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Population-based analysis of Actinobacillus pleuropneumoniae ApxIVA for use as a DIVA antigen
Abstract
APXIVA is an RTX toxin of Actinobacillus pleuropneumoniae that is a candidate antigen to differentiate infected from vaccinated animals (DIVA). Insertion of ISApl1 into the apxIVA gene is known to compromise an APXIVA-based DIVA approach, as is potentially a TGG to TGA mutation in the apxIVA gene. ISApl1 was found in 63/349 (18.1%) A. pleuropneumoniae isolates from England and Wales including serovars 2, 3, 6-8 and 12. No ISApl1 insertions into apxIVA were found. Only two serovar 3 isolates contained the TGG to TGA mutation. We conclude that an ApxIVA-based DIVA approach would potentially be viable in England and Wales.
... Although our APP-RPA gave positive amplification for all 19 serovars, the apxIVA gene is not 100% conserved between isolates, and insertions such as ISApl1 have been detected in the apxIVA gene in some isolates, which could affect detection (34). When tested with isolates that have been found to have a transposase adjacent to or within their apxIVA genes, we found the isolate MIDG3936 with a WP_005599960 transposon flanking the apxIVA gene duplication (apxIVS') and a fragmented apxIVA gene, had a similar detection time to the reference serovar 2 strain. ...
Actinobacillus pleuropneumoniae (APP) is the causative agent of porcine pleuropneumonia, resulting in high economic impact worldwide. There are currently 19 known serovars of APP, with different ones being predominant in specific geographic regions. Outbreaks of pleuropneumonia, characterized by sudden respiratory difficulties and high mortality, can occur when infected pigs are brought into naïve herds, or by those carrying different serovars. Good biosecurity measures include regular diagnostic testing for surveillance purposes. Current gold standard diagnostic techniques lack sensitivity (bacterial culture), require expensive thermocycling machinery (PCR) and are time consuming (culture and PCR). Here we describe the development of an isothermal point-of-care diagnostic test - utilizing recombinase polymerase amplification (RPA) for the detection of APP, targeting the species-specific apxIVA gene. Our APP-RPA diagnostic test achieved a sensitivity of 10 copies/μL using a strain of APP serovar 8, which is the most prevalent serovar in the UK. Additionally, our APP-RPA assay achieved a clinical sensitivity and specificity of 84.3 and 100%, respectively, across 61 extracted clinical samples obtained from farms located in England and Portugal. Using a small subset (n = 14) of the lung tissue samples, we achieved a clinical sensitivity and specificity of 76.9 and 100%, respectively) using lung imprints made on FTA cards tested directly in the APP-RPA reaction. Our results demonstrate that our APP-RPA assay enables a suitable rapid and sensitive screening tool for this important veterinary pathogen.
... To date, ISApl1 has been identified only in serovars 2, 3, 6-8, 12, and 13. 11,12,16,18 The TSD nucleotide sequences, which were probably generated by insertion of ISApl1, differed between the 2 insertion types (i.e., the sequence was GC in strains QAS68, QAS93. and QAS102, and AA in strain FH24-1; Fig. 3). ...
The aim of our study was to reveal the molecular basis of the serological nontypeability of 2 Actinobacillus pleuropneumoniae field isolates. Nine field strains of A. pleuropneumoniae, the causative agent of porcine pleuropneumonia, were isolated from pigs raised on the same farm and sent to our diagnostic laboratory for serotyping. Seven of the 9 strains were identified as serovar 15 strains by immunodiffusion tests. However, 2 strains, designated FH24-2 and FH24-5, could not be serotyped with antiserum prepared against serovars 1–15. Strain FH24-5 showed positive results in 2 serovar 15–specific PCR tests, whereas strain FH24-2 was only positive in 1 of the 2 PCR tests. The nucleotide sequence analysis of gene clusters involved in capsular polysaccharide biosynthesis of the 2 nontypeable strains revealed that both had been rendered nontypeable by the action of ISApl1, a transposable element of A. pleuropneumoniae belonging to the IS30 family. The results showed that ISApl1 of A. pleuropneumoniae can interfere with both the serological and molecular typing methods, and that nucleotide sequence analysis across the capsular gene clusters is the best means of determining the cause of serological nontypeability in A. pleuropneumoniae.
... One such instance led to the discovery of an insertion element named ISApl1, which prevented the ApxIV-based detection of A. pleuropneumoniae due to its stable insertion within its cognate gene (Tegetmeyer et al., 2008). Additionally, a Trp to stop (TGG to TGA) mutation in the coding region of the apxIVA gene that may lead to a truncated protein has been described in some serotype three strains (O'Neill et al., 2010). Discrepancies between the identification of A. pleuropneumoniae isolates by biochemical tests and PCR detection have also been reported (Urbaniak & Markowska-Daniel, 2011). ...
Bacterial respiratory diseases are responsible for considerable mortality, morbidity and economic losses in the swine industry. Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, is one of the most important disease agents, but its identification and surveillance can be impaired by the existence of many other related bacteria in normal swine microbiota. In this work, we have evaluated a BOX-A1R-based repetitive extragenic palindromic-PCR (BOX-PCR) Sequence Characterized Amplified Region (SCAR) marker for the specific identification of A. pleuropneumoniae and its use in a multiplex-PCR to detect additionally Haemophilus parasuis and Pasteurella multocida, two other major respiratory pathogens of pigs that are members of the family Pasteurellaceae. PCRs based on the BOX-SCAR fragment developed were rapid, sensitive and differentiated A. pleuropneumoniae from all swine-related members of the Pasteurellaceae family tested. Single and multiplex BOX-SCAR fragment-based PCRs can be used to identify A. pleuropneumoniae from other bacterial swine pathogens and will be useful in surveillance and epidemiological studies. This article is protected by copyright. All rights reserved.
... The expression of the apxIV has long been considered to be strictly induced in vivo, so pigs immunized with inactivated vaccines would not generate antibodies against ApxIV (11). Therefore, ApxIV antigen has been used to differentiate infected from vaccinated animals (DIVA), as it is immunogenic, specific to A. pleuropneumoniae, and encoded by all serovars (44). However, recently, an ApxIVA protein was identified for the first time from an in vitro growth of A. pleuropneumoniae as part of a subunit vaccine (45). ...
Surveillance for the presence of Actinobacillus pleuropneumoniae infection in a population plays a central role in controlling the disease. In this study, a 4-plex fluorescent microbead-based
immunoassay (FMIA), developed for the simultaneous detection of IgG antibodies to repeat-in-toxin (RTX) toxins (ApxI, ApxII,
ApxIII, and ApxIV) of A. pleuropneumoniae, was evaluated using (i) blood serum samples from pigs experimentally infected with each of the 15 known A. pleuropneumoniae serovars or with Actinobacillus suis, (ii) blood serum samples from pigs vaccinated with a bacterin containing A. pleuropneumoniae serovar 1, 3, 5, or 7, and (iii) blood serum samples from pigs with an unknown A. pleuropneumoniae exposure status. The results were compared to those obtained in a previous study where a dual-plate complement fixation test
(CFT) and three commercially available enzyme-linked immunosorbent assays (ELISAs) were conducted on the same sample set.
On samples from experimentally infected pigs, the 4-plex Apx FMIA detected specific seroconversion to Apx toxins as early
as 7 days postinfection in a total of 29 pigs inoculated with 14 of the 15 A. pleuropneumoniae serovars. Seroconversion to ApxII and ApxIII was detected by FMIA in pigs inoculated with A. suis. The vaccinated pigs showed poor humoral responses against ApxI, ApxII, ApxIII, and ApxIV. In the field samples, the humoral
response to ApxIV and the A. pleuropneumoniae seroprevalence increased with age. This novel FMIA (with a sensitivity of 82.7% and a specificity of 100% for the anti-ApxIV
antibody) was found to be more sensitive and accurate than current tests (sensitivities, 9.5 to 56%; specificity, 100%) and
is potentially an improved tool for the surveillance of disease and for monitoring vaccination compliance.
... It known to promote an immunogenic response when given recombinantly as a subunit vaccine [21] and inactivation of its cognate gene allows its use in attenuated vaccines to differentiate infected from vaccinated animals (DIVA) [22]. ApxIV was an abundant and immunogenic antigen in a vaccine derived from detergent washes of serotypes 1, 2 and 5 [23,24]. ZnuA is a known A. pleuropneumoniae immunogenic protein [24,25] and is required for virulence [26]. ...
... The ApxIVA ELISA is widely used for the evaluation of A. pleuropneumoniae infection. The applicability of ApxIV for differentiating infected from vaccinated animals is not affected by the appearance of natural ApxIVA inactivation at low frequency (31). However, the generation of ApxIVA antibody is slow (32), and in our study the serum of only 2 animals out of 20 was weakly positive in the ApxIVA ELISA 2 wk after primary vaccination with A. pleuropneumoniae live vaccine. ...
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.
... ApxIVA has been considered as a DIVA antigen as it is immunogenic and encoded by most A. pleuropneumoniae isolates [27][28][29]. It was shown for serotype 7 strain AP76 that the apxIVA gene is interrupted by an insertion element preventing in vivo expression of apxIVA [29]. ...
Protection of pigs by vaccination against Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, is hampered by the presence of 15 different serotypes. A DIVA subunit vaccine comprised of detergent-released proteins from A. pleuropneumoniae serotypes 1, 2 and 5 has been developed and shown to protect pigs from clinical symptoms upon homologous and heterologous challenge. This vaccine has not been characterized in-depth so far. Thus we performed i) mass spectrometry in order to identify the exact protein content of the vaccine and ii) cross-serotype 2-D immunoblotting in order to discover cross-reactive antigens. By these approaches we expected to gain results enabling us to argue about the reasons for the efficacy of the analyzed vaccine.
We identified 75 different proteins in the vaccine. Using the PSORTb algorithm these proteins were classified according to their cellular localization. Highly enriched proteins are outer membrane-associated lipoproteins like OmlA and TbpB, integral outer membrane proteins like FrpB, TbpA, OmpA1, OmpA2, HgbA and OmpP2, and secreted Apx toxins. The subunit vaccine also contained large amounts of the ApxIVA toxin so far thought to be expressed only during infection. Applying two-dimensional difference gel electrophoresis (2-D DIGE) we showed different isoforms and variations in expression levels of several proteins among the strains used for vaccine production. For detection of cross-reactive antigens we used detergent released proteins of serotype 7. Sera of pigs vaccinated with the detergent-released proteins of serotypes 1, 2, and 5 detected seven different proteins of serotype 7, and convalescent sera of pigs surviving experimental infection with serotype 7 reacted with 13 different proteins of the detergent-released proteins of A. pleuropneumoniae serotypes 1, 2, and 5.
A detergent extraction-based subunit vaccine of A. pleuropneumoniae was characterized by mass spectrometry. It contained a large variety of immunogenic and virulence associated proteins, among them the ApxIVA toxin. The identification of differences in expression as well as isoform variation between the serotypes implied the importance of combining proteins of different serotypes for vaccine generation. This finding was supported by immunoblotting showing the induction of cross-reactive antibodies against several surface associated proteins in immunized animals.
Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a respiratory disease leading to severe economic losses in the swine industry. The most widely used commercial vaccines are bacterins comprising inactivated whole cells of A. pleuropneumoniae but these have only been partially effective in preventing disease. Innovative immuno-prophylactic preparations of A. pleuropneumoniae based on ApxI, ApxII, ApxIII, ApxIV toxins and outer membrane proteins, among others (i.e. RnhB, GalU, GalT, HflX, ComL, LolB, LppC), have high protective efficacy in mice and pigs. Some vaccine preparations have efficacy against homologous and heterologous A. pleuropneumoniae serovars, which constitute an important advance to control porcine pleuropneumonia. In this arena, subunit vaccines based on toxins are one of the most advanced and promising developments. Many research groups have focussed on the development of live attenuated vaccines comprising strains with inactivated Apx toxins and/or other virulence factors, their protective efficacy being determined in mouse and/or swine models. Other innovative approaches such as bacteria, yeast and plants as production and oral delivery platforms have been explored in animal models and the definitive host with encouraging results. In addition, further research into A. pleuropneumoniae-based DNA and nano-vaccines, as well as bioencapsulation of antigens in plants, is envisaged. Here, the recent findings and future trends in innovative vaccine development against A. pleuropneumoniae are reviewed and placed in perspective.
The introduction into a naïve herd of animals sub-clinically infected with Actinobacillus pleuropneumoniae (App) is frequently the cause of clinical pleuropneumonia and the identification of such infected herds is a priority in the control of disease. Different serological tests for App have been developed and a number of these are routinely used. Some are species-specific whereas others identify more specifically the serotype/serogroup involved which requires updated information about important serotypes recovered from diseased pigs in a given area/country. Serotyping methods based on molecular techniques have been developed lately and are ready to be used by most diagnostic laboratories. When non-conclusive serological results are obtained, direct detection of App from tonsils is sometimes attempted. This review addresses different techniques and approaches used to monitor herds sub-clinically infected by this important pathogen.
Copyright © 2015 Elsevier Ltd. All rights reserved.
Porcine pleuropneumonia is an infectious disease caused by the Actinobacillus pleuropneumoniae. The identification of A. pleuropneumoniae genes, specially expressed in vivo is a useful tool to reveal the mechanism of infection. IVIAT was used in this work to identify antigens which are expressed in vivo during A. pleuropneumoniae infection using sera from individuals with chronic porcine pleuropneumonia. Sequencing of DNA inserts from positive clones showed eleven open reading frames with high homology to A. pleuropneumoniae genes. Base on sequence analysis, proteins encoded by these genes involved in metabolism, replication, transcription regulatory, signal transduction. Moreover, three function unknown protein were also indentified in this work. Expression analysis using quantitative real-time PCR showed that most of the genes tested were up-regulated in-vivo relative to their expression levels in-vitro. IVI (in vivo-induced) genes were amplified by PCR in different A. pleuropneumoniae strains showed that these genes could be detected in almost all of the strains. It is demonstrated that the identified IVI antigen may have important roles in the infection of A. pleuropneumoniae.
Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia, an economically important endemic bacterial disease of pigs worldwide ([Bosse and others 2002][1]). Epidemiologically, serotyping is the gold standard method, with A pleuropneumoniae being classified into 16
Actinobacillus pleuropneumoniae is a Gram-negative pathogen. It is the aetiological agent of porcine contagious pleuropneumonia (PCP), a severe and highly contagious and severe respiratory disease of swine. Four sets of RTX (repeats in toxin) exotoxins have been described in A. pleuropnuemoniae and three of them have been characterized as important virulence determinants. The aim of this study was to determine the pathogenicity of the invivo induced RTX toxin ApxIVA during infection of piglets with A. pleuropnuemoniae. An A. pleuropnuemoniae apxIVA mutant was obtained based on an A. pleuropnuemoniae apxIIC-deleted mutant strain. An experimental infection assay was performed to evaluate the virulence of ApxIVA in piglets. Clinical signs, lung lesion scores, blood biochemical parameters and histopathologic changes in the piglets were recorded. The results indicated that the pathogenicity of A. pleuropnuemoniae was greater when ApxIVA was present, suggesting that ApxIVA is essential for expression of the full virulence of A. pleuropnuemoniae.
Clonal, noniridescent mutants of Actinobacillus pleuropneumoniae serotypes 1 and 5 were isolated following chemical mutagenesis with ethyl methanesulfonate. The absence of any detectable capsule was confirmed by inhibition radioimmunoassay. There were no differences between the parent and mutant strains in lipopolysaccharide or protein electrophoretic profiles or in hemolytic activity. There was no detectable reversion to the encapsulated phenotype in vitro after passage in mice or pigs or in microporous capsules that were implanted subcutaneously in pigs for 6 weeks. The mutants were able to survive for more than 1 week in pigs following subcutaneous inoculation, which resulted in a strong immune response to whole cells and Apx toxins I and II. Intratracheal challenge of pigs with the serotype 5 mutant at a dose 1 log greater than the 50% lethal dose for the parent resulted in no clinical disease or lesions except in one pig that had slight pneumonia and pleuritis. Twenty-four hours after challenge, A. pleuropneumoniae could not be recovered from the respiratory tracts of any of the challenged pigs except for the one infected pig; this isolate remained noncapsulated. Immunization of pigs with one or both serotypes of noncapsulated mutants protected all pigs against clinical disease following intratracheal challenge with the virulent homologous or heterologous serotype. Nonimmunized control pigs and pigs immunized with a commercial bacterin died or had to be euthanized within 24 h of challenge. Thus, live noncapsulated mutants of A. pleuropneumoniae may provide safe and cost-effective protection against swine pleuropneumonia. These observations support the possibility that noncapsulated mutants of other encapsulated, toxin-producing bacteria may also prove to be efficacious live-vaccine candidates.
A fourth type of RTX determinant was identified in Actinobacillus pleuropneumoniae and was designated apxIVA. When expressed in Escherichia coli, recombinant ApxIVA showed a weak haemolytic activity and co-haemolytic synergy with the sphingomyelinase (beta-toxin) of Staphylococcus aureus. These activities required the presence of an additional gene, ORF1, that is located immediately upstream of apxIVA. The apxIVA gene product could not be detected in A. pleuropneumoniae cultures grown under various conditions in vitro; however, pigs experimentally infected with A. pleuropneumoniae serotypes 1, 5 and 7 started to produce antibodies that reacted with recombinant ApxIVA 14 d post-infection, indicating that apxIVA is expressed in vivo. In addition, sera from pigs naturally and experimentally infected with any of the serotypes all reacted with recombinant ApxIVA. The apxIVA gene from the serotype 1 A. pleuropneumoniae type strain Shope 4074T encodes a protein with a predicted molecular mass of 202 kDa which has typical features of RTX proteins including hydrophobic domains in the N-terminal half and 24 glycine-rich nonapeptides in the C-terminal half that bind Ca2+. The glycine-rich nonapeptides are arranged in a modular structure and there is some variability in the number of modules in the ApxIVA proteins of different serotypes of A. pleuropneumoniae. The deduced amino acid sequences of the ApxIVA proteins have significant similarity with the Neisseria meningitidis iron-regulated RTX proteins FrpA and FrpC, and to a much lesser extent with other RTX proteins. The apxIVA gene could be detected in all A. pleuropneumoniae serotypes and seems to be species-specific. Although the precise role of this new RTX determinant in pathogenesis of porcine pleuropneumonia needs to be determined, apxIVA is the first in vivo induced toxin gene that has been described in A. pleuropneumoniae.
A total of 90 Actinobacillus pleuropneumoniae field strains from pigs were serotyped by slide agglutination and analyzed for the presence of the apxIV gene by polymerase chain reaction. Of the 90 isolates serotyped, serotypes 2 (47 isolates) and 5 (25 isolates) were the most common, followed by serotype 6 (10 isolates). Three isolates belonged to serotype 7, and 5 isolates could not be typed. All 90 A. pleuropneumoniae field isolates tested carried the apxIV gene. This gene is species specific rather than serotype specific. Therefore, the ApxIV toxin has potential value for use both in vaccines and in diagnostic tests.
Vaccination against Actinobacillus pleuropneumoniae is hampered by the lack of vaccines inducing reliable cross-serotype protection. In contrast, pigs surviving natural infection
are at least partially protected from clinical symptoms upon reinfection with any serotype. Thus, we set out to construct
an attenuated A. pleuropneumoniae live vaccine allowing the differentiation of vaccinated from infected animals (the DIVA concept) by successively deleting
virulence-associated genes. Based on an A. pleuropneumoniae serotype 2 prototype live negative marker vaccine (W. Tonpitak, N. Baltes, I. Hennig-Pauka, and G.-F. Gerlach, Infect. Immun.
70:7120-7125, 2002), genes encoding three enzymes involved in anaerobic respiration and the ferric uptake regulator Fur were
deleted, resulting in a highly attenuated sixfold mutant; this mutant was still able to colonize the lower respiratory tract
and induced a detectable immune response. Upon a single aerosol application, this mutant provided significant protection from
clinical symptoms upon heterologous infection with an antigenically distinct A. pleuropneumoniae serotype 9 challenge strain and allowed the serological discrimination between infected and vaccinated groups.
Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia, a highly contagious and often fatal disease. We have previously reported the construction and characterization of a single gene apxIIC deletion mutant HB04C(-) based on A. pleuropneumoniae serovar 7 which produces ApxII toxin and ApxIV. A precisely defined DeltaapxIICDeltaapxIVA double-deletion mutant of A. pleuropneumoniae was constructed based on HB04C(-) by transconjugation and counterselection, and the levels of virulence of the DeltaapxIIC single mutant and DeltaapxIICDeltaapxIVA double mutant were compared in an experimental infection in mice and pigs. The results demonstrated that the DeltaapxIICDeltaapxIVA double mutant strain was less virulent than HB04C(-). Despite attenuation of virulence, the DeltaapxIICDeltaapxIVA double mutant remains immunogenic and conferred a similar level of protective immunity to pigs against challenge with a lethal dose of a heterologous fully virulent standard serovar 1 strain of A. pleuropneumoniae. The results of the virulence study suggest that ApxIV is a critical virulence factor of A. pleuropneumoniae serovar 7 and is able to induce clinical disease, but it not required for efficient vaccination of pigs against A. pleuropneumoniae infection. Two weeks after the booster immunization, animals vaccinated with HB04C(-) were positive in the ApxIVAM-ELISA based on a recombinant GST-fusion protein GST-ApxIVAM as the solid-phase antigen while animals vaccinated with the DeltaapxIICDeltaapxIVA double mutant were negative. These data demonstrate that the double mutant DeltaapxIICDeltaapxIVA can be used as an effective live marker vaccine allowing serological differentiation between vaccinated and infected animals.
We describe a highly sensitive and specific multiplex PCR, based on capsular loci and the species specific apxIV gene, that unequivocally differentiates serovar 3, 6, and 8 Actinobacillus pleuropneumoniae strains that are cross-reactive in conventional immunological tests.
Actinobacillus pleuropneumoniae is the etiologic agent of porcine contagious pleuropneumonia, a cause of considerable world wide economic losses in the swine industry. We sequenced the complete genome of A. pleuropneumoniae, JL03, an isolate of serotype 3 prevalent in China. Its genome is a single chromosome of 2,242,062 base pairs containing 2,097 predicted protein-coding sequences, six ribosomal rRNA operons, and 63 tRNA genes. Preliminary analysis of the genomic sequence and the functions of the encoded proteins not only confirmed the present physiological and pathological knowledge but also offered new insights into the metabolic and virulence characteristics of this important pathogen. We identified a full spectrum of genes related to its characteristic chemoheterotrophic catabolism of fermentation and respiration with an incomplete TCA system for anabolism. In addition to confirming the lack of ApxI toxin, identification of a nonsense mutation in apxIVA and a 5'-proximal truncation of the flp operon deleting both its promoter and the flp1flp2tadV genes have provided convincing scenarios for the low virulence property of JL03. Comparative genomic analysis using the available sequences of other serotypes, probable strain (serotype)-specific genomic islands related to capsular polysaccharides and lipopolysaccharide O-antigen biosyntheses were identified in JL03, which provides a foundation for future research into the mechanisms of serotypic diversity of A. pleuropneumoniae.
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.
Serotypes 3 and 8 of Actinobacillus pleuropneumoniae, the aetiological agent of porcine pleuropneumonia, have been reported to predominate in the UK. Direct serotyping of isolates of the organism is typically determined by the immunological reactivity of rabbit serum to its surface polysaccharides, but the method has limitations, for example, cross-reactions between serotypes 3, 6 and 8. This study describes the development of a serotype 3-specific pcr, based on the capsule locus, which can be used in a multiplex format with the organism's specific gene apxIV. The pcr test was evaluated on 266 strains of A pleuropneumoniae and 121 strains of other organisms, including all the major respiratory bacterial pathogens of pigs. The test was highly specific and sensitive and should be useful for differentiating strains of serotypes 3, 6 and 8, and in seroprevalence and epidemiological surveys in regions where serotype 3 is prevalent, such as the UK.
Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, which causes significant losses in industrialized swine production worldwide. Bacterial diagnosis of contagious porcine pleuropneumonia is generally done by bacteriological isolation and cultivation of A. pleuropneumoniae, followed by serological typing which differentiates 15 serotypes (1–3). There are significant differences in virulence among the 15 serotypes. Serotypes 1,5, and also 9 and 11 are involved in particularly severe outbreaks of disease with high mortality and severe pulmonary lesions. Serotypes 2-4, 6-8, 12, and 15 are generally less virulent, causing moderate mortality but relatively strong lung lesions. Serotype 3 seems to be mostly of low epidemiological importance, while the remaining serotypes are isolated only very rarely. The degree of virulence seems to be mainly due to the presence of one or two of the pore-forming RTX toxins ApxI, ApxII, and ApxIII, found in A. pleuropneumoniae, which characterize the different serotypes of A. pleuropneumoniae (4,5) (see Table 1). Toxin ApxI is encoded by the genes apxICABD, with gene A specifying the structural protein toxin, C coding for its activator, and B and D coding for the corresponding type I secretion pathway. ApxI is produced and secreted by the highly pathogenic serotypes 1, 5, 9, and 11, and also in serotype 10 and 14 of A. pleuropneumoniae. ApxII, encoded by apxIICA, is produced by all serotypes except 10 and 14. Table 1 Recipe for Preparation of pH 4–8 IPG IEF Gel Presence of the Various apx genes in Different Serotypes of A. pleuropneumoniae and Apx Toxin that is Actively Secreted Reference apxI apxII pxIII Secreted Serotype strain operon operon operon Apx toxin 1 4074 C A B D C A I + II 2 S1536 B D C A C A B D II + III 3 S1421 C A C A B D III 4 M62 B D C A C A B D II + III 5a K17 C A B D C A I + II 5b L20 C A B D C A I + II 6 Fem B D C A C A B D II + III 7 WF83 B D C A II 8 405 B D C A C A B D II + III 9 CVI 13261 C A B D C A I + II 10 13039 C A B D I 11 56153 C A B D C A I + II 12 8329 B D C A II 13 N273 B D C A II 14 3906 C A B D I 15 HS143 B D C A C A B D II + III
The objective of this study was to characterize Actinobacillus pleuropneumoniae (App) strains which have been isolated in Poland by using the following genotyping techniques: restriction endonuclease analysis (REA), pulsed-field gel electrophoresis (PFGE) and rRNA gene restriction analysis (ribotyping). In total, 60 field and 13 reference strains of App have been used in this study. The results of REA have shown that the patterns of field isolates of a given serotype were all similar to one another and to their respective reference strains. Additionally, numerous common fragments have caused the technique of low value to be a sensitive epidemiological tool for the study of App. Pulsed-field gel electrophoresis was demonstrated to be capable of providing useful information on the relationship amongst field isolates of App belonging either to the same serotype or different serological types of that pathogen. Ribotyping was either as sensitive or specific, in epidemiological terms, as PFGE. This technique has been able to subdivide apparently related isolates of App into genetic subtypes. Moreover, the strains that have different ribotyping patterns can be confidently regarded as having a different source. In conclusion, we have discovered that PFGE and ribotyping have the highest discriminatory power for differentiating strains of Actinobacillus pleuropneumoniae and should be recommended as tools in the epidemiological tracing of that pathogen.
This paper discusses the possible role of virulence factors of Actinobacillus pleuropneumoniae in pathogenesis and protection. Special attention is paid to the Apx-exotoxins and to adhesins.
This brief review deals with the effect of diva (Differentiating Infected from VAccinated individuals) vaccines (also termed marker vaccines) on transmission of herpesviruses and pestiviruses in swine and cattle. Pseudorabies and bovine herpesvirus 1 diva vaccines have been demonstrated to reduce transmission of wild-type virus in populations of pigs and cattle in the laboratory as well as in the field. A subunit diva vaccine based on the immunodominant E2 protein of classical swine fever virus that is expressed in the baculovirus system may reduce transmission of wild-type virus among pigs and also transmission from mother to foetuses. A similar diva vaccine against bovine virus diarrhoea infections protected sheep against transplacental transmission of antigenically homologous wild-type virus. Diva vaccines along with their companion diagnostic tests can play a role in control of infections, ultimately leading to eradication of viruses.
The apxIVA gene, a recently discovered RTX determinant of Actinobacillus pleuropneumoniae, was shown to be species-specific. DNA hybridization experiments using probes for various regions of apxIVA revealed that the 3'-terminus of this gene was present in all 14 serotypes of A. pleuropneumoniae but absent from phylogenetically related species. A primer pair spanning this region specifically amplified a 422bp fragment in PCR experiments with DNA from the reference strains of the 14 serotypes and 194 field strains isolated from various geographic locations worldwide. DNA sequence analysis of PCR products derived from all serotypes were identical except in serotypes 3, 8, and 10, which showed minor differences. The PCR did not amplify any product when DNA from 17 different bacterial species closely related to A. pleuropneumoniae was used as template. In addition, the PCR was negative with DNA of several Actinobacillus sp. which were initially characterized as A. pleuropneumoniae using routine phenotypic and serological analyses but which were subsequently shown by 16S rRNA sequence analysis to belong to yet undefined Actinobacillus species. The sensitivity of the PCR was determined to be 10pg of A. pleuropneumoniae DNA. A set of nested primers amplified a 377bp fragment specifically with A. pleuropneumoniae DNA. DNA titration experiments using the flanking and nested primer pairs showed an improved level of sensitivity to approximately 10fg of genomic DNA. The nested PCR was used to monitor the spread of A. pleuropneumoniae in pigs experimentally infected with a virulent serotype 1 strain and housed in a controlled environment facility. A. pleuropneumoniae DNA could be detected by nested PCR in nasal swab samples of infected pigs receiving either a high dose (5x10(5)) or a low dose (1x10(4)) challenge and in unchallenged cohorts that were contact-infected by the inoculated animals. Furthermore, PCR confirmed the presence of A. pleuropneumoniae in 16/17 homogenates from necrotic lung lesions, while the bacterium was successfully recovered from 13 of these lesions by culture.
Actinobacillus pleuropneumoniae causes porcine pleuropneumonia, a highly contagious disease for which there is no effective vaccine. This review considers how adhesins, iron-acquisition factors, capsule and lipopolysaccharide, RTX cytotoxins and other potential future vaccine components contribute to colonisation, to avoidance of host clearance mechanisms and to damage of host tissues.
We have investigated the use of a natural transformation protocol to introduce mutations into Actinobacillus pleuropneumoniae serotypes 1 and 5b. For both strains tested, we recovered 1 in 10(8) transformants during culture in rich medium, a result that was independent of the growth phase. This low frequency of transformation of A. pleuropneumoniae did not increase when bacteria were grown under conditions known to be optimal for induction of competence in Haemophilus influenzae. Using linearised plasmid DNA containing a kanamycin cassette inserted into the sodC gene of A. pleuropneumoniae serotype 1, we showed that natural transformation can be used as a simple method for introducing allele replacements into this bacterium, and can be used to transfer mutations from one serotype to another.
A genetic typing method utilizing PCR for the identification of Actinobacillus pleuropneumoniae serotype 2 isolates has been developed based on the in vitro amplification of a 1.4 kb DNA segment of the serotype 2 capsular polysaccharide genes cps2AB. The assay was tested with all serotype reference strains and a collection of 92 different A. pleuropneumoniae strains of all 15 serotypes of both biovars I and II, originating from 18 different countries worldwide. The cps2 based PCR identified the serotype 2 reference strain and all 12 serotype 2 collection strains contained in this set. DNA was not amplified from the remaining A. pleuropneumoniae reference and collection strains, indicating the PCR assay was highly specific. Furthermore, the PCR method detected all 31 A. pleuropneumoniae serotype 2 field isolates from diseased pigs that were identified in parallel as serotype 2 by agar gel diffusion. The serotype 2 PCR assay proved to be highly specific and reliable for the identification of serotype 2 isolates of A. pleuropneumoniae.
Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, which causes worldwide severe losses in pig farming. The virulence of the 15 serotypes of A. pleuropneumoniae is mainly determined by the three major RTX toxins ApxI, ApxII and ApxIII, which are secreted by the different serotypes in various combinations. A fourth RTX toxin, ApxIV, is produced by all 15 serotypes only during infection of pigs, but not under in vitro conditions. Pigs infected with A. pleuropneumoniae show specific antibodies directed against ApxIV. In contrast, antibodies against the other three toxins ApxI, ApxII and ApxIII are also found in pigs free of A. pleuropneumoniae. The antibodies to the three latter might result from other, less pathogenic Actinobacillus species such as A. rossii and A. suis. We used a recombinant protein based on the N'-terminal part of ApxIV to serologically detect A. pleuropneumoniae infections in pigs by immunoblot analysis. The analysis of sera of experimentally infected pigs revealed that ApxIV-immunoblots detected A. pleuropneumoniae infections in the second to third week post infection. We developed an indirect ELISA based on the purified recombinant N'-terminal moiety of ApxIV. The analysis of sera from pigs that were experimentally or naturally infected by A. pleuropneumoniae, and of sera of pigs that were free of A. pleuropneumoniae, revealed that the ELISA had a specificity of 100% and a sensitivity of 93.8%. The pre-validation study of the ApxIV-ELISA revealed that the latter was able to detect A. pleuropneumoniae-positive herds, even when clinical and pathological signs of porcine pleuropneumonia were not evident. Pigs vaccinated with a subunit vaccine Porcilis App were serologically negative in the ApxIV-ELISA.
This paper discusses what can be expected with regard to efficacy of antibacterial vaccines used in swine, based on the present knowledge of pathogen-host interactions. First, vaccination against bacteria that mainly cause disease by production of exotoxins is considered. Vaccines containing the inactivated toxin or a non-toxic but antigenic recombinant protein derived from the exotoxin can be expected to provide protection against disease. The degree of protection induced by such vaccines varies, however, depending amongst other things on the pathogenesis of the disease. Vaccination against clostridial infections, Actinobacillus pleuropneumoniae infections, progressive atrophic rhinitis and enterotoxigenic Escherichia coli, is considered. The second part of the article deals with vaccination against extracellular bacteria. Protection against these bacteria is generally mediated by antibodies against their surface antigens and certain secreted antigens, but cellular immunity may also play a role. Efficacy of vaccines against swine erysipelas, Streptococcus suis infections, Mycoplasma hyopneumoniae infections and swine dysentery is discussed. Finally, vaccination against facultatively intracellular bacteria is considered. For protection against these bacteria cell-mediated immunity plays an important role, but antibodies may also be involved. It is generally accepted that live-attenuated vaccines are more suitable for induction of cell-mediated immunity than inactivated vaccines, although this also depends on the adjuvant used in the vaccine. As an example, vaccination against Salmonella enterica serotype Typhimurium is discussed.
Recent advances in molecular biology, immunology, microbiology, genetics and microbial pathogenesis have lead to the development of a wide variety of new approaches for developing safer and more effective vaccines based on designs such as subunit vaccines, gene deleted vaccines, live vectored vaccines, and DNA mediated vaccines. Technology tools can be as basic as identifying naturally occurring strains with deletions that support differentiating infected from vaccinated animal (DIVA) needs or be based on higher technology developments such as improved protein expression and purification methods, transgenic plant- and plant virus-based antigen production, and novel adjuvants that target specific immune responses. These new approaches, when applied to the development of marker vaccines and companion diagnostic test kits hold tremendous potential for developing improved tools for eradication and control programs.
Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia which leads to high economic losses in the swine industry worldwide. Vaccination against this pathogen is hampered by the occurrence of 15 serotypes, and commonly used whole cell bacterin vaccines are not sufficiently cross-serotype protective. In addition, for generating and maintaining specified pathogen-free herds it is desirable to use DIVA (differentiating infected from vaccinated animals) vaccines. Based on a detergent wash extraction of outer membrane associated proteins and secreted proteins we developed a DIVA vaccine using the immunogenic ApxII toxin which is present in 13 of the 15 A. pleuropneumoniae serotypes as the DIVA antigen. The apxIIA gene was deleted in one strain each of serotypes 1, 2, and 5 using a single-step transconjugation system, and equal parts of detergent washes from these strains served as the vaccine antigen. After intramuscular immunisation all pigs developed a strong humoral immune response to the vaccine antigen and showed no reactivity in an ApxIIA ELISA. Upon challenge all pigs were completely protected from clinical symptoms in trials with a homologous (serotype 2) as well as with a heterologous strain (serotype 9); in addition, colonisation of the challenge strain was clearly reduced but not abolished completely. As a result of the highly efficient protection, however, immunised pigs did not develop antibodies to the DIVA-antigen at levels detectable by ELISA but only by a more sensitive Western blotting approach, thereby demonstrating the challenge in developing appropriate marker vaccines for the livestock industry.
Actinobacillus pleuropneumoniae, a gram-negative rod of the Pasteurellaceae family, causes pleuropneumonia in pigs. Establishing A. pleuropneumoniae free herds is difficult due to the occurrence of persistently infected animals. The ApxIV toxin is expressed by A. pleuropneumoniae in vivo and an ELISA based on the toxin is used to detect infection and to differentiate between infected and vaccinated animals. In this study, we have identified a 1070bp insertion element of the IS30 family, designated ISApl1, in the A. pleuropneumoniae serotype 7 strain AP76. ISApl1 contains a 924bp ORF encoding a transposase, which is flanked by 27bp inverted repeats showing six mismatches. We investigated the occurrence of ISApl1 in other A. pleuropneumoniae strains, and its possible interference with virulence associated factors. Four insertion sites were identified in AP76: within the apxIVA toxin ORF, within a putative autotransporter adhesin ORF, upstream of a capsular polysaccharide biosynthesis gene cluster, and downstream of a beta-lactamase gene. ISApl1 is also present in some serotype 7 field isolates, but not in reference or field strains of other serotypes. In A. pleuropneumoniae AP76, the transposase gene is transcribed in vitro. The insertion in the apxIVA toxin gene remains stable after animal passage. Since this insertion should disrupt toxin expression, we tested 7 pigs infected with AP76 at day 21 post-infection. All were negative in the ApxIV ELISA but four out of seven were positive in an ApxII toxin ELISA. These results show that insertion elements can affect the detection of A. pleuropneumoniae infected animals.
A PCR assay for simultaneous species identification and separation of Actinobacillus pleuropneumoniae serovars 1, 7 and 12 was developed. Primers specific for genes involved in biosynthesis of the capsular polysaccharides (cps genes) of serovars 1, 7, and 12 were combined with a species-specific PCR test based on the omlA gene. The PCR test was evaluated with the serovar reference strains of A. pleuropneumoniae as well as 183 Danish field isolates. For all typable strains, a complete correspondence was found between results obtained with the multiplex PCR test and results from the traditional serotyping methods. Among eight serologically cross-reacting strains designated K1:O7, seven isolates produced amplicons of similar sizes as serovar 1 and one isolate produced amplicons of similar sizes as serovar 7. The species specificity of the assay was evaluated using a collection of 126 strains representing 25 different species within the family Pasteurellaceae including 45 field strains of the phylogenetically affiliated species Actinobacillus lignieresii. All these isolates tested negative for the cps genes by the multiplex PCR test except for 6 isolates of A. lignieresii. Five of these isolates produced an amplicon identical to the cps gene of serovar 7, whereas one isolate produced an amplicon identical to the cps gene of serovar 1. In addition, four isolates of Actinobacillus genomospecies 1 tested positive for the omlA gene but negative for the cps genes. The test represents a convenient and specific method for serotyping A. pleuropneumoniae in diagnostic laboratories.
Actinobacillus pleuropneumoniae
- Dj Taylor
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Taylor DJ, Gottschalk M. Actinobacillus pleuropneumoniae. In: Straw BE, Zim-merman JZ, D'Allaire S, Taylor DJ, editors. Diseases of swine. Ames: Iowa State University Press; 2006. p. 563–76.
Actinobacillus pleuropneumoniae serovar prevalence in England and Wales
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- Jt Bossé
- Cm Watson
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O'Neill C, Jones SCP, Bossé JT, Watson CM, Williamson SM, Rycroft AN, et al. Actinobacillus pleuropneumoniae serovar prevalence in England and Wales. Vet Rec, in press. C. O'Neill et al. / Vaccine 28 (2010) 4871–4874
Actinobacillus pleuropneumoniae strains
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Harnessing natural transformation in Actinobacillus pleuropneumoniae: a simple method for allelic replacements
- Bossé