ABSTRACT
Actinobacillus pleuropneumoniae is responsible for porcine pleuropneumonia, a highly contagious lung infection. The control of this respiratory disease remains heavily reliant on antibiotics, with phenicols being one of the primary classes of antibiotics used in pig farming. In the present study, we describe three isolates (B2278, B2176 and B2177) of A. pleuropneumoniae resistant to florfenicol attributed to the presence of the floR gene, which were obtained from two pig farms in Italy. Florfenicol susceptibility tests indicated that B2176 exhibited an intermediate susceptibility profile, while B2177 and B2278 were resistant. All three isolates belonged to serovar 6 and tested positive for the presence of the floR gene. Whole genome sequencing analysis revealed that isolates B2176, B2177 and B2278 harbored genes encoding the toxins ApxII and ApxIII, characteristic of strains with moderate virulence. Moreover, phylogenetic analysis demonstrated that these isolates were closely related, with single nucleotide polymorphisms (SNPs) ranging from 8 to 19. The floR gene was located on a novel 5588â¯bp plasmid, designated as pAp-floR. BLASTN analysis showed that the pAp-floR plasmid had high nucleotide identity (99â¯%) and coverage (60â¯%) with the pMVSCS1 plasmid (5621â¯bp) from Mannheimia varigena MVSCS1 of porcine origin. Additionally, at least under laboratory conditions, pAp-floR was stably maintained even in the absence of direct selective pressure, suggesting that it does not impose a fitness cost. Our study underscores the necessity of monitoring the spread of florfenicol-resistant A. pleuropneumoniae isolates in the coming years.
Subject(s)
Actinobacillus Infections , Actinobacillus pleuropneumoniae , Anti-Bacterial Agents , Drug Resistance, Bacterial , Swine Diseases , Thiamphenicol , Animals , Actinobacillus pleuropneumoniae/drug effects , Actinobacillus pleuropneumoniae/genetics , Actinobacillus pleuropneumoniae/isolation & purification , Actinobacillus pleuropneumoniae/classification , Thiamphenicol/analogs & derivatives , Thiamphenicol/pharmacology , Swine , Italy/epidemiology , Swine Diseases/microbiology , Anti-Bacterial Agents/pharmacology , Actinobacillus Infections/microbiology , Actinobacillus Infections/veterinary , Drug Resistance, Bacterial/genetics , Phylogeny , Microbial Sensitivity Tests , Whole Genome Sequencing , Farms , Pleuropneumonia/microbiology , Pleuropneumonia/veterinary , Plasmids/genetics , Bacterial Proteins/genetics , Polymorphism, Single Nucleotide , Virulence/geneticsABSTRACT
Actinobacillus pleuropneumoniae (APP) causes significant economic losses to the swine industry. Antibiotic treatment can be challenging due to its clinical urgency and the turnover of antimicrobial susceptibility results from the diagnostic laboratory. The aim of this study was to evaluate the vertical transmission of APP within integrated systems as a criterion for optimising antimicrobial treatment in the field, using whole genome sequencing (WGS). Additionally, the genetic variability of Spanish APP isolates has been assessed to decipher antimicrobial resistance (AMR) determinants, toxin presence, serotype, and phenotype/genotype concordance of AMR. A total of 169 isolates from clinical cases of porcine pleuropneumonia with known antimicrobial susceptibility profiles were sequenced. Additionally, 48 NCBI assemblies were included to perform a phylogenetic analysis. Phylogenetic analysis revealed high association between phylogenetic clusters, serotypes, and presence of toxins that are associated within vertically integrated systems by its epidemiological link. Concordance between presence of AMR determinants (genotype) vs in-vitro antimicrobial susceptibility pattern (phenotype) was acceptable for amoxicillin, florfenicol, oxytetracycline, and enrofloxacin using epidemiological cut-off values (ECOFFs), but low concordance was observed for doxycycline and trimethoprim-sulfamethoxazole (T/S). On the other hand, using CLSI clinical breakpoints (CBPs), concordance was acceptable for florfenicol and enrofloxacin and not evaluated for doxycycline, oxytetracycline, trimethoprim-sulfamethoxazole (T/S), and amoxicillin because no CBP are available for them. Finally, WGS has demonstrated the clonality between isolates that shared a common origin (grandmother's farm) and resistance phenotype, suggesting vertical transmission of this pathogen and supporting the use of the epidemiological approach as a good criterion to optimise the antimicrobial use.
Subject(s)
Actinobacillus Infections , Actinobacillus pleuropneumoniae , Anti-Bacterial Agents , Microbial Sensitivity Tests , Phylogeny , Swine Diseases , Whole Genome Sequencing , Actinobacillus pleuropneumoniae/genetics , Actinobacillus pleuropneumoniae/drug effects , Actinobacillus pleuropneumoniae/classification , Actinobacillus pleuropneumoniae/isolation & purification , Swine , Animals , Actinobacillus Infections/microbiology , Actinobacillus Infections/veterinary , Actinobacillus Infections/transmission , Swine Diseases/microbiology , Swine Diseases/transmission , Anti-Bacterial Agents/pharmacology , Pleuropneumonia/microbiology , Pleuropneumonia/veterinary , Genotype , Genome, Bacterial , Drug Resistance, Bacterial/genetics , Spain/epidemiologySubject(s)
Actinobacillus pleuropneumoniae , Serogroup , Streptococcal Infections , Streptococcus suis , Swine Diseases , Animals , Swine Diseases/microbiology , Swine , Quebec/epidemiology , Streptococcus suis/isolation & purification , Streptococcal Infections/veterinary , Streptococcal Infections/microbiology , Actinobacillus pleuropneumoniae/isolation & purification , Actinobacillus pleuropneumoniae/classification , Actinobacillus pleuropneumoniae/genetics , Actinobacillus Infections/veterinary , Actinobacillus Infections/microbiologyABSTRACT
Actinobacillus pleuropneumoniae causes porcine pleuropneumonia, an important disease in the pig industry. Accurate and sensitive diagnostics such as DNA-based diagnostics are essential for preventing or responding to an outbreak. The specificity of DNA-based diagnostics depends on species-specific markers. Previously, an insertion element was found within an A. pleuropneumoniae-specific gene commonly used for A. pleuropneumoniae detection, prompting the need for additional species-specific markers. Herein, 12 marker candidates highly conserved (99 - 100% identity) among 34 A. pleuropneumoniae genomes (covering 13 serovars) were identified to be A. pleuropneumoniae-specific in silico, as these sequences are distinct from 30 genomes of 13 other Actinobacillus and problematic [Actinobacillus] species and more than 1700 genomes of other bacteria in the Pasteurellaceae family. Five marker candidates are within the apxIVA gene, a known A. pleuropneumoniae-specific gene, validating our in silico marker discovery method. Seven other A. pleuropneumoniae-specific marker candidates within the eamA, nusG, sppA, xerD, ybbN, ycfL, and ychJ genes were validated by polymerase chain reaction (PCR) to be specific to 129 isolates of A. pleuropneumoniae (covering all 19 serovars), but not to four closely related Actinobacillus species, four [Actinobacillus] species, or seven other bacterial species. This is the first study to identify A. pleuropneumoniae-specific markers through genome mining. Seven novel A. pleuropneumoniae-specific DNA markers were identified by a combination of in silico and molecular methods and can serve as additional or alternative targets for A. pleuropneumoniae diagnostics, potentially leading to better control of the disease. IMPORTANCE Species-specific markers are crucial for infectious disease diagnostics. Mutations within a marker sequence can lead to false-negative results, inappropriate treatment, and economic loss. The availability of several species-specific markers is therefore desirable. In this study, 12 DNA markers specific to A. pleuropneumoniae, a pig pathogen, were simultaneously identified. Five marker candidates are within a known A. pleuropneumoniae-specific gene. Seven novel markers can be used as additional targets in DNA-based diagnostics, which in turn can expedite disease diagnosis, assist farm management, and lead to better animal health and food security. The marker discovery strategy outlined herein requires less time, effort, and cost, and results in more markers compared with conventional methods. Identification of species-specific markers of other pathogens and corresponding infectious disease diagnostics are possible, conceivably improving health care and the economy.
Subject(s)
Actinobacillus pleuropneumoniae/genetics , Actinobacillus pleuropneumoniae/isolation & purification , Bacterial Proteins/genetics , Pathology, Molecular/methods , Pleuropneumonia/veterinary , Polymerase Chain Reaction/methods , Swine Diseases/microbiology , Actinobacillus pleuropneumoniae/classification , Animals , Genetic Markers , Genome, Bacterial , Pleuropneumonia/diagnosis , Pleuropneumonia/microbiology , Swine , Swine Diseases/diagnosisABSTRACT
The aim of this study was to investigate an isolate of Actinobacillus pleuropneumoniae, named 14-760, which was serologically not classifiable among the recognised serovars of A. pleuropneumoniae. It reacted with the antisera raised against serovars 3, 6, 8, 15 and 17 in the agar gel precipitation (AGP) test, and was positive in the capsular serovar 4-specific PCR (cps4B PCR) assay. The isolate contains a type II capsule locus similar to serovar 4 but with variations in the length of four intergeneric regions (modF-cpxA, cpxD-cpsA, cpsC-a 114 bp orf, and lysA-ydeN), and three gene sequences (modF, cpsC and ydeN). The main difference found between the K4 and K4b cps genes is the additional 35 AAs found in type 4b due to a 4 bp insert in cps4bC. The LPS O-Ag locus is highly similar to that of reference strains of serovars 3, 6, 8, 15, 17 and 19. Isolate 14-760 is biovar 1 and contains solely the structural genes required for toxin ApxII production (apxIICA), and the type I secretion system (apxIBD) for the export of ApxII. Antiserum against isolate 14-760 adsorbed with antigen prepared from serovars 8, 15 or 17 reference strains remained reactive with isolate 14-760, but not with antigens prepared from serovars 1-18. Taken together, our results indicate the existence of a subtype of A. pleuropneumoniae, serovar 4, that we called "K4b:O3â³, and we propose isolate 14-760 as the reference strain.
Subject(s)
Actinobacillus pleuropneumoniae , Bacterial Typing Techniques , Actinobacillus Infections/veterinary , Actinobacillus pleuropneumoniae/classification , Actinobacillus pleuropneumoniae/genetics , Animals , Bacterial Typing Techniques/veterinary , Genotype , Pleuropneumonia/microbiology , Pleuropneumonia/veterinary , Serogroup , Serotyping/veterinary , Swine , Swine Diseases/microbiologyABSTRACT
We report here the complete genome sequence of the widely studied Actinobacillus pleuropneumoniae serovar 8 reference strain 405, generated using the Pacific Biosciences (PacBio) RS II platform. Furthermore, we compared draft sequences generated by Illumina sequencing of six stocks of this strain, including the same original stock used to generate the PacBio sequence, held in different countries and found little genetic variation, with only three SNPs identified, all within the degS gene. However, sequences of two small plasmids, pARD3079 and p405tetH, detected by Illumina sequencing of the draft genomes were not identified in the PacBio sequence of the reference strain.
Subject(s)
Actinobacillus Infections , Actinobacillus pleuropneumoniae , Swine Diseases , Actinobacillus pleuropneumoniae/classification , Actinobacillus pleuropneumoniae/genetics , Animals , Genetic Variation , Genome, Bacterial , High-Throughput Nucleotide Sequencing , Serogroup , SwineABSTRACT
Two serologically and molecularly non-typeable isolates of the porcine lung pathogen Actinobacillus pleuropneumoniae have been identified from diseased swine in two different continents. Genome sequencing was carried out to identify their diagnostically relevant genotypes. Both isolates are biovar 1 and encode genes for production of ApxIV and ApxII (apxIICA structural genes, and apxIBD export genes). They both possess the same novel type II capsule locus (most similar to serovar 1, but with two capsule genes not previously found in A. pleuropneumoniae) but differ in their O-Ag loci. Strain 7213384-1 from Denmark, which we propose as the reference strain for serovar 19, has a serogroup 3/6/8/15 O-Ag locus; the Canadian isolate A08-013 has a serogroup 4/7 O-Ag locus. We have expanded the second of our two previously described A. pleuropneumoniae mPCRs to include capsule gene-specific primers for definitive detection of serovars 13-14 and 16-19.
Subject(s)
Actinobacillus pleuropneumoniae/classification , Bacterial Capsules/classification , Multiplex Polymerase Chain Reaction/methods , Serotyping/methods , Actinobacillus pleuropneumoniae/genetics , Bacterial Capsules/chemistry , DNA, Bacterial/genetics , Genome, BacterialABSTRACT
The RNA chaperone Hfq regulates diverse processes in numerous bacteria. In this study, we compared phenotypes (growth rate, adherence, response to different stress conditions and virulence in Galleria mellonella) of wild-type (WT) and isogenic hfq mutants of three serovars (1, 8 and 15) of the porcine pathogen Actinobacillus pleuropneumoniae. Similar growth in rich broth was seen for all strains except Ap1∆hfq, which showed slightly reduced growth throughout the 24 h time course, and the complemented Ap8∆hfqC mutant had a prolonged lag phase. Differences were seen between the three serovar WT strains regarding adherence, stress response and virulence in G. mellonella, and deletion of hfq affected some, but not all of these phenotypes, depending on serovar. Complementation by expression of cloned hfq from an endogenous promoter only restored some WT phenotypes, indicating that complex regulatory networks may be involved, and that levels of Hfq may be as important as presence/absence of the protein regarding its contribution to gene regulation. Our results support that Hfq is a pleiotropic global regulator in A. pleuropneumoniae, but serovar-related differences exist. These results highlight the importance of testing multiple strains/serovars within a given species when determining contributions of global regulators, such as Hfq, to expression of complex phenotypes.
Subject(s)
Actinobacillus pleuropneumoniae/pathogenicity , Bacterial Adhesion , Host Factor 1 Protein/metabolism , Stress, Physiological , Virulence , Actinobacillus Infections/microbiology , Actinobacillus pleuropneumoniae/classification , Animals , Disease Models, Animal , Gene Deletion , Gene Expression Regulation, Bacterial , Genetic Complementation Test , Host Factor 1 Protein/genetics , Larva/microbiology , Moths/microbiology , Phenotype , Promoter Regions, Genetic , Serogroup , SwineABSTRACT
Actinobacillus pleuropneumonia (APP) causes porcine pleuropneumoniae, resulting in severe economic losses in the swine industry. Since there are diverse serotypes of APP, it is necessary for vaccines to induce cross-protection. In this report, we developed a bivalent fusion vaccine, the L vaccine composed of ApxIA and ApxIIA fragments. According to the experimental results of the L vaccine, recombinant protein specific-IgG antibody level increased significantly as well as Apx toxin specific-IgG antibody, suggesting toxin-neutralizing effect. Also, the production of both IgG1 and IgG2a indicates this fusion vaccine induces Th1 and Th2 immune reactions. In addition, lymphocytes were proliferated and immune related-cytokines of TNF-α, IL-12, IFN-γ and IL-5 were detected in the serum after the vaccination. The L vaccine showed a perfect cross-protection against APP serovar 1 and 2 that each secrete different Apx exotoxins. These findings reveal that the fusion L vaccine induces specific humoral and cellular immunity, leading to a perfect cross-protection against A. pleuropneumoniae infections in a murine model.
Subject(s)
Actinobacillus Infections/prevention & control , Actinobacillus pleuropneumoniae/classification , Bacterial Toxins/immunology , Bacterial Vaccines/immunology , Recombinant Fusion Proteins/immunology , Actinobacillus Infections/mortality , Amino Acid Sequence , Animals , Antibodies, Bacterial/blood , Antibodies, Bacterial/immunology , Bacterial Toxins/administration & dosage , Bacterial Toxins/chemistry , Bacterial Vaccines/administration & dosage , Disease Models, Animal , Immunization , Immunoglobulin G/immunology , Lymphocytes/immunology , Lymphocytes/metabolism , Mice , Recombinant Fusion Proteins/administration & dosageABSTRACT
A total of 255 Actinobacillus pleuropneumoniae isolates were collected from 634 lung samples representing 70 swine herds in Hungary between January 2012 and June 2016. On the basis of the indirect haemagglutination test 77 independent strains were included in the evaluation after the elimination of duplicate or multiple serotypes from the same herd. In the case of 7 herds strains of two different serotypes were identified. Fourteen Hungarian A. pleuropneumoniae isolates from the culture collection of the Department of Microbiology and Infectious Diseases, isolated before 2012, were also included in the evaluation (one each from 12 herds and two each from two herds, where two serotypes occurred). Out of the altogether 91 A. pleuropneumoniae strains 72 strains belonged to biotype I and 19 strains could be allocated to biotype II. In Hungary, the most common serotypes were serotype 2 (39.5%), 13 (15.4%), 8 (8.8%) and 16 (8.8%), but serotypes 9 (5.5%), 11 (3.3%) and 12 (3.3%) were also isolated. Twelve strains (13.2%) were untypable.
Subject(s)
Actinobacillus Infections/veterinary , Actinobacillus pleuropneumoniae/classification , Actinobacillus pleuropneumoniae/genetics , Pleuropneumonia/veterinary , Serogroup , Swine Diseases/microbiology , Actinobacillus Infections/epidemiology , Actinobacillus Infections/microbiology , Animals , Hungary/epidemiology , Lung/microbiology , Pleuropneumonia/microbiology , Swine , Swine Diseases/epidemiologyABSTRACT
SXT/R391 integrative conjugative elements (ICEs) are capable of self-transfer by conjugation and highly prevalent in various aquatic bacteria and Proteus species. In the present study, a novel SXT/R391 ICE, named ICEAplChn1, was identified in the multidrug resistant (MDR) Actinobacillus pleuropneumoniae strain app6. ICEAplChn1 was composed of the typical SXT/R391 backbone and insertion DNA at eight hotspots, including HS1, HS2, HS3, HS4, HS5, VRII, VRIII and a new variation region VRVI. Many of the insertion contents were not present in other reported SXT/R391 family members, including ICEApl2, a recently identified SXT/R391 ICE from a clinical isolate of A. pleuropneumoniae. Remarkably, the VRIII region had accumulated seven resistance genes tet(A), erm(42), floR, aphA6, strB (two copies), strA and sul2. Of them, erm(42) and aphA6 emerged for the first time not only in the SXT/R391 elements but also in A. pleuropneumoniae. Phylogenetic analysis showed considerable variation of the backbone sequence of ICEAplChn1, as compared to those of other SXT/R391 ICEs. A circular intermediate form of ICEAplChn1 was detected by nested PCR. However, the conjugation experiments using different bacteria as recipients failed. These findings demonstrated that SXT/R391 ICEs are able to adapt to a broader range of host bacterial species. The presence of the MDR gene cluster in ICEAplChn1 underlines that SXT/R391 ICE could serve as an important vector for the accumulation of antibiotic resistance genes.
Subject(s)
Actinobacillus Infections/veterinary , Actinobacillus pleuropneumoniae/genetics , Bacterial Proteins/genetics , Conjugation, Genetic , Drug Resistance, Multiple, Bacterial/genetics , Actinobacillus Infections/microbiology , Actinobacillus pleuropneumoniae/classification , Animals , DNA Transposable Elements , DNA, Bacterial/genetics , Multigene Family , Phylogeny , Swine/microbiology , Swine Diseases/microbiologyABSTRACT
Problems with serological cross-reactivity have led to development of a number of PCRs (individual and multiplex) for molecular typing of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia. Most of these assays were developed for detection of specific amplicons within capsule biosynthetic genes before the availability of complete sequences for the different serovars. Here we describe comparative analysis of the complete capsular loci for all 18 serovars of A. pleuropneumoniae, and development of two multiplex PCRs for comprehensive capsule typing of this important pig pathogen.
Subject(s)
Actinobacillus pleuropneumoniae/genetics , Bacterial Capsules/classification , Bacterial Capsules/genetics , Multiplex Polymerase Chain Reaction/methods , Polysaccharides, Bacterial/genetics , Sequence Analysis , Swine Diseases/diagnosis , Actinobacillus Infections/diagnosis , Actinobacillus Infections/microbiology , Actinobacillus pleuropneumoniae/classification , Actinobacillus pleuropneumoniae/pathogenicity , Animals , Bacterial Capsules/chemistry , Serogroup , Serotyping , Swine , Swine Diseases/microbiologyABSTRACT
GALT is an important antigen of Actinobacillus pleuropneumoniae (APP), which was shown to provide partial protection against APP infection in a previous study in our lab. The main purpose of the present study is to investigate GALT induced cross-protection between different APP serotypes and elucidate key mechanisms of the immune response to GALT antigenic stimulation. Bioinformatic analysis demonstrated that galT is a highly conserved gene in APP, widely distributed across multiple pathogenic strains. Homologies between any two strains ranges from 78.9% to 100% regarding the galT locus. Indirect enzyme-linked immunosorbent assay (ELISA) confirmed that GALT specific antibodies could not be induced by inactivated APP L20 or MS71 whole cell bacterin preparations. A recombinant fusion GALT protein derived from APP L20, however has proven to be an effective cross-protective antigen against APP sevorar 1 MS71 (50%, 4/8) and APP sevorar 5b L20 (75%, 6/8). Histopathological examinations have confirmed that recombinant GALT vaccinated animals showed less severe pathological signs in lung tissues than negative controls after APP challenge. Immunohistochemical (IHC) analysis indicated that the infiltration of neutrophils in the negative group is significantly increased compared with that in the normal control (P<0.001) and that in surviving animals is decreased compared to the negative group. Anti-GALT antibodies were shown to mediate phagocytosis of neutrophils. After interaction with anti-GALT antibodies, survival rate of APP challenged vaccinated animals was significantly reduced (P<0.001). This study demonstrated that GALT is an effective cross-protective antigen, which could be used as a potential vaccine candidate against multiple APP serotypes.
Subject(s)
Actinobacillus Infections/veterinary , Actinobacillus pleuropneumoniae/immunology , Antigens, Bacterial/immunology , Bacterial Vaccines/immunology , Pleuropneumonia/veterinary , Swine Diseases/prevention & control , UDPglucose-Hexose-1-Phosphate Uridylyltransferase/immunology , Actinobacillus Infections/prevention & control , Actinobacillus pleuropneumoniae/classification , Actinobacillus pleuropneumoniae/genetics , Animals , Antibodies, Bacterial/biosynthesis , Antibodies, Bacterial/immunology , Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Conserved Sequence , Drug Evaluation, Preclinical/veterinary , Enzyme-Linked Immunosorbent Assay , Female , Immunization, Secondary , Immunoglobulin G/biosynthesis , Immunoglobulin G/immunology , Lung/pathology , Mice , Mice, Inbred BALB C , Neutrophil Infiltration/immunology , Phagocytosis/immunology , Pleuropneumonia/pathology , Pleuropneumonia/prevention & control , Random Allocation , Sequence Alignment , Sequence Homology, Amino Acid , Serogroup , Swine , Swine Diseases/immunology , UDPglucose-Hexose-1-Phosphate Uridylyltransferase/genetics , Vaccination/veterinaryABSTRACT
The aim of this study was to investigate isolates of Actinobacillus pleuropneumoniae previously designated serologically either as non-typable (NT) or as 'K2:07', which did not produce serovar-specific amplicons in PCR assays. We used whole genome sequencing to identify the capsule (CPS) loci of six previously designated biovar 1 NT and two biovar 1 'K2:O7' isolates of A. pleuropneumoniae from Denmark, as well as a recent biovar 2 NT isolate from Canada. All of the NT isolates have the same six-gene type I CPS locus, sharing common cpsABC genes with serovars 2, 3, 6, 7, 8, 9, 11 and 13. The two 'K2:O7' isolates contain a unique three-gene type II CPS locus, having a cpsA gene similar to that of serovars 1, 4, 12, 14 and 15. The previously NT isolates share the same O-antigen genes, found between erpA and rpsU, as serovars 3, 6, 8, and 15. Whereas the 'K2:O7' isolates, have the same O-antigen genes as serovar 7, which likely contributed to their previous mis-identification. All of the NT and 'K2:O7' isolates have only the genes required for production of ApxII (apxIICA structural genes, and apxIBD export genes). Rabbit polyclonal antisera raised against representative isolates with these new CPS loci demonstrated distinct reactivity compared to the 16 known serovars. The serological and genomic results indicate that the isolates constitute new serovars 17 (previously NT) and 18 (previously 'K2:O7'). Primers designed for amplification of specific serovar 17 and 18 sequences for molecular diagnostics will facilitate epidemiological tracking of these two new serovars of A. pleuropneumoniae.
Subject(s)
Actinobacillus Infections/veterinary , Actinobacillus pleuropneumoniae/classification , Actinobacillus pleuropneumoniae/genetics , Genotype , Serogroup , Actinobacillus Infections/epidemiology , Actinobacillus pleuropneumoniae/immunology , Actinobacillus pleuropneumoniae/isolation & purification , Animals , Bacterial Capsules/genetics , Canada/epidemiology , DNA Primers/genetics , DNA, Bacterial/genetics , Denmark/epidemiology , Polymerase Chain Reaction/methods , Serotyping , Swine , Swine Diseases/epidemiology , Swine Diseases/microbiology , Whole Genome SequencingABSTRACT
We developed and made a preliminary validation of a bead-based multiplexed immunoassay for simultaneous detection of porcine serum antibodies to Actinobacillus pleuropneumoniae serovars 1, 2, 6, 7, and 12. Magnetic fluorescent beads were coupled with A. pleuropneumoniae antigens and tested with a panel of serum samples from experimentally infected pigs and with serum samples from uninfected and naturally infected pigs. The multiplex assay was compared to in-house ELISAs and complement fixation (CF) tests, which have been used for decades as tools for herd classification in the Danish Specific Pathogen Free system. Assay specificities and sensitivities as well as the corresponding cutoff values were determined using receiver operating characteristic (ROC) curve analysis, and the A. pleuropneumoniae multiplex assay showed good correlation with the in-house ELISAs and CF tests with areas under ROC curves ≥ 0.988. Benefits of multiplexed assays compared to ELISAs and CF tests include reduced serum sample volumes needed for analysis, less labor, and shorter assay time.
Subject(s)
Actinobacillus pleuropneumoniae/classification , Antibodies, Bacterial/blood , Serogroup , Actinobacillus Infections/blood , Actinobacillus Infections/diagnosis , Actinobacillus Infections/microbiology , Actinobacillus Infections/veterinary , Actinobacillus pleuropneumoniae/immunology , Animals , Complement Fixation Tests/veterinary , Enzyme-Linked Immunosorbent Assay/veterinary , Immunoassay , Sensitivity and Specificity , Swine , Swine Diseases/blood , Swine Diseases/diagnosis , Swine Diseases/microbiologyABSTRACT
Actinobacillus pleuropneumoniae causes pleuropneumonia, an economically significant lung disease of pigs. Recently, isolates of A. pleuropneumoniae that were serologically distinct from the previously characterized 15 serovars were described, and a proposal was put forward that they comprised a new serovar, serovar 16. Here we used whole-genome sequencing of the proposed serovar 16 reference strain A-85/14 to confirm the presence of a unique capsular polysaccharide biosynthetic locus. For molecular diagnostics, primers were designed from the capsule locus of strain A-85/14, and a PCR was formulated that differentiated serovar 16 isolates from all 15 known serovars and other common respiratory pathogenic/commensal bacteria of pigs. Analysis of the capsule locus of strain A-85/14 combined with the previous serological data show the existence of a sixteenth serovar-designated serovar 16-of A. pleuropneumoniae.
Subject(s)
Actinobacillus Infections/veterinary , Actinobacillus pleuropneumoniae/classification , Bacterial Capsules/genetics , Genetic Loci , Polymerase Chain Reaction/methods , Serogroup , Swine Diseases/diagnosis , Actinobacillus Infections/diagnosis , Actinobacillus pleuropneumoniae/genetics , Animals , DNA Primers/genetics , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Genome, Bacterial , Molecular Diagnostic Techniques/methods , Pleuropneumonia/microbiology , Pleuropneumonia/veterinary , Sequence Analysis, DNA , SwineSubject(s)
Actinobacillus Infections/veterinary , Actinobacillus pleuropneumoniae/classification , Actinobacillus pleuropneumoniae/isolation & purification , Serogroup , Swine Diseases/microbiology , Actinobacillus Infections/epidemiology , Actinobacillus Infections/microbiology , Animals , England/epidemiology , Swine , Swine Diseases/epidemiology , Wales/epidemiologyABSTRACT
Apx toxins produced by Actinobacillus pleuropneumoniae are essential components of new generation vaccines. In this study, apxIIA and apxIIIA genes of serovars 2, 3, 4, 6, 8 and 15 were cloned and sequenced. Amino acid sequences of ApxIIA proteins of serovars 2, 3, 4, 6, 8 and 15 were almost identical to those of serovars 1, 5, 7, 9 and 11-13. Immunoblot analysis showed that rApxIIA from serovars 2 and 15 reacts strongly with sera from animals infected with various serovars. Sequence analysis revealed that ApxIIIA proteins has two variants, one in strains of serovar 2 and the other in strains of serovars 3, 4, 6, 8 and 15. A mouse cross-protection study showed that mice actively immunized with rApxIIIA/2 or rApxIIIA/15 are protected against challenge with A. pleuropneumoniae strains of serovars 3, 4, 6, 8, 15, and 2 expressing ApxIII/15 and ApxIII/2, respectively. Similarly, mice passively immunized with rabbit anti-rApxIIIA/2 or anti-rApxIIIA/15 sera were found to be protected against challenge with strains of serovars 2 and 15. Our study revealed antigenic and sequence similarities within ApxIIA and ApxIIIA proteins, which may help in the development of effective vaccines against disease caused by A. pleuropneumoniae.
Subject(s)
Actinobacillus pleuropneumoniae/genetics , Actinobacillus pleuropneumoniae/immunology , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Hemolysin Proteins/genetics , Hemolysin Proteins/immunology , Serogroup , Actinobacillus Infections/immunology , Actinobacillus Infections/prevention & control , Actinobacillus pleuropneumoniae/classification , Amino Acid Sequence , Animals , Antibodies, Bacterial/immunology , Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Gene Expression , Hemolysin Proteins/chemistry , Hemolysin Proteins/isolation & purification , Immunization , Mice , Models, Molecular , Protein Conformation , Protein Domains , Rabbits , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/isolation & purification , SwineABSTRACT
We observed increasing unserotypable (UT) Actinobacillus pleuropneumoniae isolates using agar gel diffusion (AGD) test. To reanalyze their serovar, we performed rapid slide agglutination (RSA) test and multiplex PCR for 47 UT isolates. Of these, 25 were serovar 1 (UT-serovar 1), 20 were serovar 2 (UT-serovar 2) and 2 were serovar 15 (UT-serovar 15). We examined serotyping antigen extraction temperature to determine heat influence. UT-serovar 1 and 15 were influenced by heat, because their precipitation lines were observed in the case of low antigen extraction temperature. To investigate the relationship between antigenicity and genotype, we performed pulsed-field gel electrophoresis (PFGE) analysis using UT-serovar 2 and 15. The predominant PFGE pattern of UT-serovar 2 was identical to that of serovar 2.