Enhancement of protective immune responses by oral vaccination with Saccharomyces cerevisiae expressing recombinant Actinobacillus pleuropneumoniae ApxIA or ApxIIA in mice

We previously induced protective immune response by oral immunization with yeast expressing the ApxIIA antigen. The ApxI antigen is also an important factor in the protection against Actinobacillus pleuropneumoniae serotype 5 infection; therefore, the protective immunity in mice following oral immunization with Saccharomyces cerevisiae expressing either ApxIA (group C) or ApxIIA (group D) alone or both (group E) was compared with that in two control groups (group A and B). The immunogenicity of the rApxIA antigen derived from the yeast was confirmed by a high survival rate and an ApxIA-specific IgG antibody response (p < 0.01). The highest systemic (IgG) and local (IgA) humoral immune responses to ApxIA and ApxIIA were detected in group E after the third immunization (p < 0.05). The levels of IL-1β and IL-6 after challenge with an A. pleuropneumoniae field isolate did not change significantly in the vaccinated groups. The level of TNF-α increased in a time-dependent manner in group E but was not significantly different after the challenge. After the challenge, the mice in group E had a significantly lower infectious burden and a higher level of protection than the mice in the other groups (p < 0.05). The survival rate in each group was closely correlated to the immune response and histopathological observations in the lung following the challenge. These results suggested that immunity to the ApxIA antigen is required for optimal protection.     

Porcine CD18 mediates Actinobacillus pleuropneumoniae ApxIII species-specific toxicity

Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, produces Apx toxins that are recognized as major virulence factors. Recently, we showed that ApxIIIA-cytotoxic activity specifically targets Sus scrofa leukocytes. Since both LtxA from Aggregatibacter actinomycetemcomitans (aggressive periodontitis in humans) and LktA from Mannheimia haemolytica (pneumonia in ruminants) share this characteristic, respectively towards human and ruminant leukocytes, and because both use the CD18 subunit to interact with their respective LFA-1, we hypothesized that ApxIIIA was likely to bind porcine CD18 to exercise its deleterious effects on pig leukocytes. A β ₂−integrin-deficient ApxIIIA-resistant human erythroleukemic cell line was transfected either with homologous or heterologous CD11a/CD18 heterodimers using a set of plasmids coding for human (ApxIIIA-resistant), bovine (-resistant) and porcine (-susceptible) CD11a and CD18 subunits. Cell preparations that switched from ApxIIIA-resistance to -susceptibility were then sought to identify the LFA-1 subunit involved. The results showed that the ApxIIIA-resistant recipient cell line was rendered susceptible only if the CD18 partner within the LFA-1 heterodimer was that of the pig. It is concluded that porcine CD18 is necessary to mediate A. pleuropneumoniae ApxIIIA toxin-induced leukolysis.     

The genetic organization of the capsular polysaccharide biosynthesis region of Actinobacillus pleuropneumoniae serotype 15

Nucleotide sequence determination and analysis of the cps gene involved in the capsular polysaccharide biosynthesis of Actinobacillus pleuropneumoniae serotype 15 revealed the presence of three open reading frames, designated as cps15ABC genes. At the protein level, Cps15A and Cps15B showed considerably high homology to CpsA (67.0 to 68.7%) and CpsB (31.7 to 36.8%), respectively, of A. pleuropneumoniae serotypes 1, 4 and 12, revealing the common genetic organization of the cps among serotypes 1, 4, 12 and 15. However, Cps15C showed no homology to any proteins of A. pleuropneumoniae serotypes, indicating that cps15C may be specific to serotype 15. This study will provide the basic molecular knowledge necessary for the development of diagnostics and a vaccine for A. pleuropneumoniae serotype 15.     

The genetic organization of the capsular polysaccharide biosynthesis region of Actinobacillus pleuropneumoniae serotype 14

The genetic organization of the gene involved in the capsular polysaccharide (CPS) biosynthesis of Actinobacillus pleuropneumoniae serotype 14 has been determined. The DNA region for the CPS biosynthesis of serotype 14 (cps14) comprised 9 open reading frames, designated as cps14AB₁B₂B₃CDEFG genes, encoding Cps14A to Cps14G protein, respectively. Cps14A was similar to CpsA of A. pleuropneumoniae serotypes 1, 4 and 12; the Cps14B₁ and Cps14B₂ were similar to CpsB of A. pleuropneumoniae serotypes 1, 4 and 12, suggesting that CPS structure of A. pleuropneumoniae serotype 14 would belong to Group I including A. pleuropneumoniae serotypes 1, 4, 12 and 15. Surprisingly, the overall nucleotide sequence, deduced amino acid sequence, and the genetic organization of the cps14 were nearly identical to those of Actinobacillus suis. This study will provide the molecular basic knowledge for development of diagnostics and vaccine of A. pleuropneumoniae serotype 14.     

Elucidating the role of ApxI in hemolysis and cellular damage by using a novel apxIA mutant of Actinobacillus pleuropneumoniae serotype 10

Exotoxins produced by Actinobacillus (A.) pleuropneumoniae (Apx) play major roles in the pathogenesis of pleuropneumonia in swine. This study investigated the role of ApxI in hemolysis and cellular damage using a novel apxIA mutant, ApxIA336, which was developed from the parental strain A. pleuropneumoniae serotype 10 that produces only ApxI in vitro. The genotype of ApxIA336 was confirmed by PCR, Southern blotting, and gene sequencing. Exotoxin preparation derived from ApxIA336 was analyzed for its bioactivity towards porcine erythrocytes and alveolar macrophages. Analysis results indicated that ApxIA336 contained a kanamycin-resistant cassette inserted immediately after 1005 bp of the apxIA gene. Phenotype analysis of ApxIA336 revealed no difference in the growth rate as compared to the parental strain. Meanwhile, ApxI production was abolished in the bacterial culture supernatant, i.e. exotoxin preparation. The inability of ApxIA336 to produce ApxI corresponded to the loss of hemolytic and cytotoxic bioactivity in exotoxin preparation, as demonstrated by hemolysis, lactate dehydrogenase release, mitochondrial activity, and apoptosis assays. Additionally, the virulence of ApxIA336 appeared to be attenuated by 15-fold in BALB/c mice. Collectively, ApxI, but not other components in the exotoxin preparation of A. pleuropneumoniae serotype 10, was responsible for the hemolytic and cytotoxic effects on porcine erythrocytes and alveolar macrophages.     

Immunological study of an attenuated Salmonella Typhimurium expressing ApxIA,ApxIIA, ApxIIIA and OmpA of Actinobacillus pleuropneumoniae in a mouse model

Salmonella Typhimurium strain expressing the Actinobacillus pleuropneumoniae antigens, ApxIA, ApxIIA, ApxIIIA and OmpA, was previously constructed as a vaccine candidate for porcine pleuropneumonia. This strain was a live attenuated (∆lon∆cpxR∆asd)Salmonella as a delivery host and contained a vector containing asd. An immunological study of lymphocyte proliferation, T-lymphocyte subsets and cytokines in the splenocytes of a mouse model was carried out after stimulation with the candidate Salmonella Typhimurium by intranasal inoculation. The splenic lymphocyte proliferation and the levels of IL-4, IL-6 and IL-12 of the inoculated mice were significantly increased, and the T- and B-cell populations were also elevated. Collectively, the candidate may efficiently induce the Th1- and Th2-type immune responses.     

Evaluation of a single dose versus a divided dose regimen of amoxycillin in treatment of Actinobacillus pleuropneumoniae infection in pigs

The theory of a time-dependent effect of amoxycillin was examined in a model of porcine Actinobacillus pleuropneumoniae (Ap)-infection using clinically relevant dosage regimens. Twenty hours after infection of fourteen pigs, when clinical signs of pneumonia were present, one group of pigs received a single dose of amoxycillin (20 mg/kg, i.m.), whereas another group received four doses of 5 mg/kg injected at 8-h intervals. A similar AUC of the plasma amoxycillin concentration versus time curve was obtained in the two groups, whereas the maximum concentration was threefold higher using the single high dose. Plasma amoxycillin was above the MIC for twice as long using the fractionated dosage scheme. The condition of the animals was evaluated by clinical and haematological observations combined with quantification of biochemical infection markers: C-reactive protein, zinc and ascorbic acid. Within 48 h of treatment, the pigs in both treatment groups recovered clinically. No significant differences in the time-course of clinical observations or plasma concentrations of the biomarkers of infection were observed between the two treatments. In conclusion, the efficacy of these two dosage regimens of amoxycillin was not significantly different in treatment of acute Ap-infection in pigs.     

Porcine mononuclear phagocyte subpopulations in the lung,blood and bone marrow: dynamics during inflammation induced by Actinobacillus pleuropneumoniae

Mononuclear phagocytes (MP) are cells of nonspecific immunity, playing an essential role in defense against bacterial pathogens. Although various MP subpopulations have been described in the pig, relations among these populations in vivo are unknown to date. The present study was aimed at describing porcine MP subpopulations infiltrating inflamed tissue of pigs under in vivo conditions. Actinobacillus pleuropneumoniae (APP) infection was used to induce an inflammatory response. CD172α, CD14, CD163, MHCII and CD203α cell surface molecules were used to identify MP by flow cytometry. Changes in MP subpopulations in the peripheral blood (PB) and bone marrow (BM) compartments along with the analysis of MP appearing in the inflamed lungs were assessed to elucidate the possible origin and maturation stages of the infiltrating MP. The MP population migrating to the inflamed lungs was phenotype CD14⁺ CD163⁺ CD203α^+/− MHCII^+/−. Concomitantly, after APP infection there was an increase in the PB MP CD14⁺ CD163⁺ CD203α⁻ MHC II⁻ population, suggesting that these cells give rise to inflammatory monocytes/macrophages. The CD203α and MHCII molecules appear on these cells after leaving the PB. In healthy animals, the BM MP precursors were represented by CD14⁻ CD163⁻ cells maturing directly into CD14⁺ CD163⁻ that were then released into the PB. After infection, an altered maturation pathway of MP precursors appeared, represented by CD14⁻ CD163⁻ CD203α⁻ MHCII⁻ MP directly switching into CD14⁺ CD163⁺ CD203α⁻ MHCII⁻ MP. In conclusion, two different MP maturation pathways were suggested in pigs. The use of these pathways differs under inflammatory and noninflammatory conditions.     

Evaluation of a single dose versus a divided dose regimen of danofloxacin in treatment of Actinobacillus pleuropneumoniae infection in pigs

A single versus a divided dose regimen of danofloxacin was evaluated in treatment of porcine Actinobacillus pleuropneumoniae infection using clinical observations combined with biochemical infection markers: C-reactive protein, zinc and ascorbic acid. Twenty hours after experimental infection, the 18 pigs received danofloxacin intravenously as a single dose of 2.5mg/kg or four doses of 0.6 mg/kg administered at 24h intervals. These dosage regimens resulted in similar AUCs of the plasma danofloxacin vs time curve. The maximum concentration was 3.5-fold higher using the single dose regimen, while the time with concentrations above the MIC was 2.5-fold longer using the fractionated regimen. Using the single dose regimen, temperature was normalised 32 h post-infection. In contrast, normalisation was delayed until 44 h post-infection using four low doses and a relapse with elevated temperatures at 52 and 68 h was observed. No other significant differences between the treatments were found, neither regarding clinical, haematological nor biochemical observations. The use of the more convenient single dose regimen was appropriate, as it was at least equivalent to the fractionated regimen.     

Evaluating the immune responses of mice to subcutaneous immunization with Helicobacter pylori urease B subunit

Background

Helicobacter pylori, a gram-negative bacterial pathogen that expresses a strong urease activity, is associated with the development of gastroduodenal disease. Urease B subunit, one of the two structural subunits of urease, was expressed in E. coli BL21 (DE3) strain. The objective of this study was to evaluate the effects of Helicobacter pylori urease B subunit on the immune responses in mice by subcutaneous immunization.

Methods

The mice were immunized and boosted with Helicobacter pylori urease B subunit antigen subcutaneously three times with 2-wk intervals between the immunizations and boosters. The mice in the control group were immunized with PBS. The adjuvant group received PBS containing complete/incomplete freund’s adjuvant identical to antigen group without Helicobacter pylori urease B subunit antigen. Four weeks after the final booster, all the mice were sacrificed. Blood was collected on d 0, 14, 28 and 56 before immunization, booster and sacrifice, respectively. Immediately after sacrifice, gastric liquid and spleen were collected for antibody and cytokine analyses.

Results

Urease B subunit increased the concentrations of serum and gastric anti-urease B antigen specific IgG, and the levels of interleukin-4 and interferon-γ in splenocytes of the mice (P < 0.05).

Conclusions

This study demonstrated that recombinant urease B subunit can induce systemic and local immune responses in mice by subcutaneous immunization, which might be used as the effective component of vaccine against Helicobacter pylori.     

Putative biomarkers for evaluating antibiotic treatment: an experimental model of porcine Actinobacillus pleuropneumoniae infection

Biomarkers of infection were screened for their possible role as evaluators of antibiotic treatment in an aerosol infection model of porcine pneumonia caused by Actinobacillus pleuropneumoniae (Ap). Following infection of 12 pigs, clinical signs of pneumonia developed within 20 h, whereafter the animals received a single dose of either danofloxacin (2.5mg/kg) or tiamulin (10 mg/kg). To test the discriminative properties of the biomarkers, the dosage regimens were designed with an expected difference in therapeutic efficacy in favour of danofloxacin. Accordingly, the danofloxacin-treated pigs recovered clinically within 24h after treatment, whereas tiamulin-treated animals remained clinically ill until the end of the study, 48 h after treatment. A similar picture was seen for the biomarkers of infection. During the infection period, plasma C-reactive protein (CRP), interleukin-6 and haptoglobin increased, whereas plasma zinc, ascorbic acid and alpha-tocopherol decreased. In the danofloxacin-treated animals, CRP, interleukin-6, zinc, ascorbic acid and alpha-tocopherol reverted significantly towards normalisation within 24h of treatment. In contrast, signs of normalisation were absent (CRP, zinc and ascorbic acid) or less marked (interleukin-6 and alpha-tocopherol) in the tiamulin-treated animals. Plasma haptoglobin remained elevated throughout the study in both groups. This indicates that CRP, zinc, ascorbic acid and to a lesser extent interleukin-6 and alpha-tocopherol might be used to evaluate antibiotic treatment of acute Ap-infection in pigs. The present model provides a valuable tool in the evaluation of antibiotic treatments, offering the advantage of clinical and pathological examinations combined with the use of biochemical infection markers.     

Evaluation of glycoproteins purified from adult and larval camel ticks (Hyalomma dromedarii) as a candidate vaccine

In order to identify antigens that can help prevent camel tick infestations, three major glycoproteins (GLPs) about 97, 66 and 40 kDa in size were purified from adult and larval Egyptian ticks, Hyalomma (H.) dromedarii, using a single-step purification method with Con-A sepharose. The purified GLPs were evaluated as vaccines against camel tick infestation in rabbits. The rabbits received three intramuscular inoculations of GLPs (20 µg/animal) on days 0, 14, and 28. In the immunoblot analysis, Sera from the immunized rabbits recognized the native GLPs and other proteins from larval and adult H. dromedarii ticks along with those from other tick species such as Rhipicephalus sanguineus but not Ornithodoros moubata. The effects of immunity induced by these GLPs were determined by exposing rabbits to adult H. dromedarii ticks. These results demonstrated that GLP immunization led to a slightly decreased reproductive index and significantly reduced rates of egg hatchability. These results demonstrated that immunization with the purified GLPs can provide protection against infestation by H. dromedarii and some other tick species. Further studies are needed to confirm the effectiveness of immunization with GLPs against other tick species.     

A proteomic reference map for pig serum proteins as a prerequisite for diagnostic applications

A reference protein map for pig serum was set up using two-dimensional electrophoresis (2-DE). Thirty-nine protein chains or spots deriving from 26 different proteins were identified by immunological and mass spectrometric methods. Thus, the positions of most medium to higher abundance serum proteins could be determined on the 2-DE gels. The plasma protein fibrinogen was also included in our study. The overall pig protein pattern differs in some respect to serum/plasma maps of other mammalian species, e.g. in levels and properties of single proteins such as haptoglobin or IgM or in species-specific proteins like pig major acute phase protein. Serum protein maps are a useful tool to get an overview on expressed proteins, and to monitor changes in concentration as well as isotype distribution of the identified proteins. As a consequence, more detailed knowledge on protein pattern changes may give deeper insights into the metabolic development of some pathologic conditions and may lead to putative biomarkers for further investigation. Selected examples for protein pattern changes in pigs infected by a viral (porcine circovirus type 2) and a bacterial (Actinobacillus pleuropneumoniae) pathogen illustrate the usefulness of the method.     

The role of rpoS,hmp, and ssrAB in Salmonella enterica Gallinarum and evaluation of a triple-deletion mutant as a live vaccine candidate in Lohmann layer chickens

Salmonella enterica Gallinarum (SG) causes fowl typhoid (FT), a septicemic disease in avian species. We constructed deletion mutants lacking the stress sigma factor RpoS, the nitric oxide (NO)-detoxifying flavohemoglobin Hmp, and the SsrA/SsrB regulator to confirm the functions of these factors in SG. All gene products were fully functional in wild-type (WT) SG whereas mutants harboring single mutations or a combination of rpoS, hmp, and ssrAB mutations showed hypersusceptibility to H₂O₂, loss of NO metabolism, and absence of Salmonella pathogenicity island (SPI)-2 expression, respectively. A triple-deletion mutant, SGΔ3 (SGΔrpoSΔhmpΔssrAB), was evaluated for attenuated virulence and protection efficacy in two-week-old Lohmann layer chickens. The SGΔ3 mutant did not cause any mortality after inoculation with either 1 × 10⁶ or 1 × 10⁸ colony-forming units (CFUs) of bacteria. Significantly lower numbers of salmonellae were recovered from the liver and spleen of chickens inoculated with the SGΔ3 mutant compared to chickens inoculated with WT SG. Vaccination with the SGΔ3 mutant conferred complete protection against challenge with virulent SG on the chickens comparable to the group vaccinated with a conventional vaccine strain, SG9R. Overall, these results indicate that SGΔ3 could be a promising candidate for a live Salmonella vaccine against FT.     

Protection of chickens from Newcastle disease with a recombinant baculovirus subunit vaccine expressing the fusion and hemagglutinin-neuraminidase proteins

Recombinant baculoviruses containing the fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein gene of the viscerotropic velogenic (vv) Newcastle disease virus (NDV) isolate, Kr-005/00, and a lentogenic La Sota strain of the NDV were constructed in an attempt to develop an effective subunit vaccine to the recent epizootic vvNDV. The level of protection was determined by evaluating the clinical signs, mortality, and virus shedding from the oropharynx and cloaca of chickens after a challenge with vvNDV Kr-005/00. The recombinant ND F (rND F) and recombinant HN (rND HN) glycoproteins derived from the velogenic strain provided good protection against the clinical signs and mortality, showing a 0.00 PI value and 100% protection after a booster immunization. On the other hand, the combined rND F + HN glycoprotein derived from the velogenic strain induced complete protection (0.00 PI value and 100% protection) and significantly reduced the amount of virus shedding even after a single immunization. The rND F and rND HN glycoproteins derived from the velogenic strain had a slightly, but not significantly, greater protective effect than the lentogenic strain. These results suggest that the combined rND F + HN glycoprotein derived from vvNDV can be an ideal subunit marker vaccine candidate in chickens in a future ND eradication program.     

Potent immune responses induced by a Salmonella ghost delivery system that expresses the recombinant Stx2eB,FedF, and FedA proteins of the Escherichia coli-producing F18 and Shiga toxin in a murine model and evaluation of its protective effect as a porcine vaccine candidate

Background: In the pathogenicity of porcine edema disease (ED), which is caused by the Escherichia coli-producing F18 and Shiga toxin, F18⁺ fimbrial adhesins and Shiga toxin 2e (Stx2e) play pivotal roles in the colonization and enterotoxicity of this pathogen.

Objective: To develop a vaccine candidate against ED by combining three selected antigens of F18⁺ E. coli.

Methods: Genetically engineered Salmonella Typhimurium (ST) ghosts that express Stx2eB, FedF, and FedA were individually inserted in a ghost plasmid cassette, and the resultant plasmids were transformed into an attenuated ST (JOL912). The individual expression of Stx2eB, FedF, and FedA in JOL912 was validated by using an immunoblotting assay.

Results: Immunization of the ghosts in BALB/c mice led to a significant increase in antigen-specific secretory IgA and serum IgG. Significantly marked elevation of the CD3⁺CD4⁺ T cell subpopulation and lymphocyte proliferating activity in the primed splenocytes were also observed. Furthermore, mRNA of IL-4 and IFN-γ were highly upregulated in in vitro stimulated splenic T cells. Subsequently, the immunized mice showed significant protection efficacy against a lethal dose 50 of a virulent strain, resulting in approximately 85% and 92% survival rates in mice with a single- and double-dose immunization, respectively, compared to only 40% of the non-immunized controls.

Conclusion: A mixture of the ghosts expressing these three antigens is a potential vaccine candidate for protection against the porcine edema disease.     

Comparison of the immune responses induced by oral immunization of mice with Lactobacillus casei-expressing porcine parvovirus VP2 and VP2 fused to Escherichia coli heat-labile enterotoxin B subunit protein

The major structural protein VP2 of porcine parvovirus (PPV) was used as the model parvovirus antigen, which has been expressed in Lactobacillus casei fusing with Escherichia coli heat-labile enterotoxin B subunit (LTB) as mucosal adjuvant. The VP2-LTB DNA fragment was cloned into vector pPG611 or pPG612 to generated inducible surface-displayed and secretion expression systems based on xylose promoter, designated as rLc:pPG611-VP2-LTB (recombinant L. casei) and rLc:pPG612-VP2-LTB, respectively. Expression of the fusion protein was verified by SDS-PAGE, Western blot immunofluorescence and electron microscopy. It was observed that the level of IgG or sIgA from mice orally immunized with VP2-LTB was higher than that from mice received VP2 and negative control, which demonstrated significantly statistically different. Especially, the titer of IgG or sIgA in mice immunized with rLc:pPG612-VP2-LTB is the highest in this study. In summary, LTB as mucosal adjuvant was able to effectively facilitate induction of mucosal and systemic immunity by L. casei-expressing VP2 fusion protein.