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.     

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.     

Optimization of immune strategy for a construct of Salmonella-delivered ApxIA,ApxIIA, ApxIIIA and OmpA antigens of Actinobacillus pleuropneumoniae for prevention of porcine pleuropneumonia using a murine model

In this study, the Actinobacillus pleuropneumoniae antigens ApxIA, ApxIIA, ApxIIIA and OmpA were expressed in an attenuated strain of Salmonella (?lon?cpxR?asd) for prevention of porcine pleuropneumonia. In order to evaluate the immunization strategy of the construct, a total 60 BALB/c mice were equally divided into four groups (n?=?15). Group A mice were intranasally immunized only at 6-weeks-of-age, while group B mice were intransally primed and boosted at 6- and 9-weeks-of-age, respectively, and group C mice were intransally primed at 6-weeks-of-age and subsequently boosted twice at 9- and 12-weeks-of-age. Group D mice were used as a control, which were inoculated with sterile PBS. Groups A, B, and C showed significantly higher serum IgG and fecal IgA immune responses than those of the control group. After virulent challenge with a wild type A. pleuropneumoniae, the immunized groups A, B and C showed 33.3 %, 13.3 % and 26.7 % mortality as the control group showed 60 % mortality. These results showed that the protection against porcine pleuropneumonia using the construct can be optimized by a double intranasal vaccination.     

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.     

Generation of transgenic corn-derived Actinobacillus pleuropneumoniae ApxIIA fused with the cholera toxin B subunit as a vaccine candidate

Corn, one of the most important forage crops worldwide, has proven to be a useful expression vehicle due to the availability of established transformation procedures for this well-studied plant. The exotoxin Apx, a major virulence factor, is recognized as a common antigen of Actinobacillus (A.) pleuropneumoniae, the causative agent of porcine pleuropneumonia. In this study, a cholera toxin B (CTB)-ApxIIA#5 fusion protein and full-size ApxIIA expressed in corn seed, as a subunit vaccine candidate, were observed to induce Apx-specific immune responses in mice. These results suggest that transgenic corn-derived ApxIIA and CTB-ApxIIA#5 proteins are potential vaccine candidates against A. pleuropneumoniae infection.     

Nasal immunization with major epitope-containing ApxIIA toxin fragment induces protective immunity against challenge infection with Actinobacillus pleuropneumoniae in a murine model

Actinobacillus pleuropneumoniae is an infective agent that leads to porcine pleuropneumonia, a disease that causes severe economic losses in the swine industry. Based on the fact that the respiratory tract is the primary site for bacterial infection, it has been suggested that bacterial exclusion in the respiratory tract through mucosal immune induction is the most effective disease prevention strategy. ApxIIA is a vaccine candidate against A. pleuropneumoniae infection, and fragment #5 (aa. 439–801) of ApxIIA contains the major epitopes for effective vaccination. In this study, we used mice to verify the efficacy of intranasal immunization with fragment #5 in the induction of protective immunity against nasal challenge with A. pleuropneumoniae and compared its efficacy with that of subcutaneous immunization. Intranasal immunization of the fragment induced significantly higher systemic and mucosal immune responses measured at the levels of antigen-specific antibodies, cytokine-secreting cells after antigen exposure, and antigen-specific lymphocyte proliferation. Intranasal immunization not only efficiently inhibited the bacterial colonization in respiratory organs, but also prevented alveolar tissue damage in infectious condition similar to that of a contaminated pig. Moreover, intranasal immunization with fragment #5 provided acquired protective immunity against intranasal challenge with A. pleuropneumoniae serotype 2. In addition, it conferred cross-protection against serotype 5, a heterologous pathogen that causes severe disease by ApxI and ApxII secretion. Collectively, intranasal immunization with fragment #5 of ApxIIA can be considered an efficient protective immunization procedure against A. pleuropneumoniae infection.     

Vaccination Against Pleuropneumonia of Pigs Caused by Haemophilus pleuropneumoniae

A strain of Haemophilus pleuropneumoniae was isolated from a pig with pleuropneumonia from a herd where this condition was frequent. A formalin inactivated culture of this isolate was used as antigen in two vaccine preparations: A and B. Vaccine A had peanut oil + arlacel 80 + tween 80 as adjuvant and vaccine B had aluminum hydroxide gel as adjuvant. Twenty pigs were vaccinated twice with vaccine A and 19 with vaccine B. Twenty additional pigs were not vaccinated. All pigs were transferred to the herd. Eleven pigs in the nonvaccinated group developed pneumonia and seven of these died within eight days after exposure. None of the vaccinated pigs had signs of pneumonia. It is concluded that the vaccines prevented the acute form of pleuropneumonia due to H. pleuropneumoniae.     

Detection of Actinobacillus pleuropneumoniae Infection in Pigs

It is difficult to control the spread of porcine haemophilus pleuropneumonia caused by Actinobacillus pleuropneumoniae because there is no sensitive and specific way to accurately determine whether or not a pig herd is infected. This paper reports bacteriological and serological techniques used to detect A. pleuropneumoniae infection in pigs from a herd with endemic disease.

The bacteria were isolated from the anterior nasal mucosa of grower pigs, but not from younger or older pigs. Bacteriological culture of several tissues from the respiratory tract showed that nine of ten young finishing pigs were infected, but culture of lung tissue from slaughtered hogs detected infection in only 39 of 288 (13.5%). Both cooler storage temperature and use of selective medium prolonged the time that lung tissue could be stored and the organism still recovered. An enzyme-linked immunosorbent assay detected serotype-specific antibodies in serum of infected pigs.

     

Type IV fimbrial subunit protein ApfA contributes to protection against porcine pleuropneumonia

ABSTRACT: Porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae accounts for serious economic losses in the pig farming industry worldwide. We examined here the immunogenicity and protective efficacy of the recombinant type IV fimbrial subunit protein ApfA as a single antigen vaccine against pleuropneumonia, or as a component of a multi-antigen preparation comprising five other recombinant antigens derived from key virulence factors of A. pleuropneumoniae (ApxIA, ApxIIA, ApxIIIA, ApxIVA and TbpB). Immunization of pigs with recombinant ApfA alone induced high levels of specific serum antibodies and provided partial protection against challenge with the heterologous A. pleuropneumoniae serotype 9 strain. This protection was higher than that engendered by vaccination with rApxIVA or rTbpB alone and similar to that observed after immunization with the tri-antigen combination of rApxIA, rApxIIA and rApxIIIA. In addition, rApfA improved the vaccination potential of the penta-antigen mixture of rApxIA, rApxIIA, rApxIIIA, rApxIVA and rTbpB proteins, where the hexa-antigen vaccine containing rApfA conferred a high level of protection on pigs against the disease. Moreover, when rApfA was used for vaccination alone or in combination with other antigens, such immunization reduced the number of pigs colonized with the challenge strain. These results indicate that ApfA could be a valuable component of an efficient subunit vaccine for the prevention of porcine pleuropneumonia.     

Evaluation of a Killed Vaccine Against Porcine Pleuropneumonia Due to Haemophilus pleuropneumoniae

A bacterin containing serotypes 1 and 5 of Haemophilus pleuropneumoniae was developed for the prevention and the control of porcine pleuropneumonia. It was injected intramuscularly into three groups of ten piglets, the first group with one dose, the second one with two doses and the third one with three doses at two-week intervals. Another group of ten piglets did not receive the vaccine. All the piglets were then challenged by an aerosol of mixed suspensions of H. pleuropneumoniae serotypes 1 and 5. Two and three injections of vaccine completely prevented mortality, whereas half of the control piglets and of those receiving only one dose of vaccine died. All surviving piglets, both control and vaccinated, had severe signs of respiratory disease for at least 36 hours after exposure to challenge. Moreover, vaccination did not induce the production of antibodies at high titers. Local reactions were not noted after vaccination and at postmortem; ten weeks after the challenge, there were no signs of abscess formation or induration.     

Systemic and mucosal immunity induced by attenuated Salmonella enterica serovar Typhimurium expressing ORF7 of porcine reproductive and respiratory syndrome virus

Oral administration of attenuated Salmonella vaccine may provide valuable advantages such as low cost, easy preparation, and safety. Attenuated Salmonella vaccines also serve as carriers of foreign antigens and immunomodulatory cytokines. Presently, an attenuated Salmonella enterica serovar Typhimurium strain was used as a carrier for open reading frame 7 (ORF7) protein of porcine reproductive and respiratory syndrome virus (PRRSV), a swine pathogen of significant global economic importance. Initially, an attenuated S. enterica serovar Typhimurium expressing ORF7 gene derived from PRRSV Korean isolate was constructed. Following oral administration of a single dose of the attenuated Salmonella vaccine expressing PRRSV ORF7, humoral and cell-mediated immune responses specific for ORF7 were induced at both systemic and mucosal sites including spleen, mesenteric lymph node, Peyer's patch, and laminar propria, as evaluated by determining serum ORF7-specific IgG and mucosal IgA responses, as well as Th1- and Th2-type cytokine production from antigen-stimulated T cells. The induced humoral responses were sustained for at least 12 weeks post-immunization. In particular, the immunized mice displayed immune responses to both the foreign ORF7 antigen and Salmonella itself. The results indicate the value of attenuated S. enterica serovar Typhimurium as an oral carrier of PRRSV antigenic proteins to induce effective systemic and mucosal immunity.     

Characteristics of Haemophilus pleuropneumoniae Isolates and Some Epidemiological Findings on Porcine Haemophilus Pleuropneumonia in Saskatchewan

Thirty isolates of Haemophilus pleuropneumoniae from clinical and slautherhouse cases of porcine Haemophilus pleuropneumonia in Saskatchewan as well as six isolates from British Columbia and Ontario were subjected to cultural, biochemical, serological and antibiotic sensitivity tests. All strains were Gram-negative pleomorphic rods or coccobacilli which grew only in the presence of V factor and all produced porphyrin from delta-aminolaevulinic acid. Biochemically, the organism was positive for urease, O-nitrophenyl-β-D-galactopyranosidase and the fermentation of sucrose, mannitol, dextrose, lactose and xylose, but was usually negative for indole. Most strains of H. pleuropneumoniae were sensitive to chloramphenicol, furamazone, carbenicillin and ampicillin, but only about 50% were sensitive to tetracycline. Serotype 5 was more common than serotype 1 or the untyped strains among Saskatchewan isolates. In addition, serotype 3 was identified from British Columbia.

Retrospective epidemiological studies showed that Haemophilus pleuropneumonia occurred and recurred on farms in the Saskatoon and adjoining districts, serviced by the diagnostic laboratories of the Western College of Veterinary Medicine and that the disease was more common among three month old pigs during the fall-winter season.

     

Immune response to an Actinobacillus pleuropneumoniae vaccine in swine

Piglets vaccinated with an inactivated tetravalent vaccine (Pleurovac 4) against Actinobacillus pleuropneumoniae serotypes 1, 2, 5 and 7, produced circulating antibodies after a first intramuscular injection and showed an anamnestic reaction after a second. The rise in antibody levels in vaccinated piglets was statistically significant when compared with those of the control group. The administration of 1 or 2 mL of vaccine did not lead to significantly different antibody levels. The specificity of the immune response is demonstrated by an increase in titer to all four serotypes in pigs given the tetravalent vaccine, but an increase in titer to only two serotypes in pigs given a bivalent vaccine (Pleurovac).     

Differences in immune responses of pigs vaccinated with Salmonella Typhimurium and S. Choleraesuis strains and challenged with S. Choleraesuis

S. Choleraesuis (Choleraesuis) and S. Typhimurium (Typhimurium) cause salmonellosis in pigs and humans. The effects of vaccine strains pSV-less Typhimurium OU5048 and Choleraesuis OU7266 and SPI-2-mutant Choleraesuis SC2284 on the immune responses of pigs against Typhimurium, Choleraesuis, and S. Enteritidis (Enteritidis) with or without the virulence plasmid (pSV) were determined. After oral vaccination of three vaccine groups and challenge with Choleraesuis CN36, the level of Salmonella-specific IgG in sera and the bactericidal effects and superoxide generation of peripheral blood mononuclear cells (PBMCs) and polymorphonuclear leukocytes (PMNs) against the above strains were determined using ELISA and NBT assay, respectively. Among three vaccine strains tested, OU7266 stimulated the highest Salmonella-specific IgG levels. Complement inactivation increased IgG concentration, while E. coli absorption reduced IgG levels. The pSV-containing strains were less resistant to serum killing than the pSV-less strains, and Enteritidis exhibited the lowest resistance to serum killing. Serovars tested, vaccine strains, and timeline periods postvaccination and challenge were important factors affecting superoxide production. The two Choleraesuis vaccine strains stimulated greater levels of superoxide from PMNs and PBMCs than the Typhimurium strains. The PMNs and PBMCs in challenged and vaccinated pigs reduced more superoxide than those in challenged hosts. In vaccinated hosts, pSV-less Salmonella strains triggered lower levels of PMN/PBMC-generated superoxide upon challenge than strains with pSV against Enteritidis and Choleraesuis. Overall, Choleraesuis OU7266 may be better than the other vaccine strains in generating the greatest IgG levels, serum bactericidal activity and superoxide levels. The pSV likely influences the immune responses.     

Efficacy of tilmicosin in the control of experimentally induced Actinobacillus pleuropneumoniae infection in swine

The efficacy of tilmicosin administered in the feed to control Actinobacillus pleuropneumoniae infections in pigs was evaluated through a multisite, multitrial study. For each of 6 trials, 48 pigs (stratified by weight and sex) were randomly assigned to 6 to 8 pens. Medicated feed containing tilmicosin (200 g/t) and unmedicated feed were randomly assigned at the pen level and were provided ad libitum from day −7 to trial termination (day 14). Seeder pigs (inoculated intranasally with A. pleuropneumoniae serotype 1 and showing signs of clinical disease) were introduced to each pen on day 0. Rates of death, gross lesions, and culture of A. pleuropneumoniae at necropsy, clinical scores, average daily gain in weight, and average body temperature were compared between the medicated and unmedicated pigs. Compared with the unmedicated pigs, significantly fewer (P < 0.05) pigs given tilmicosin had lesions typical of A. pleuropneumoniae or had A. pleuropneumoniae isolated from their tissues at necropsy. Together with a significant reduction (P < 0.05) in the average percentage of pneumonic lung involvement (both visually and by weight), there were reductions in the numbers of pigs with moderate and severe pneumonic lung lesions and with A. pleuropneumoniae associated mortality. With tilmicosin treatment, the average daily weight gain, daily temperature, abdominal appearance, attitude, and respiration were also significantly better (P < 0.05). The results of this study demonstrate the in vivo effectiveness of tilmicosin (200 g/t) in controlling pleuropneumonia among swine experimentally infected with A. pleuropneumoniae.     

Passive protection of mice pups through oral or intranasal immunization of dams with recombinant Lactobacillus casei vaccine against ETEC F41

Mucosal immunization is advantageous over other routes of antigen delivery because it can induce both mucosal and systemic immune responses. In this study, we have developed fimbriae protein of enterotoxigenic Escherichia coli (ETEC) F41 was stably expressed on the surface Lactobacillus casei 525. The method of expressing vaccine antigens in L. casei induces both systemic and mucosal immunity after oral or intranasal administration. We demonstrate that an oral or intranasal vaccine based on live recombinant L. casei 525 protects infant mice from ETEC F41 infection. This platform technology can be applied to design oral or intranasal vaccine delivery vehicles against several microbial pathogens.     

Protective immunity following immunisation of pigs with aerosol of Actinobacillus pleuropneumoniae serotype 2

To introduce antigen to the respiratory mucosa, killed Actinobacillus pleuropneumoniae with quil A as adjuvant was administered to pigs as an aerosol. Immunisation by this aerosol induced a marked IgA response in the bronchoalveolar and nasal fluids, and in the serum. Following challenge with live bacteria two weeks after the last exposure to the aerosol, the immunised pigs were protected from the severe pleuropneumonia which developed in non-immunised pigs. The immunised pigs had lower antibody titres in the mucosal fluids and serum after exposure to the challenge. The immune response after experimental infection of non-immunised animals was a weak IgA antibody response in the bronchoalveolar and nasal fluids, whereas the systemic immune response after challenge included both IgA and IgG antibodies.     

Protective potential of an attenuated Pasteurella multocida,which expresses only the N-terminal truncated fragment of P. multocida toxin

Pasteurella multocida serogroup D causes progressive atrophic rhinitis in pigs and produces a potent, intracellular, mitogenic toxin known as P. multocida toxin (PMT), which is encoded by the toxA gene. Highly toxic to cells, PMT is a poor antigen and becomes more immunogenic after its native structure has been destroyed. Previously, we found that the N-terminal fragment of PMT (N-PMT) can induce a strong immune response that is protective against wild-type challenge. Here, an attenuated P. multocida mutant expressing only N-PMT was developed and its protective effect was evaluated. The mutant provides protective immune responses against bacterial and toxin challenges, and so is a good live vaccine candidate.     

Characterization of colonization-deficient mutants of Actinobacillus suis

Actinobacillus suis is an important opportunistic pathogen of swine that can cause disease in pigs of all ages, especially in high-health status herds. Although A. suis shares many virulence factors in common with Actinobacillus pleuropneumoniae and can cause a haemorrhagic pleuropneumonia similar to that caused by A. pleuropneumoniae, A. suis most often causes septicaemia and diseases such as arthritis and meningitis that are sequelae to septicaemia. In a recent signature-tagged transposon mutagenesis study, 30 colonization-essential genes of A. suis were identified. In the current study, the attachment and invasion patterns of strains harboring Tn10 insertions in ompA, pfhaB1, lcbB, and cpxR were evaluated using porcine palatine tonsil organ cultures, the swine kidney epithelial cell line, SK6, and a porcine brain microvascular endothelial cell line, PBMEC/C1-2. All of these mutants attached in lower numbers than wild type to the tonsillar explants and to the SK6 cells. The ompA mutant attached in significantly lower numbers than wild type to the porcine tonsil cells (P = 0.02) and to PBMEC (P = 0.0008) at 60 min time point. As well, the ompA mutant showed significantly greater sensitivity than wild type to chemical stressors and to swine serum. Using fluorescent microscopy, a GST-OmpA fusion protein could be demonstrated to interact with the crypt epithelial cells of porcine palatine tonsil.     

Modulation of systemic and mucosal immunity against an inactivated vaccine of Newcastle disease virus by oral co-administration of live attenuated Salmonella enterica serovar Typhimurium expressing chicken interleukin-18 and interferon-α

Newcastle disease (ND) is a highly contagious disease of chickens causing significant economic losses worldwide. Due to limitations in the efficacy against currently circulating ND viruses, existing vaccination strategies require improvements, and incorporating immunomodulatory cytokines with existing vaccines might be a novel approach. Here, we investigated the systemic and mucosal immunomodulatory properties of oral co-administration of chicken interleukin-18 (chIL-18) and chicken interferon-α (chIFN-α) using attenuated Salmonella enterica serovar Typhimurium on an inactivated ND vaccine. Our results demonstrate that oral administration of S. enterica serovar Typhimurium expressing chIL-18 or chIFN-α provided enhanced systemic and mucosal immune responses, as determined by serum hemagglutination inhibition antibody and NDV Ag-specific IgG as well as NDV Ag-specific IgA in lung and duodenal lavages of chickens immunized with inactivated ND vaccine via the intramuscular or intranasal route. Notably, combined oral administration of S. enterica serovar Typhimurium expressing chIL-18 and chIFN-α significantly enhanced systemic and mucosal immunity in ND-vaccinated chickens, compared to single administration of S. enterica serovar Typhimurium expressing chIL-18 or chIFN-α. In addition, oral co-administration of S. enterica serovar Typhimurium expressing chIL-18 and chIFN-α provided enhanced NDV Ag-specific proliferation of peripheral blood mononuclear cells and Th1-biased cell-mediated immunity, compared to single administration of either construct. Therefore, our results provide valuable insight into the modulation of systemic and mucosal immunity by incorporation of immunomodulatory chIL-18 and chIFN-α using Salmonella vaccines into existing ND vaccines.