions typical of post-weaning multisystemic wasting syndrome (PMWS), a disease associated to PCV2 infection. However, IHC and/or PCR analyses showed that clinically silent PCV2 infection developed in five of the eight inoculated pigs, regardless of the administration of the vaccine. In particular, PCV2 DNA and/or antigen were detected in most of the tissues examined in the two pigs with the lowest titer of maternal PCV2 antibodies at the beginning of the trial. PPV DNA was not detected in any of the samples examined. The five pigs with PCR and/or IHC evidence of PCV2 infection had a mean weight gain during the experiment lower than that of the inoculated PCR-negative pigs considered together and that of the control pigs. In conclusion, it would appear that passive immunity against PCV2 can play a role in preventing the development of PMWS, but is not able to prevent the establishing of clinically silent PCV2 infections. The dissemination and persistence of the virus in the tissues may depend on the level of PCV2 antibodies at the time of inoculation.

An experimental model using 3-day-old snatch-farrowed colostrum-deprived piglets co-infected with porcine circovirus type 2 (PCV2) and porcine parvovirus (PPV) is at present one of the best methods to study factors affecting development of postweaning multisystemic wasting syndrome (PMWS). A Swedish isolate of PCV2 (S-PCV2) retrieved in 1993 from a healthy pig has been used in this model to reproduce PMWS in pigs from Northern Ireland. This virus has been present in the Swedish pig population for at least a decade without causing any known PMWS disease problems, despite its potential pathogenicity. The reasons for this are unknown, but could be related to genetics, absence of triggers for PCV2 upregulation (infectious agent and/or management forms) within Swedish pig husbandry. In order to confirm the pathogenicity of S-PCV2, Swedish and Danish pigs were experimentally infected with this isolate according to the established model. Swedish pigs were also infected with a reference isolate of PCV2 (PCV2-1010) to compare the severity of disease caused by the two isolates in Swedish pigs. Both Danish and Swedish pigs developed PMWS after the experimental infection with S-PCV2. Antibodies to PCV2 developed later and reached lower levels in serum from pigs infected with S-PCV2 than in pigs inoculated with PCV2-1010. In general, pigs infected with S-PCV2 showed more severe clinical signs of disease than pigs infected with PCV2-1010, but pigs from all PCV2-inoculated groups displayed gross and histological lesions consistent with PMWS. All pigs inoculated with PPV, alone or in combination with PCV2, displayed interleukin-10 responses in serum while only pigs infected with PPV in combination with PCV2 showed interferon-alpha in serum on repeated occasions. Thus, the pathogenicity of S-PCV2 was confirmed and a role for cytokines in the etiology of PMWS was indicated.

Controlling the cross-presentation of exogenous Ags to CD8+ T cells represents a major step for designing new vaccination strategies. Whereas several recombinant pseudo-viral particles have been used as delivery systems for triggering potent CTL responses to heterologous exogenous Ags, the adjuvant properties of virus-like particles (VLPs) themselves were little questioned. Here, we analyzed the contribution of the porcine parvovirus (PPV)-VLPs to the induction of protective cellular responses to exogenous Ags carried by an independent delivery system. Microspheres, which are known to transfer exogenous Ags into the MHC class I pathway, were chosen for delivering the immunodominant OVA(257-264) CD8+ T cell epitope (B-OVAp). This delivery system fulfills the requirements in terms of cross-presentation, but fails to induce cross-priming of specific CD8+ T cells. Coinjection of PPV-VLPs with B-OVAp results in the priming of potent CTL responses and type 1-biased immunity in a CD4- and CD40-independent manner, as efficiently as the recombinant PPV-VLPs carrying the same epitope (PPV-OVAp). Furthermore, vaccination with PPV-VLPs and B-OVAp was fully efficient to protect mice against the development of OVA-bearing melanoma. These findings indicate that PPV-VLPs act not only as a delivery system but also as a strong adjuvant when independently provided with exogenous Ag. Thus, dissociation between delivery system and adjuvant would provide a more flexible and reliable system to induce potent and protective CTL.

Porcine parvovirus (PPV) is widespread among swine. Our objective was to determine the prevalence of loosely housed sow herds in Finland with at least one animal with high (infection level) PPV antibodies and to gather basic knowledge about vaccination practices. In addition, selected factors associated with high antibody levels found in sows were examined. Altogether, 247 animals were sampled in 21 randomly chosen loosely housed sow herds. Samples were analysed with the haemagglutination inhibition (HI) test. PPV proved to be common; in 17 farms (81%) at least one animal had a high titre (>1 : 512), and 44% of all animals sampled had a high titre. The vaccination programmes had many shortcomings. In the generalised estimation equations (GEE) population-averaged model developed, the factors found to have a significant (p < or = 0.05) effect on HI titres were herd size, parity of two or greater and storage of the vaccine vial after use. Non-returning rate, re-breeding interval and litter size did not differ between herds with no high HI titres (n = 4) and those with at least one high HI titre (n = 17).

The authors prepared experimental lipoid adjuvant of the oil-in-water (O/W) type, based on squalene (ESA: experimental squalene adjuvant), intended for liquid inactivated veterinary vaccines. The final concentration of squalene in the vaccine was 5% (w/v). Detergents Poloxamer 105 in the concentration of 4% (w/v) and Abil-Care in the concentration of 2% were used as emulsifiers. The water component was inactivated virus suspension. Emulsification at decreased temperatures (up to 37 degrees C) fails to denaturate the immunization antigen. The effectiveness of experimental squalene adjuvant was tested using a liquid inactivated adjuvant rabies vaccine and a liquid porcine parvovirus vaccine in comparison with adjuvant-free vaccines or vaccines potentiated with aluminium hydroxide. The squalene adjuvant increased the immunogenic activity of non-potentiated rabies vaccine approximately 1.8-fold, while aluminium hydroxide increased the effectiveness of rabies vaccine only 1.08-fold. The antigenic activity, i.e. the level of specific antibodies after vaccination was significantly increased by using both adjuvants; ESA was more efficient than aluminium hydroxide.

The objectives of this study were to determine if coinfection of segregated early weaned (SEW) pigs with porcine circovirus type 2 (PCV2) and porcine parvovirus (PPV) induces an increase in the incidence of post-weaning multisystemic wasting syndrome (PMWS) compared to singular PCV2 infection, and to determine if vaccination against PPV protects pigs against PMWS associated with PCV2/PPV coinfection in SEW pigs. Seventy, 3-week-old, SEW pigs were randomly assigned to one of the five groups. Pigs in group 1 (n = 14) served as the negative controls, group 2 pigs (n = 14) were inoculated with PCV2, group 3 pigs (n = 12) were inoculated with PPV, groups 4 (n = 16) and 5 (n = 14) pigs were inoculated with both PCV2 and PPV. Pigs in groups 1-3 and 5 were vaccinated with two doses of a killed parvovirus-leptospira-erysipelothrix (PLE) vaccine prior to inoculation. The PCV2/PPV-coinfected pigs (groups 4 and 5) had significantly (P < 0.05) higher and more persistent fevers than the singular PCV2-infected pigs. One pig in each of the coinfected groups developed clinical disease (fever, respiratory disease, jaundice, weight loss) consistent with PMWS. Lymphoid depletion was significantly (P < 0.05) more severe in the dually-infected pigs at 42 days post-inoculation (DPI). Vaccinated, coinfected pigs (group 5) remained viremic significantly (P < 0.05) longer and had higher copy numbers of genomic PCV2 DNA in sera at 28, 35, and 42 DPI compared to the unvaccinated coinfected pigs (group 4). PPV-viremia was detected only in the unvaccinated group 4 pigs. PLE-vaccination prevented PPV-viremia but did not prevent clinical PMWS or reduce the severity of lymphoid depletion in PCV2/PPV-coinfected pigs. Evidence of increased incidence of clinical PMWS due to vaccination was not observed in this model.

Porcine circovirus type 2 (PCV2) is now recognised as the causal agent of porcine multisystemic wasting syndrome (PMWS), an economically important wasting disease of young pigs [J. Vet. Diagn. Invest. 12 (2000) 3]. Gross lesions of PMWS include generalised lymphadenopathy, hepatitis, nephritis and pneumonia and typical histological lesions include lymphocytic depletion and multinucleated giant cell formation in lymph nodes, degeneration and necrosis of hepatocytes, and multifocal lymphohistocytic interstitial pneumonia. This communication will review the results of experimental infections of gnotobiotic (GN), colostrum-deprived (CD) and colostrum-fed (CF) pigs within our group, and elsewhere, with PCV2 and the conclusions that can be drawn from this work.

Recombinant parvovirus-like particles (PPV-VLPs) are particulate exogenous Ags that induce strong CTL response in the absence of adjuvant. In the present report to decipher the mechanisms responsible for CTL activation by such exogenous Ag, we analyzed ex vivo and in vitro the mechanisms of capture and processing of PPV-VLPs by dendritic cells (DCs). In vivo, PPV-VLPs are very efficiently captured by CD8alpha- and CD8alpha+ DCs and then localize in late endosomes of DCs. Macropinocytosis and lipid rafts participate in PPV-VLPs capture. Processing of PPV-VLPs does not depend upon recycling of MHC class I molecules, but requires vacuolar acidification as well as proteasome activity, TAP translocation, and neosynthesis of MHC class I molecules. This study therefore shows that in vivo DCs can cross-present PPV-VLPs using an endosome-to-cytosol processing pathway.

A comparative serologic and virologic study was performed in pigs from 5 herds with postweaning multisystemic wasting syndrome (PMWS) and 2 herds without PMWS in Quebec. In each herd, 60 blood samples were collected at 4-wk intervals from pigs from 3 to 23 wk of age. The serum was evaluated for the presence of antibodies to porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV), as well as for the presence of nucleic acid of PCV2, PRRSV, an porcine parvovirus (PPV), by means of the polymerase chain reaction (PCR). Serologic profiles for PCV2 were very similar in 6 of the 7 herds, including the 2 without PMWS, and were characterized by a gradual decrease in antibody titres from 3 until 11 wk of age, followed by seroconversion at 15 wk, and high PCV2 antibody titres thereafter in all pigs. Only starting at 11 to 15 wk of age could PCV2 viremia be detected, except in 1 herd, in which clinical signs were observed at 6 to 7 wk of age. A PCV2 viremia could be detected within the same pigs for a minimum of 8 wk, and the virus could still be detected in 41% of the serum samples obtained at 23 wk of age. The antibody level did not appear to influence the occurrence of disease, since titres were similar in pigs in the herds with or without PMWS. Infection with PRRSV, as demonstrated by PCR and seroconversion, preceded that of PCV2 by at least 1 mo in both types of herd. Both PRRSV and PCV2 were detected in some pigs in 5 of the 7 herds, including 1 herd without PMWS. Porcine parvovirus could be detected in serum by PCR in 2 herds with PMWS after the onset of clinical signs and also in 1 herd without PMWS. Genomic analysis of PCV2 strains identified in the herds without PMWS indicated complete or very high homology (99.4% to 100%) with the PCV2 strains identified in 4 herds with PMWS. In our field study, the triggering of PMWS in the herds could not be linked to coinfection with either PRRSV or PPV or to the use of a specific immunostimulant, such as vaccines, or to particular genomic differences between the PCV2 strains identified.

The dry extract prepared from the Panax ginseng C.A. Meyer-root (total ginseng (T-ginseng)) contain ginsenosides (G-des) which were shown to have adjuvant properties as demonstrated by: (a) injecting guinea pigs with a mixture of T-ginseng and inactivated porcine parvovirus (PPV) as a conventional vaccine; (b)injecting PPV-antigen and T-ginseng simultaneously but separately at different sites on the animal and (c)injecting only the T-ginseng 1 or 2 weeks prior to immunisation with the PPV-antigen. Using a haemagglutination inhibition (HI) test in the antibody titration, it was found that the mean HI-titre for the animals injected with PPV-antigen only was 320 +/- 0. By comparison, the mean titre value was 2026 +/- 1206 for the sera from the animals injected with the same vaccine but adjuvanted with 4 mg of T-ginseng, while the antibody titre induced by a vaccine containing Al(OH)(3)-gel was 2986 +/- 1596. Interestingly, the T-ginseng and Al(OH)(3) acted synergistically and further improved the antibody response to the PPV-antigen to 6826 +/- 2413, i.e. more than 20 times the HI titre of the non-adjuvanted PPV-vaccine. Immunisations using PPV-vaccines adjuvanted with single purified G-des demonstrated that the ginseng fractions Rb1 and Rg1 are potent adjuvants inducing higher or similar antibody titres than the vaccine adjuvanted with Al(OH)(3), e.g. Rb1 tested at a concentration of 830 microg per dose induced a significantly (P = 0.009) higher antibody titre than the one adjuvanted with Al(OH)(3). Nevertheless, different than the mixture Al(OH)(3)-T-ginseng; Rb1 and Rg1 act antagonistically and partially inhibit each other. The G-des adjuvanted vaccines induced significantly (P = 0.0011) higher titres of IgG2 antibodies compared with IgG1., (Copyright 2002 Elsevier Science Ltd.)

Ginseng, the dry extract prepared from the Panax ginseng C.A. Mayer-root contain immunomodulators named ginsenosides, which in the pig enhance the antibody response to viral and bacterial antigens. The enhancing effect of ginseng was demonstrated vaccinating pigs against porcine parvovirus (PPV) and Erysipelothrix rhusiopathiae infections, using commercially available vaccines. The potency of the licensed, aluminium hydroxide adjuvanted; vaccines were compared with those supplemented with ginseng. The antibody response to PPV was measured by the haemagglutination inhibition (HI) test whereas the mouse potency test and ELISA evaluated the immune response to E. rhusiopathiae. Antibodies to the 64-66 kDa glycoprotein of the E. rhusiopathiae were demonstrated by immunoblotting. The qualitative antibody responses were evaluated by means of ELISA(s) using monoclonal antibodies to swine IgG1 and IgG2. The addition of 2mg ginseng per vaccine dose, potentiate the antibody response of the commercial vaccines without altering their safety. Significantly higher (P<0.001) antibody titres were achieved to both PPV and to E. rhusiopathiae by the supplementation with ginseng. Aluminium hydroxide adjuvanted vaccines favoured the production of IgG1 antibodies. Interestingly, the vaccines supplemented with ginseng favoured IgG2. The vaccines used in the evaluations varied in their immunogenic potency. However, after the addition of ginseng the less immunogenic vaccine proved to be as potent as the better one without ginseng. Thus, the use of ginseng as a co-adjuvant provides a simple, safe and cheap alternative for improving the potency of aluminium hydroxide adjuvanted vaccines.

The aim of the study was to investigate the effects of a number of environmental, behavioural and biological factors on passive immunization of piglets as assessed by transfer of porcine parvovirus (PPV) antibodies (ab) from the colostrum of PPV vaccinated mothers to the serum of the piglets. Twenty primiparous sows were housed in pens with peat, straw and branches for nest building. Half the sows were prevented from achieving feedback from a completed farrowing nest by repeated removal of the nest from 10 to 12 h after nest building had begun, whereas the other half kept their nests. Sow serum PPV-ab titres were positively related to colostrum PPV-ab titres at birth of the first piglet (BFP) (P < 0.001). Litter average piglet PPV-ab titre was positively related to both sow serum and colostrum PPV-ab titres (both P < 0.001). In addition, in the individual piglets. PPV-ab titres were reduced as time from BFP to birth and time from birth to first sucking increased and time spent sucking decreased (all P < 0.01). There were no effects of treatment, time spent in lateral recumbency by the sow, number of times the sow stood or piglet weight on day 1 on piglet serum PPV-ab titres. Preventing prolonged farrowing, while at the same time ensuring the piglets' access to the udder, is important for transfer of maternal immunity. Measurements of specific antibodies in sow serum during the periparturient period and in piglet serum at 28 days of age may provide a practical tool for evaluating transfer of maternal immunity from sow to piglets.

Porcine parvovirus (PPV) virus-like particles (VLPs) constitute a potential vaccine for prevention of parvovirus-induced reproductive failure in gilts. Here we report the development of a large scale (25 l) production process for PPV-VLPs with baculovirus-infected insect cells. A low multiplicity of infection (MOI) strategy was efficiently applied avoiding the use of an extra baculovirus expansion step. The optimal harvest time was defined at 120 h post-infection at the MOI used, with the cell concentration at infection being 1.5x10(6) cells/ml. An efficient purification scheme using centrifugation, precipitation and ultrafiltration/diafiltration as stepwise unit operations was developed. The global yield of the downstream process was 68%. Baculovirus inactivation with Triton X-100 was successfully integrated into the purification scheme without an increase in the number of process stages. Immunogenicity of the PPV-VLPs tested in guinea pigs was similar to highly purified reference material produced from cells cultured in the presence of serum-containing medium. These results indicate the feasibility of industrial scale production of PPV-VLPs in the baculovirus system, safety of the product, and the potency of the product for vaccine application.

Recombinant porcine parvovirus virus-like particles (PPV-VLPs) are particulate exogenous antigens that induce a strong, specific cytotoxic T lymphocyte (CTL) response in the absence of adjuvant. In the present report, we demonstrate in vivo that dendritic cells (DCs) present PPV-VLPs to CD8+ T cells after intracellular processing. PPV-VLPs are captured by DCs with a high efficacy, which results in the delivery of these exogenous antigens to 50% of the whole spleen DC population. In vivo, a few hours after injection, PPV-VLPs are presented exclusively to CD8+ T cells by CD8alpha- DCs, whereas 15 hours later they are presented mainly by CD8alpha+ DCs. After PPV-VLPs processing, a fraction of CD11b+ DCs undergo phenotypic changes, i.e., the up-regulation of CD8alpha and CD205 and the loss of CD4 molecules on their surface. The failure to detect mRNA coding for CD8alpha in CD11b+ DCs suggests that CD8alpha expression by these cells is not due to de novo synthesis. In recombination-activating gene knockout mice (Rag-/-), CD11b+ DCs did not express CD8alpha and PPV-VLPs presentation by CD8alpha+ DCs was severely diminished. These results indicate that both CD8alpha- and CD8alpha+ DCs play an important role in the induction of CTL responses by exogenous antigens, such as VLP.

Porcine parvovirus (PPV) is an ubiquitous pathogen causing reproductive failure in swine. Protection against reproductive failure caused by acute PPV infection has commonly been related to the presence of specific antibodies in the dam. However, the role of cell-mediated immunity during chronic PPV infection remains to be elucidated, and may be relevant to the pathogenesis of novel diseases such as postweaning multisystemic wasting syndrome (PMWS), which may be triggered by coinfection with PPV and porcine circovirus type 2 (PCV2). To investigate whether pigs infected with PPV generate a cell-mediated immune response, a longitudinal infection experiment was performed, using swine leukocyte antigens (SLA) class I characterized growing pigs (haplotype H7/H7). Pigs were intranasally inoculated with PPV at 0, 80, and 136 days. At predetermined time points, peripheral blood mononuclear cells (PBMC) were isolated, and virus-specific lymphoproliferative responses and the cytolytic activities of cytotoxic T-lymphocytes (CTL) and natural killer (NK) cells were examined. Cytolytic assays were performed by the chromium release method, using as targets a syngeneic porcine kidney cell line established for the purpose (CTL assays) and K562 cells (NK assays). A specific proliferative response of PBMC from virus-infected pigs to PPV was observed from day 101 onwards. In contrast, PBMC from mock-infected pigs did not proliferate in response to PPV. Flow cytometric analysis indicated that the CD4+CD8+ T-cell subset of PBMC proliferated in response to virus antigen, in keeping with the assumed role for these cells in immunological memory. This is, to our knowledge, the first indication of a cellular immune response following PPV infection. A weak CTL activity, which peaked on days 80 and 87, was observed in PPV-infected pigs. In vitro restimulation of PBMC with live PPV did not induce further CTL activity. A pronounced NK cell activity was detected in both virus-infected and control pigs throughout the experiment, and may have negatively affected the sensitivity of the CTL assay. In conclusion, the findings of a late lymphoproliferative response together with weak CTL activity are in keeping with an effective control of acute PPV infection by humoral immunity, but open the possibility that cellular immunity may play a role in controlling PPV reinfection. Finally, we find that the established experimental model using SLA characterized pigs may constitute a valuable tool for future studies of CTL activity in pigs.