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.     

Protection of mice against the lethal effect of an intraperitoneal infection with Haemophilus (Actinobacillus) pleuropneumoniae after vaccination with capsular proteins

Haemophilus (Actinobacillus) pleuropneumoniae Serotypes 5 and 7 capsular antigens (CA-1) were precipitated from culture supernatants with N-cetyl-N,N,N,-trimethylammonium bromide (Cetavlon). CA-1 contained a carbohydrate to protein ratio of 2:1 for Serotype 5 and 3:1 for Serotype 7. Glucosamine and uronic acid were detected in CA-1 from both serotypes suggesting that the capsule contained hyaluronic acid. All mice immunized intraperitoneally with CA-1 vaccine were protected from death when challenged with 10X the LD50 of the homologous but not the heterologous serotype. Oil adjuvants and the use of young (6 h) cultures were necessary for CA-1 vaccines to be protective. Deproteinization of CA-1 with chloroform and butanol followed by pronase treatment resulted in failure to protect mice from death. The protective capsular protein antigen in CA-1 vaccine may not originate from the outer membrane (OM) since repeated washing of the OM to elute the capsular protein antigen rendered the OM vaccine completely nonprotective for mice. Vaccines prepared from cell-wall lipopolysaccharide also were nonprotective for mice. Passive immunization of mice with anti-CA-1 antibody produced in rabbits to Serotype 5 was highly protective (P less than 0.01) for mice when challenged with 10X the LD50 of the homologous serotype.     

Differential expression of non-cytoplasmic Actinobacillus pleuropneumoniae proteins induced by addition of bronchoalveolar lavage fluid

Actinobacillus (A.) pleuropneumoniae is the causative agent of a porcine pleuropneumonia occurring worldwide. In order to identify novel non-cytoplasmic putative virulence-associated proteins, we prepared fractions enriched in surface-associated proteins for differential proteome analysis by two-dimensional (2D) gel electrophoresis and quadrupole time-of-flight mass spectrometry (Q-Tof MS). Bacteria grown under standard culture conditions were compared to an ex vivo model based on the addition of bronchoalveolar lavage fluid (BALF) to the culture media. Twelve proteins were found to be upregulated upon induction with BALF, among them a superoxide dismutase, a parvulin-like peptidy-prolyl isomerase, a polynucleotide phosphorylase and the highly immunogenic lipoprotein OmlA. Four of the proteins upregulated by BALF were additionally constitutively expressed by an isogenic A. pleuropneumoniae fur deletion mutant and could be identified by Q-Tof MS as the heat shock protein GroES, a putative dipeptide transporter, a putative metal ion transporter and a conserved protein of unknown function. In silico analysis of the putative promoter regions of the encoding genes revealed putative Fur boxes upstream of two genes, one of which encodes part of a putative metal ion transporter. An isogenic mutant with a deletion in this protein was constructed and designated as A. pleuropneumoniae Deltafui. Analysis of the mutant in an aerosol infection model revealed symptoms indistinguishable from those seen upon infection with wild type A. pleuropneumoniae. This result implies that not all proteins upregulated by BALF are directly involved in A. pleuropneumoniae virulence.     

Cloning, expression, and characterization of TonB2 from Actinobacillus pleuropneumoniae and potential use as an antigenic vaccine candidate and diagnostic marker

In this study the tonB2 gene was cloned from Actinobacillus pleuropneumoniae JL01 (serovar 1) and expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli BL21(DE3). The GST fusion protein was recognized by antibodies in serum positive for A. pleuropneumoniae by Western blot analysis. Purified soluble GST-TonB2 was assessed for its ability to protect BALB/c mice against A. pleuropneumoniae infection. Mice were vaccinated with GST-TonB2 subcutaneously and challenged intraperitoneally with either ~4.0 × 10(5) colony-forming units (CFU) or ~1.0 × 10(6) CFU of A. pleuropneumoniae 4074. They were examined daily for 7 d after challenge. The survival rate of the TonB2-vaccinated mice was significant higher than that of the mice given recombinant GST or adjuvant alone. These results demonstrate that A. pleuropneumoniae TonB2 is immunogenic in mice and should be further assessed as a potential candidate for a vaccine against A. pleuropneumoniae infection. In addition, an indirect enzyme-linked immunosorbent assay (ELISA) based on the GST-TonB2 recombinant protein was developed. Compared with the ApxIVA ELISA, the TonB2 ELISA provided earlier detection of antibodies in pigs at various times after vaccination with A. pleuropneumoniae live attenuated vaccine. When compared with an indirect hemagglutination test, the sensitivity and specificity of the TonB2 ELISA were 95% and 88%, respectively. The TonB2 ELISA provides an alternative method for rapid serologic diagnosis of A. pleuropneumoniae infection through antibody screening, which would be especially useful when the infection status or serovar is unknown.     

Actinobacillus pleuropneumoniae vaccines: from bacterins to new insights into vaccination strategies

With the growing emergence of antibiotic resistance and rising consumer demands concerning food safety, vaccination to prevent bacterial infections is of increasing relevance. Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a respiratory disease leading to severe economic losses in the swine industry. Despite all the research and trials that were performed with A. pleuropneumoniae vaccination in the past, a safe vaccine that offers complete protection against all serotypes has yet not reached the market. However, recent advances made in the identification of new potential vaccine candidates and in the targeting of specific immune responses, give encouraging vaccination perspectives. Here, we review past and current knowledge on A. pleuropneumoniae vaccines as well as the newly available genomic tools and vaccination strategies that could be useful in the design of an efficient vaccine against A. pleuropneumoniae infection.     

Protective efficacy of capsule extracts of Haemophilus pleuropneumoniae in pigs and mice

Capsular extracts of Haemophilus pleuropneumoniae, Serotype 1, were mixed with AL(OH)3 gel (3 parts extract + 1 part Al(OH)3) and used as vaccines in pigs and mice. Four preparations were tested in Experiment I: NaCl and Cetavlon (hexadecyltrimethyl ammonium bromide) extracts of both low in vitro passage (LP) and high in vitro passage (HP) culture, respectively. Four pigs vaccinated with the NaCl extract of the LP strain survived, whereas one of four from each of the remaining vaccine groups and five of six from the control group died. All vaccines induced complement-fixing antibodies. No apparent boosting of titres occurred as a result of challenge with live bacteria. Mice were vaccinated in Experiment II with NaCl and Cetavlon extracts of the LP strain. Both were protective, although the Cetavlon vaccine appeared more efficacious than the NaCl extract. The use of Al(OH)3 adjuvant improved the efficacy of the NaCl vaccine in mice. In Experiment III six gnotobiotic pigs were vaccinated with a combined NaCl and Cetavlon vaccine and seven animals were given placebo. In Experiment IV seven specific pathogen-free (SPF) pigs were given the combined vaccine and eight pigs received placebo treatment. Both of these experiments indicated that the extract vaccines did not completely protect but reduced the mortality in pigs challenged with homologous virulent H. pleuropneumoniae bacteria. The results indicate that capsular antigens of H. pleuropneumoniae have some protective immunogenic efficacy in pigs and mice.     

An on-farm study of the epidemiology of Actinobacillus pleuropneumoniae infection in pigs as part of a vaccine efficacy trial

Thirty cohort pigs were followed from birth to slaughter to study epidemiological patterns of porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae. The study was conducted within a larger 380-animal study of vaccination against Mycoplasma hyopneumoniae and A. pleuropneumoniae in a 340-sow farrow-to-finish piggery with 4-month weaning, operating a continuous system of intensive production in the North Island of New Zealand. The cohort pigs were randomly allocated into two equal groups: vaccinated and control. Pigs in the first group were vaccinated at 2 and 4 weeks of age with both M. hyopneumoniae vaccine and A. pleuropneumoniae vaccine at separate vaccination sites. A series of nasal swabs was taken at 4, 8, 10, 11, 12, 14, 16 and 18 weeks of age. Each swab was streaked onto the surface of a selective medium on the farm and the plates were immediately transported to a laboratory and incubated at 37 degrees C for 5 days. After the trial, pigs were slaughtered at an average of 132 days of age, lungs were examined and samples taken for bacteriological culture and isolation. Thirty-five out of 256 samples produced haemolytic colonies which were Gram-negative, V-factor-dependent and positive to the CAMP test. A. pleuropneumoniae was first isolated at 4 weeks of age from one vaccinated pig. This finding suggests that piglets became infected in the farrowing pen and the source of infection might be a carrier sow. The interval-specific cumulative incidence of A. pleuropneumoniae infection reached a maximum of 54% and 40% at 11 weeks of age in the vaccinated and control groups, respectively. Infection status of the litter is considered to be a factor influencing morbidity in infected herds during weaner and grower periods. Our results suggest that simultaneous vaccination with M. hyopneumoniae and A. pleuropneumoniae vaccines at 2 and 4 weeks of age might lessen the prevalence but cannot absolutely prevent A. pleuropneumoniae infection during the weaner or grower-finisher periods.     

胸膜肺炎放线杆菌溶血外毒素研究进展

猪传染性胸膜肺炎是由胸膜肺炎放线杆菌(APP)引起的一种呼吸道传染性疾病.根据荚膜多糖和脂多糖抗原表位不同,将胸膜肺炎放线杆菌分为15个血清型,不同血清型的菌株毒力大小不同,致病性也有强弱之别.研究发现与APP致病性有关的毒力因子包括荚膜多糖、脂多糖、外膜蛋白、转铁结合蛋白、溶血外毒素等,其中溶血外毒素是APP引起猪致病最主要的毒力因子,也是主要的保护性抗原.论文从猪传染性胸膜肺炎溶血外毒素的分子结构、致病机制、免疫原性几个方面对其进行综述,为获得以Apx为抗原制备减毒活疫苗提供参考资料.     

An evaluation of the role of antibodies to Actinobacillus pleuropneumoniae serovar 1 and 15 in the protection provided by sub-unit and live streptomycin-dependent pleuropneumonia vaccines

OBJECTIVE: To evaluate the serological response of pigs receiving either the Porcilis APP vaccine or a modified live vaccine based on a streptomycin-dependent (SD) strain of Actinobacillus pleuropneumoniae, and then challenged with an Australian isolate of A. pleuropneumoniae of either serovar 1 or 15 as a means of understanding the protection provided by both vaccines against serovar 1 but not against serovar 15. DESIGN: The serological tests evaluated were serovar-specific polysaccharide ELISA tests (for serovar 1 and 15), ELISA tests for antibodies to three A. pleuropneumoniae toxins (ApxI, ApxII and ApxIII) as well as to a 42 kDa outer membrane protein (OMP), a haemolysin neutralisation (HN) assay and immunoblotting. The tests were used to detect antibodies in vaccinated pigs that had been shown to be protected against serovar 1 but not serovar 15. RESULTS: In the polysaccharide antigen ELISA assays, both vaccines resulted in a significant rise in the titre in the serovar 1 ELISA but not the serovar 15 ELISA. The Porcilis APP vaccinated pigs showed a significant response in the ApxI, ApxIII and 42 kDa OMP ELISA. In the ApxII ELISA, all pigs tested (the Porcilis APP vaccinates and the controls) were positive on entry to the trial. In the HN assay, the Porcilis APP vaccinated pigs showed a significant response after one dose while the SD vaccinated pigs required two doses of vaccine before a marked rise in titre was induced. Immunoblotting revealed that neither vaccine generated antibodies that recognised the ApxIII produced by serovar 15. CONCLUSIONS: The failure of these vaccines to provide protection against serovar 15 may be due to novel virulence factors possessed by serovar 15, significant differences between the ApxIII toxin of serovar 15 and those present in the Porcilis APP vaccine or failure by both vaccines to induce antibodies to the serovar 15 specific polysaccharide.     

A field evaluation of the efficacy of two vaccines against ovine pneumonic pasteurellosis

Two vaccines against pneumonic pasteurellosis were evaluated for efficacy in lambs transported by sea from New Zealand to Saudi Arabia. One vaccine contained whole cell antigens of Pasteurella haemolytica A2 grown under iron restricted conditions. The other contained Pasteurella haemolytica A1 cell surface and leucotoxin antigens. There was no clear evidence of either vaccine leading to a lower pneumonia death or lesion rate than for the control group.     

Improved protection of swine from pleuropneumonia by vaccination with proteinase K-treated outer membrane of Actinobacillus (Haemophilus) pleuropneumoniae

The immunogenic and protective potentials of an outer membrane-enriched fraction (OM) from a serotype 5 strain of Actinobacillus (Haemophilus) pleuropneumoniae (APP) and the same OM degraded with proteinase K or periodate were evaluated in swine. Groups of pigs were vaccinated with two doses of OM, proteinase K-treated OM (P-OM), periodate-treated OM (PI-OM), or placebo vaccine and challenged intranasally with the homologous strain of APP. Results from triplicate experiments indicated that proteinase K treatment of OM resulted in an improved efficacy. This improved efficacy of P-OM vaccine over untreated OM vaccine was evidenced not only by less severe lung lesions in P-OM vaccinated pigs but also by significant reduction (P less than 0.05) in the number of P-OM vaccinated pigs which developed lung lesions upon challenge with APP. Assessment of sera from vaccinated animals by immunoblotting, complement fixation test, or ELISA indicated that the immunogenicity of some but not all protein or carbohydrate components were reduced (or eliminated) by proteinase K and periodate treatments respectively.     

Comparison of the efficacy of a subunit and a live streptomycin-dependent porcine pleuropneumonia vaccine

Objective To evaluate the efficacy of two new-generation porcine pleuropneumonia vaccines when challenged with Australian isolates of Actinobacillus pleuropneumoniae of serovars 1 and 15.
Design The Porcilis APP vaccine and an experimental streptomycin-dependent strain of A pleuropneumoniae were evaluated in a standardised pen trial. Each vaccine/challenge group consisted of 10 pigs.
Results With the serovar 1 challenge, the Porcilis APP vaccine and the live vaccine, compared with the control group, gave significant protection in terms of clinical signs, lung lesions, re-isolation scores and average daily gain (ADG) postchallenge. Only the Porcilis APP vaccine provided significant protection against mortality. In the serovar 15 challenged pigs, the only significant difference detected was that the Porcilis APP vaccinated pigs had a better postchallenge ADG than the controls. None of the Porcilis APP vaccinated pigs showed signs of depression postvaccination and none were euthanased after challenge with either serovar 1 or 15. The pigs vaccinated with the live vaccine showed obvious depression after each vaccination and a total of 3 pigs were euthanased after challenge (one with serovar 1 and two with serovar 15).
Conclusions Both of the vaccines provided significant protection against a severe challenge with serovar 1 A pleuropneumoniae. Neither vaccine was effective against a serovar 15 A pleuropneumoniae challenge. There was evidence that the Porcilis APP vaccine did provide some protection against the serovar 15 challenge because the ADG, after challenge of pigs given this vaccine, was greater than the control pigs.     

Immune responses to a Staphylococcus aureus GapC/B chimera and its potential use as a component of a vaccine for S. aureus mastitis

Bovine mastitis caused by strains of S. aureus is the most economically important disease affecting the dairy industry worldwide. Commercially available vaccines show various degrees of success and work in research laboratories with experimental vaccines suggests that in part, the failure of these vaccines lies in the limited antigenic repertoire contained in the vaccine formulations. Since it seems impractical to produce a vaccine containing antigens from all major S. aureus mastitis isolates, we took the approach of using two surface antigens GapB and GapC that appear to be conserved and constructed a GapC/B chimera as the basis for a vaccine. The humoral and cellular immune responses to GapC/B were compared to the responses to the individual proteins, alone or in combination. The GapC/B protein elicited strong humoral and cellular responses in mice as judged by the levels of total IgG, IgG1, IgG2a, and number of IL-4- and IFN-gamma-secreting cells. These results suggest that this chimeric protein could be an attractive target for further vaccine efficacy studies.     

Evaluation of immunogenicity and protective efficacy of Actinobacillus pleuropneumoniae HB04C(-) mutant lacking a drug resistance marker in the pigs

Previously, we reported the construction and characterization of a genetically defined Actinobacillus pleuropneumoniae (A. pleuropneumoniae) apxIIC gene mutant, HB04C(-), which conferred protection to mice against infection with A. pleuropneumoniae. In this study, we further evaluated HB04C(-) for safety and its ability to elicit protective immunity in pigs. It was demonstrated that a dose of 2 x 10(8) CFU HB04C(-) was safe to the pigs via intranasal or intramuscular injection. Immunization with a dose of 2 x 10(8) HB04C(-) by both intranasal and intramuscular routine could yield equal protective efficacy and elicited significant protection against experiment challenge with homologous or heterologous serotypes of a virulent A. pleuropneumonia. Taken together, HB04C(-) might serve as a promising vaccine candidate against infection with A. pleuropneumoniae.     

Vaccines against veterinary helminths

The majority of attempts to develop commercial vaccines for veterinary helminths have focussed on identifying protein antigens, which could be formulated as protective vaccines. Notable successes have been achieved for some cestode parasites, where recombinant proteins have been developed into highly effective vaccines. Although effective protection can also be obtained using some nematode proteins in their native forms, it has not yet been possible to formulate commercially successful vaccines for other helminth parasites of veterinary significance. Increasing evidence suggests that parasite glycan moieties may provide an alternative source of vaccine antigens, and increased attention is now being given to this class of compounds. In addition to identifying candidate protective antigen(s), an increased research effort is needed to develop appropriate strategies for the formulation and delivery of helminth vaccines.     

Factors involved in immunity against Actinobacillus pleuropneumoniae in mice.

Active and passive immunization studies in mice were undertaken to examine the protective efficiency of vaccines prepared from different components of Actinobacillus pleuropneumoniae, or combinations thereof. Subcutaneous immunization using either washed formalinized whole cells, capsular polysaccharide, lipopolysaccharide or purified hemolysin I (105 kDa protein) partially protected mice against intranasal challenge with a lethal dose of homologous or heterologous A. pleuropneumoniae serotypes. However, full protection was obtained if the formalinized whole cells were supplemented with purified hemolysin. Similar protection was obtained when mice were immunized simultaneously with a sublethal dose of live cells by the intranasal route and with formalinized whole cells subcutaneously. Passive immunization using rabbit hyperimmune serum against formalinized whole cells provided almost total protection whereas hyperimmune serum against capsular polysaccharide, lipopolysaccharide or hemolysin alone provided only a partial protection. Cell mediated immunity as detected by the foot pad test may not be implicated significantly in the protein against acute A. pleuropneumoniae infection. However, humoral immune response seems to play an important role in protection. All the antigenic components examined may contribute to the protection to some extent. However, heat-labile components such as hemolysin and outer membrane proteins may play a crucial role in protection against acute challenge infection.     

Evaluation of an indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to the Apx toxins of Actinobacillus pleuropneumoniae

The reference strains of the 12 serotypes of Actinobacillus pleuropneumoniae express one or two of three different RTX exotoxins designated Apx I, Apx II and Apx III. The toxins are important virulence factors. In the present study, ELISAs with purified Apx I, Apx II and Apx III, respectively, as antigen were evaluated as candidates for serological diagnosis of Actinobacillus pleuropneumoniae infection in pigs. The pigs were inoculated with biotype 1, serotypes 1-12, and biotype 2, serotype 14, respectively. A strong humoral antibody response was seen to all the three antigens in most pigs irrespective of the serotype used for inoculation. However, titers to the exotoxins secreted by the serotype used for inoculation were generally highest. The results show that toxin proteins of Actinobacillus pleuropneumoniae are antigenically related and that a correlation between serotype and secretion of exotoxin is not revealed serologically in the ELISA test.     

Study of the virulence of Actinobacillus pleuropneumoniae in finishing pigs as a basis for vaccination development

For vaccine licensing data about efficiency and duration of protection are essential. Within the scope of the developement of a new subunit vaccine against Actinobacillus pleuropneumoniae (A.pp.) the protective efficiency over the whole length of the fattening period must be proven. This required infection experiments in finishing pigs. Eight pigs in the age of six months were infected experimentally into the trachea with an A.pp. serotyp 2 strain. To our knowledge data about the susceptibility of pigs of this age do not exist, so that the infectious dose for pigs of this age and this route of infection had to be determined. Two pigs each were infected with different doses of 10(10), 6 x 10(5), 8 x 10(3) and 2 x 10(3) CFU (colony forming units). The aim of the study was to produce a typical pleuropneumonia with fever and severe respiratory symptoms as well as characteristic pathomorphological lung alterations without loss of animals during the acute stage of infection. The pathogen should be cultivated from lung tissue. The recommended dose for testing the efficiency of vaccines turned out to be approximately 10(3) CFU A.pp. serotyp 2.     

Characterization of the in vitro core surface proteome of Mycoplasma mycoides subsp. mycoides, the causative agent of contagious bovine pleuropneumonia

Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides (Mmm) is a severe cattle disease, present in many countries in sub-Saharan Africa. The development of improved diagnostic tests and vaccines for CBPP control remains a research priority. Polyacrylamide gel electrophoresis and mass spectrometry were used to characterize the Triton X-114 soluble proteome of nine Mmm strains isolated from Europe or Africa. Of a total of 250 proteins detected, 67 were present in all strains investigated. Of these, 44 were predicted to be lipoproteins or cytoplasmic membrane-associated proteins and are thus likely to be members of the core in vitro surface membrane-associated proteome of Mmm. Moreover, the presence of all identified proteins in other ruminant Mycoplasma pathogens were investigated. Two proteins of the core proteome were identified only in other cattle pathogens of the genus Mycoplasma pointing towards a role in host–pathogen interactions. The data generated will facilitate the identification and prioritization of candidate Mycoplasma antigens for improved control measures, as it is likely that surface-exposed membrane proteins will include those that are involved in host–pathogen interactions.