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

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

The genetic organisation of the capsule biosynthesis region of Actinobacillus pleuropneumoniae serotypes 1, 6, 7, and 12

The aim of the present study was to investigate the organisation of the genes (cps) involved in biosynthesis the capsular polysaccharide (CPS) of Actinobacillus pleuropneumoniae serotypes 6, 7, and 12 and to compare these to the corresponding genes previously described in other A. pleuropneumoniae serotypes. In serotypes 6 and 7 the sequenced DNA regions comprised five and four open reading frames, respectively, designated cps6ABCDE and cps7ABCD, whereas the sequenced DNA region in serotype 12 comprised only two open reading frames designated cps12AB. At the amino acid level, CpsA, CpsB, and CpsC of A. pleuropneumoniae serotypes 2, 6, 7, and 8 contained a high degree of homology. At the amino acid level Cps6D revealed a high degree of homology to Cps8D, whereas Cps7D contained a high degree of homology to the Cps2D. The deduced gene product of the partially sequenced cps6E gene showed no homology to any deduced gene products of any cps genes of A. pleuropneumoniae investigated so far. None of the deduced gene products of the cps genes involved in encapsulation of A. pleuropneumoniae serotypes 2, 6, 7, and 8 revealed homology to the deduced gene products of the cps genes of serotypes 1, 5A, and 12. For some genes, a local homology was found to genes probably involved in teichoic acid synthesis in other bacteria. The results obtained revealed a high degree of homology among the genes involved in CPS biosynthesis for serotypes 2, 6, 7, and 8 and a different group of homologous cps genes for serotypes 1 and 12. In some serotype 7 strains, including the serotype 7 reference strain, WF83, the cps genes were not located adjacent to the genes responsible for CPS export (cpx), probably due to genetic rearrangements.     

Differentiation of Twelve Actinobacillus pleuropneumoniae Serotypes by Outer Membrane Lipoprotein Gene‐based Restriction Fragment Length Polymorphism

Twelve Actinobacillus pleuropneumoniae serotypes were differentiated by restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR)‐amplified fragments from the outer membrane lipoprotein (omlA) gene. All 12 reference serotypes and 80 field isolates produced the expected 950‐base pair (bp) fragment of the omlA gene by PCR. Combining the RFLP patterns obtained with SfaNI, Bst71I, AluI, NciI, nine distinct patterns were observed in the 12 serotype reference strains. The PCR‐based RFLP analysis of omlA genes allows differentiation among the 12 serotypes, with the exception of group 1 (serotypes 1, 9 and 11), and group 2 (serotypes 2 and 8). When the PCR products from the 70 field isolates were subjected to RFLP analysis, 68 showed the same RFLP patterns as their respective serotype reference strain. Two isolates that could not be typed had the same RFLP patterns as those of serotype 5. These results suggest that PCR‐based RFLP analysis of the omlA genes may be of value in differentiating among 12 A. pleuropneumoniae serotypes.     

DNA microarray-based identification and typing of Actinobacillus pleuropneumoniae

A DNA microarray system was prepared and shown to facilitate identification and typing of Actinobacillus pleuropneumoniae. The DNA microarray, composed of 18 DNA polymerase chain reaction (PCR) amplicons printed on glass slides and arranged in 3 subarrays, was developed. These target DNA included 1 or multiple fragments of the outer membrane lipoprotein, apx toxin, capsular polysaccharide, and disulfide bound formation protein E (dsbE)-like genes of A. pleuropneumoniae. These arrayed target DNA retained their expceted hybridization properties. The hybridization signal intensities ranged from the least-intense to the most-intense, 4626 to 9789 arbitrary fluorescence units, respectively. Cy3-probes of A. pleuropneumoniae strains labeled with multiplex PCR were hybridized to the DNA microarray. A total of 51 different A. pleuropneumoniae strains representing serotype 1 to 12 reference strains and clinical isolates were detected and typed by the DNA microarray. Twelve reference serotypes produced 11 distinct target DNA hybridization patterns, and hybridization patterns of serotypes 1 (n = 7), 3 (n = 5), and 7 (n = 6) field isolates were identical to hybridization patterns of reference serotypes 1, 3, and 7, respectively. Non-serotyped isolates 4, 6, and 11 (out of 21) from diseased pigs had identical hybridization patterns to reference serotypes 3, 7, and 1, respectively. The results show that the DNA microarray system described in the present study is a valuable tool for identifying and typing reference strains and isolates of A. pleuropneumoniae, and enables relatively rapid identification of non-serotyped isolates.     

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.     

Long-chain LPS-based enzyme-linked immunosorbent assay to detect swine herds infected by Actinobacillus pleuropneumoniae serotype 17

Actinobacillus pleuropneumoniae serotype 17, one of the two most recent serotypes described, has been isolated from diseased pigs in North America. Yet, no serological test for surveillance has been developed so far. An enzyme-linked immunosorbent assay (ELISA) using the long-chain lipopolysaccharide antigen (LC-LPS) of this serotype is described. As predicted by previous genetic data on the O-antigen locus, cross reactions were observed between this serotype and serotypes 3, 6, 8, and 15. Although animals infected by serotype 17 would be detected using the current serotype 3 LC-LPS ELISA, better results may be obtained when plates are coated with the antigen purified from the homologous serotype.     

Evaluation and application of ribotyping for epidemiological studies of Actinobacillus pleuropneumoniae in Denmark

The aim of the present study was to evaluate ribotyping as an epidemiological tool for Actinobacillus pleuropneumoniae and apply the method in studies of A. pleuropneumoniae infections in Danish pig herds. The evaluation of ribotyping was based on the 13 international reference strains and 106 epidemiologically unrelated Danish field strains representing the nine serotypes of biotype 1 (1, 2, 5A/B, 6, 7, 8, 10, 12, and K2:O7) and one serotype 14 of biotype 2. Enzymes CfoI and HindIII were chosen for generation of ribotype patterns. Ribotyping of the reference strains resulted in 10 CfoI types and 11 HindIII types. Ribotyping of the Danish strains resulted in 17 different CfoI ribotypes and 24 different HindIII ribotypes. Combining HindIII- and CfoI-ribotyping divided the Danish strains into 26 different types. The stability, reproducibility and typability of ribotype patterns were good, and the discriminatory power was between 0.85–0.89. The relatively low discriminatory power was caused by four predominant types, containing 61% of the isolates. The typing system was applied in studies of routes of infection of specific pathogen-free (SPF) pig herds and included 112 strains of A. pleuropneumoniae. Airborne transmission from neighboring conventional pig farms was investigated in 12 cases of infected SPF herds. Transmission via vehicles transporting pigs between SPF herds was investigated in nine cases while transmission by trading of pigs between SPF herds was investigated in two cases. Serotype 2 was isolated from all SPF herds included in this study, except one, emphasizing the high prevalence of this serotype in Denmark. By ribotyping, airborne transmission was indicated in five of 12 cases, transmission via pig transporting vehicle was indicated in six of nine cases, and transmission via trading was indicated in one of two cases. In many cases findings of predominant ribotypes made interpretations of suspected routes of transmission difficult. The relationship of strains based on ribotypes was calculated using Dices coefficient and clustered by UPGMA. HindIII ribotypes of serotype 2 strains were closely related, though only showing 43% similarity to HindIII ribotypes of remaining serotypes.     

Identification and preliminary characterization of a 75-kDa hemin- and hemoglobin-binding outer membrane protein of Actinobacillus pleuropneumoniae serotype 1

The reference strains representing serotypes 1 to 12 of Actinobacillus pleuropneumoniae biotype 1 were examined for their ability to utilize porcine hemoglobin (Hb) or porcine hemin (Hm) as iron sources for growth. In a growth promotion assay, all of the reference strains were able to use porcine Hb, and all strains except 2 were able to use porcine Hm. Using a preliminary characterization procedure with Hm- or Hb-agarose, Hm- and Hb-binding outer membrane proteins (OMPs) of approximately 75 kDa were isolated from A. pleuropneumoniae serotype 1 strain 4074 grown under iron-restricted conditions. Matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) analysis revealed a number of common tryptic peptides between the Hb-agarose- and Hm-agarose-purified 75 kDa OMPs, strongly suggesting that these peptides originate from the same protein. A database search of these peptide sequences revealed identities with proteins from various Gram-negative bacteria, including iron-regulated OMPs, transporter proteins, as well as TonB-dependent receptors. Taken together, our data suggest that A. pleuropneumoniae synthesizes potential Hm- and Hb-binding proteins that could be implicated in the iron uptake from porcine Hb and Hm.     

Role of the capsular polysaccharide as a virulence factor for Streptococcus suis serotype 14

Streptococcus suis is an important swine pathogen and a zoonotic agent causing meningitis and septicemia. Although serotype 2 is the most virulent type, serotype 14 is emerging, and understanding of its pathogenesis is limited. To study the role of the capsular polysaccharide (CPS) of serotype 14 as a virulence factor, we constructed knockout mutants devoid of either cps14B, a highly conserved regulatory gene, or neu14C, a gene coding for uridine diphospho-N-acetylglucosamine 2-epimerase, which is involved in sialic acid synthesis. The mutants showed total loss of the CPS with coagglutination assays and electron microscopy. Phagocytosis assays showed high susceptibility of mutant Δcps14B. An in vivo murine model was used to demonstrate attenuated virulence of this non-encapsulated mutant. Despite the difference in the CPS composition of different serotypes, this study has demonstrated for the first time that the CPS of a serotype other than 2 is also an important antiphagocytic factor and a critical virulence factor.     

Molecular heterogeneity of plpE gene in Indian isolates of Pasteurella multocida and expression of recombinant PlpE in vaccine strain of P. multocida serotype B: 2

Outer membrane proteins of Pasteurella (P.) multocida have been known to be protective immunogens. Pasteurella lipoprotein E (PlpE) has been reported to be an important cross reactive outer membrane protein in P. multocida. The gene encoding the PlpE of P. multocida serotypes A: 3, B: 2 and D: 1 was amplified from the genomic DNA. The amplified products were cloned and the nucleotide sequence was determined. Sequence analysis of the recombinant clones revealed a single open reading frame of 1,011 bp, 1,008 bp and 1,017 bp encoding a protein with a calculated molecular mass of 37.829 kDa, 37.389 kDa and 37.965 kDa for serotypes A: 3, B: 2 and D: 1 respectively. The comparison of the plpE sequence in different capsular types revealed a high degree (>90%) of homology. Furthermore, the plpE gene of Haemorhhagic septicaemia causing serotype (B: 2) was expressed in E. coli and recombinant PlpE was strongly immunostained by antiserum against whole cell antigen, indicating that the protein is expressed in vivo.     

Serological characterization of Actinobacillus pleuropneumoniae biotype 2 strains isolated from pigs in two Danish herds

Eight Actinobacillus pleuropneumoniae biotype 2 strains were isolated in pure culture from lungs of pigs originating from two Danish herds with growing and finishing pigs. The antigenic properties were studied by indirect haemagglutination (IHA) and immunodiffusion (ID) tests using soluble surface antigens and by enzyme-linked immunosorbent assays (ELISA) using capsular enriched fractions and LPS. In all tests the strains proved antigenically homogeneous and serologically distinct from the known biotype 1 and 2 serotypes. Thus, the strains represent a new serotype which is provisionally proposed as biotype 2 serotype 14.     

Serological characterization and antimicrobial susceptibility of Streptococcus suis isolates from diagnostic samples in Denmark during 1995 and 1996

At the Danish Veterinary Laboratory Streptococcus suis infections in pigs were diagnosed in 114 cases in 1995 and in 151 cases in 1996. Isolates were serotyped using specific antisera against type 1 through 28 and a total of 67 cases from 1995 and 113 cases in 1996 were tested for resistance to 11 antimicrobial agents. The majority of cases were lung diseases (57%), followed by septicaemia (16%), meningitis (15%) and endocarditis (8%). Almost 96% of the isolates could be typed using the 28 antisera. The most common serotype was serotype 2 (29%), followed by serotype 7 (17%), and serotypes 3, 4 and 8 (9–10%). The remaining serotypes were observed in frequencies of less than 5%. Serotype 7 was more commonly isolated from septicaemia than the other serotypes. Serotype 2 was more commonly isolated from pigs older than 4 weeks compared to the other serotypes. Most isolates were susceptible to amoxycillin+clavulanate, ampicillin, ceftiofur, enrofloxacin, penicillin, spectinomycin, tiamulin and trimethoprim+sulphadiazine. A high frequency (>30%) of resistance to tetracycline was observed. Among isolates of serotype 2, 9.7% were resistant to lincomycin and 12.9% to spiramycin. Among other serotypes 56.8% were resistant to lincomycin and spiramycin. The differences in susceptibility between isolates of serotype 2 and the other serotypes were statistically significant. Compared to a previous Danish study the distribution of serotypes of S. suis causing infections among pigs in Denmark has changed during the last 15 years.     

Disease patterns and immune responses in the offspring to sows with high or low antibody levels to Actinobacillus pleuropneumoniae serotype 2

The serum antibody responses to Actinobacillus pleuropneumoniae and the secondary invader Pasteurella multocida were monitored from birth until slaughter in the offspring to sows with high or low levels of serum antibodies to A. pleuropneumoniae.Serum antibody concentrations to A. pleuropneumoniae were higher from birth to the age of 9 weeks in piglets delivered by high responding sows. In contrast, antibody levels to P. multocida were similar in both groups during this period. From the age of 20 and 15 weeks, antibody levels to A. pleuropneumoniae and P. multocida, respectively, were higher in the offspring to high responding sows.This implies that the offspring to sows with high levels of antibodies may be better protected during the first period of life because of a higher level of passively derived immunity. These piglets will also mount a higher antibody response when later infected, indicating a heritability of the humoral immune response.     

PCR-based identification of serotype 2 isolates of Actinobacillus pleuropneumoniae biovars I and II

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.     

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).     

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.     

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.     

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.     

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.