Actinobacillus Pleuropneumoniae Strains Research Articles

Coinfections and Phenotypic Antimicrobial Resistance in Actinobacillus pleuropneumoniae Strains Isolated From Diseased Swine in North Western Germany-Temporal Patterns in Samples From Routine Laboratory Practice From 2006 to 2020.

Actinobacillus pleuropneumoniae (APP) is one major bacterial porcine respiratory tract pathogen causing disease outbreaks worldwide, although effective commercial vaccines are available. Due to frequent failure of this preventive measure, treatment with antimicrobials is indispensable to prevent animal losses within an outbreak situation. To preserve the effectivity of antimicrobial substances to fight APP should therefore be the primary aim of any interventions. In this study, the temporal development of antimicrobial resistance in APP was analyzed retrospectively in the time period 2006–2020 from a routine diagnostic database. In parallel, frequent coinfections were evaluated to identify most important biotic cofactors as important triggers for disease outbreaks in endemically infected herds. The proportion of APP serotype 2 decreased over time but was isolated most often from diseased swine (57% in 2020). In ~1% of the cases, APP was isolated from body sites outside the respiratory tract as brain and joints. The lowest frequencies of resistant isolates were found for cephalothin and ceftiofur (0.18%), florfenicol (0.24%), tilmicosin (2.4%), tiamulin (2.4%), enrofloxacin (2.7%), and spectinomycin (3.6%), while the highest frequencies of resistant isolates were found for gentamicin (30.9%), penicillin (51.5%), and tetracycline (78.2%). For enrofloxacin, tiamulin, tilmicosin, and tetracycline, significantly lower frequencies of resistant isolates were found in the time period 2015–2020 compared to 2006–2014, while gentamicin-resistant isolates increased. In summary, there is only a low risk of treatment failure due to resistant isolates. In maximum, up to six coinfecting pathogens were identified in pigs positive for APP. Most often pigs were coinfected with Porcine Circovirus 2 (56%), Streptococcus suis (24.8%), or the Porcine Reproductive and Respiratory Syndrome Virus (23.3%). Potential synergistic effects between these pathogens published from experimental findings can be hypothesized by these field data as well. To prevent APP disease outbreaks in endemically infected herds more efficiently in the future, next to environmental trigger factors, preventive measures must also address the coinfecting agents.

BIOCHEMICAL PROPERTIES OF ACTINOBACILLUS PLEUROPNEUMONIAE MUSEUM STRAINS

The article provides data on the biochemical properties of museum strains of Actinobacillus pleuropneumoni-ae. The belonging of the strains of Actinobacillus pleuropneumoniae was confirmed in the polymerase chain reaction using the developed RUE «Institute of experimental veterinary medicine nam. of S.N. Wyshelessky» test system for detecting the genome of Actinobacillus pleuropneumoniae.

Recombinant tandem epitope vaccination provides cross protection against Actinobacillus pleuropneumoniae challenge in mice

Actinobacillus pleuropneumoniae (A. pleuropneumoniae/APP) is the pathogen that causes porcine contagious pleuropneumonia. Actinobacillus pleuropneumoniae is divided into 18 serovars, and the cross protection efficacy of epitopes is debatable, which has resulted in the slow development of a vaccine. Consequently, epitope-based vaccines conferring Actinobacillus pleuropneumoniae cross protection have rarely been reported. In this study, B cell epitopes in the head domain of trimeric autotransporter adhesin were predicted, and 6 epitopes were selected. Then, the predicted epitopes (Ba1, Bb5, C1, PH1 and PH2) were connected by linkers to construct a recombinant tandem antigen (rta) gene. The RTA protein encoded by the recombinant rta gene was expressed, and finally the ICR mice were immunized with the RTA protein with or without inactivated Actinobacillus pleuropneumoniae (serovars 1 and 5b) and challenged with Actinobacillus pleuropneumoniae to evaluate the protective effect of the epitope-based vaccine and combined vaccine. The mice in the RTA-immunized group and RTA plus inactivated Actinobacillus pleuropneumoniae vaccine group had a significant improvement in clinical symptoms and a higher level of antibody in the serum than those in the control group. The RTA immune group had a 40% survival rate after Actinobacillus pleuropneumoniae infection, whereas the combination of RTA and inactivated Actinobacillus pleuropneumoniae produced very strong cross immune protection in mice, at least 50% (RTA IB1 + C5) and at most 100% (RTA IB5 + C1), whereas no cross immunoprotection was found in the solo Actinobacillus pleuropneumoniae immune group. Overall, the combination of the RTA protein and inactivated bacteria significantly enhanced the cross protection effects. This implies that RTA protein in combination with a suitable inactivated Actinobacillus pleuropneumoniae strain could be a candidate vaccine for porcine contagious pleuropneumonia.

Post-antibiotic effect of marbofloxacin, enrofloxacin and amoxicillin against selected respiratory pathogens of pigs

The post-antibiotic effect is defined as the period of bacterial growth suppression that persists after a limited exposure of organisms to antimicrobials and knowledge of its duration is important in establishing and optimising current dosing schedules for the treatment of bacterial infections. The post-antibiotic effect of marbofloxacin, enrofloxacin and amoxicillin were evaluated in vitro for Actinobacillus pleuropneumoniae, Haemophilus parasuis and Pasteurella multocida strains which originated from clinical samples of diseased pigs and were confirmed as susceptible to all tested antimicrobials based on determination of minimal inhibitory concentrations. The post-antibiotic effect for individual antimicrobials was monitored at five and ten times minimum inhibitory concentrations for one and two hours. The duration of the post-antibiotic effect for tested antimicrobials was found to exhibit the following order for all tested pathogens: marbofloxacin > enrofloxacin > amoxicillin. The longest duration of post-antibiotic effect of all tested antimicrobials was found in A. pleuropneumoniae and the shortest post-antibiotic effect duration was detected in P. multocida. No statistical differences in post-antibiotic effect duration were found within marbofloxacin and enrofloxacin in A. pleuropneumoniae and H. parasuis strains. In P. multocida strains there was a statistically significant difference (P = 0.0189). On the other hand, the differences between amoxicillin and marbofloxacin or enrofloxacin were statistically significant in all cases (P-values ranged between 0.0058 and 0.008). The prolonged post-antibiotic effect of fluoroquinolones and amoxicillin on important Gram-negative swine pathogens was confirmed. The results can be used to clarify the effect and mechanism of action of antimicrobial drugs in veterinary medicine.

Antimicrobial resistance, biofilm formation and virulence reveal Actinobacillus pleuropneumoniae strains' pathogenicity complexity

Porcine pleuropneumonia is an important cause of lowered productivity and economic loss in the pig industry worldwide, associated primarily with Actinobacillus pleuropneumoniae infection. Its colonization and persistence within the upper respiratory tract of affected pigs depends upon interactions between a number of genetically controlled virulence factors, such as pore-forming repeats-in-toxin exoproteins, biofilm formation, and antimicrobial resistance. This study investigated correlations between biofilm-forming capacity, antimicrobial resistance, and virulence of A. pleuropneumoniae obtained from clinical outbreaks of disease, using a Galleria mellonella alternative infection model. Results suggest that virulence is diverse amongst the 21 strains of A. pleuropneumoniae examined and biofilm formation correlated with genetic control of antimicrobial resistance.

ICEAplChn1, a novel SXT/R391 integrative conjugative element (ICE), carrying multiple antibiotic resistance genes in Actinobacillus pleuropneumoniae

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.

Differential cellular immune response of Galleria mellonella to Actinobacillus pleuropneumoniae.

In the present work, we have investigate the cellular immune response of Galleria mellonella larvae against three strains of the gram-negative bacterium Actinobacillus pleuropneumoniae: low-virulence (780), high-virulence (1022) and the serotype 8 reference strain (R8). Prohemocytes, plasmatocytes, granulocytes, oenocytoids and spherulocytes were distinguished according to their size and morphology, their molecular markers and dye-staining properties and their role in the immune response. Total hemocyte count, differential hemocyte count, lysosome activity, autophagic response, cell viability and caspase-3 activation were determined in circulating hemocytes of naive and infected larvae. The presence of the autophagosome protein LC3 A/B within the circulating hemocytes of G. mellonella was dependent on and related to the infecting A. pleuropneumoniae strain and duration of infection. Hemocytes treated with the high-virulence strain expressed higher levels of LC3 A/B, whereas treatment with the low-virulence strain induced lower expression levels of this protein in the cells. Moreover, our results showed that apoptosis in circulating hemocytes of G. mellonella larvae after exposure to virulent bacterial strains occurred simultaneously with excessive cell death response induced by stress and subsequent caspase-3 activation.

Antimicrobial susceptibility of Actinobacillus pleuropneumoniae strains isolated from pig's clinical samples in Serbia

Porcine pleuropneumonia, caused by Actinobacillus pleuropneumoniae is one of the most important bacterial diseases of the pig respiratory tract. One hundred and forty eight Actinobacillus pleuropneumoniae strains isolated from pig's clinical samples were tested for their susceptibility to 11 antimicrobials by disk diffusion method. Isolates were obtained from the pathoanatomically altered pig's lung that died with signs of respiratory disease. Antimicrobial discs with following concentration were used: ampicillin 10 µg, amoxicillin 10 µg, amoxicillin/clavulanic acid 20/10 µg, cefotaxime 30 µg, gentamicin 10 µg, penicillin 10 IU, streptomycin 10 µg, tetracycline 30 µg, enrofloxacin 5µg, lincospectin 15/200 µg, florfenicol 30 µg and trimethoprim/sulfamethoxazole 1.25/23.75 µg. Isolated strains showed various susceptibility to tested antimicrobials. Seventy-five percent of isolates were resistant to one or more drugs frequently used in the treatment of pig's diseases at farms in Serbia. All 148 isolates were susceptible to cefotaxime, enrofloxacin and florfenicol, while 96% of them were susceptible to amoxicillin/clavulanic acid and lincospectin. Susceptibility of isolates to amoxicillin, gentamicin and trimethoprim/sulfamethoxazole were 91%, 77% and 64%, respectively. The highest resistance rates were determined for tetracycline and streptomycin, 34% and 31%, respectively. When compared to our previous results, we noticed a decrease in resistance to tetracyclines and trimethoprim/sulfamethoxazole. Our results indicate that A. pleuropneumoniae strains isolated from pig's clinical samples in Serbia show high susceptibility to antimicrobial agents which are most often used in the pig industry.

Serovar and Antimicrobial Resistance Profiles of <i>Actinobacillus pleuropneumoniae</i> Isolated in Japan from 2006 to 2011

Serovar and Antimicrobial Resistance Profiles of <i>Actinobacillus pleuropneumoniae</i> Isolated in Japan from 2006 to 2011

Identification of a proposed new serovar of Actinobacillus Pleuropneumoniae: Serovar 16.

Five Actinobacillus pleuropneumoniae strains isolated from pathological lesions of porcine pleuropneumonia in Hungary could not be assigned to any of the accepted 15 serovars. Using hyperimmune serum raised against these unty-pable-serovar A. pleuropneumoniae strains in rabbits, indirect haemagglutination tests proved that they form a distinct group and there is no cross-reaction between them and the type strains of A. pleuropneumoniae. All five strains harboured the toxin-associated genes for the production (apxIA) and secretion (apxIB) of ApxI, the gene for the expression of ApxII and the largest-size (2800 bp) apxIV gene. The carbon source utilisation pattern and the sequence analysis of the 16S rRNA gene confirmed the species identification of the suggested type strain, A. pleuropneumoniae A-85/14. A new serovar of A. pleuropneumoniae - serovar 16 - is proposed with A. pleuropneumoniae A-85/14 as reference strain.

Prevalence and Characterization ofActinobacillus pleuropneumoniaeIsolated from Korean Pigs

Actinobacillus pleuropneumoniae causes porcine pleuropneumoniae which is one of severe threats to the swine industry. In total, 54 strains of Actinobacillus pleuropneumoniae were isolated from 443 pigs between 2012 and 2013 in Korea. Isolates were classified into serotypes 1, 2, 5, 7, 12, and unclassified by multiplex PCR. Genotypes of isolates were divided into three groups according to the sequence of the omlA gene. The antimicrobial resistance rate of serotype 1 was slightly higher than that of serotype 5. In conclusion, to block and treat porcine pleuropneumonia, it is necessary to conduct ongoing characterization of A. pleuropneumoniae isolated from pigs.

Experimental Identification of Actinobacillus pleuropneumoniae Strains L20 and JL03 Heptosyltransferases, Evidence for a New Heptosyltransferase Signature Sequence

We experimentally identified the activities of six predicted heptosyltransferases in Actinobacillus pleuropneumoniae genome serotype 5b strain L20 and serotype 3 strain JL03. The initial identification was based on a bioinformatic analysis of the amino acid similarity between these putative heptosyltrasferases with others of known function from enteric bacteria and Aeromonas. The putative functions of all the Actinobacillus pleuropneumoniae heptosyltrasferases were determined by using surrogate LPS acceptor molecules from well-defined A. hydrophyla AH-3 and A. salmonicida A450 mutants. Our results show that heptosyltransferases APL_0981 and APJL_1001 are responsible for the transfer of the terminal outer core D-glycero-D-manno-heptose (D,D-Hep) residue although they are not currently included in the CAZY glycosyltransferase 9 family. The WahF heptosyltransferase group signature sequence [S(T/S)(GA)XXH] differs from the heptosyltransferases consensus signature sequence [D(TS)(GA)XXH], because of the substitution of D261 for S261, being unique.

Antimicrobial susceptibility of some Actinobacillus pleuropneumoniae strains.

A total of 100 Actinobacillus pleuropneumoniae strains isolated from Hungary and Romania were tested for in vitro susceptibility to 7 antimicrobial agents by using standard disc diffusion method. Results showed that majority of A. pleuropneumoniae isolates were found susceptible to most antimicrobial agents tested.

Molecular characterization of enrofloxacin resistant Actinobacillus pleuropneumoniae isolates

Enrofloxacin (ER) resistant Actinobacillus pleuropneumoniae strains emerged in Taiwan in 2002. The mechanism of ER resistance in A. pleuropneumoniae has not yet been reported. A total of 48 A. pleuropneumoniae isolates were obtained from the lungs of pigs with pleuropneumonia in Taiwan between September 2007 and April 2008. Twenty-nine isolates were found to be resistant to enrofloxacin. To understand the mechanisms of A. pleuropneumoniae's resistance to ER, enrofloxacin susceptibility of the isolates along with the mutations of the quinolone resistance-determining region (QRDR) of DNA gyrase and topoisomerase IV, qnr genes were analyzed. Enrofloxacin resistant isolates were found to carry at least one mutation in the QRDR of gyrA, leading to amino acid changes at codon 83 or 87. Efflux pump inhibitor (Phe-Arg-β-naphthylamide) decreased enrofloxacin minimum inhibitory concentration 2–16-fold, suggesting participation of efflux in ER resistance. Plasmid mediated quinolone resistance genes qnr were not detected in these isolates. In conclusion, enrofloxacin resistance of A. pleuropneumoniae may be linked to multiple target gene mutations and active effluxs.

Effects of growth conditions on biofilm formation byActinobacillus pleuropneumoniae

Biofilm formation is an important virulence trait of many bacterial pathogens. It has been reported in the literature that only two of the reference strains of the swine pathogen Actinobacillus pleuropneumoniae, representing serotypes 5b and 11, were able to form biofilm in vitro. In this study, we compared biofilm formation by the serotype 1 reference strain S4074 of A. pleuropneumoniae grown in five different culture media. We observed that strain S4074 of A. pleuropneumoniae is able to form biofilms after growth in one of the culture conditions tested brain heart infusion (BHI medium, supplier B). Confocal laser scanning microscopy using a fluorescent probe specific to the poly-N-acetylglucosamine (PGA) polysaccharide further confirmed biofilm formation. In accordance, biofilm formation was susceptible to dispersin B, a PGA hydrolase. Transcriptional profiles of A. pleuropneumoniae S4074 following growth in BHI-B, which allowed a robust biofilm formation, and in BHI-A, in which only a slight biofilm formation was observed, were compared. Genes such as tadC, tadD, genes with homology to autotransporter adhesins as well as genes pgaABC involved in PGA biosynthesis and genes involved in zinc transport were up-regulated after growth in BHI-B. Interestingly, biofilm formation was inhibited by zinc, which was found to be more present in BHI-A (no or slight biofilm) than in BHI-B. We also observed biofilm formation in reference strains representing serotypes 3, 4, 5a, 12 and 14 as well as in 20 of the 37 fresh field isolates tested. Our data indicate that A. pleuropneumoniae has the ability to form biofilms under appropriate growth conditions and transition from a biofilm-positive to a biofilm-negative phenotype was reversible.

Analysis of an Actinobacillus pleuropneumoniae multi-resistance plasmid, pHB0503

A plasmid containing multidrug resistance genes has been discovered from a clinical Actinobacillus pleuropneumoniae strain isolated in China. The complete 15079 kb sequence of this plasmid, designated pHB0503, was analyzed with regard to the organization and evolution of multidrug resistance genes. The deduced amino acid sequences from seven open reading frames ( sul2 catA3, aacC2, strA, truncated strB ( strB′), bla ROB-1 and aph( 3′) -I) identified in pHB0503 were entirely or nearly identical to resistance genes of plasmids both within and outside of the family Pasteurellaceaea, indicating that pHB0503 arose through inter-plasmid recombination processes among them. In addition, co-transcription of the cluster of resistance genes from the promoter upstream of sul2 and bla ROB-1 was confirmed by RT-PCR. This is the first report of a complete sequence of the plasmid containing seven resistance genes from A. pleuropneumoniae.

Isolation and identification of Actinobacillus pleuropneumoniae in pig's lungs at farms and their sensitivity to antibiotics

The presence of Actinobacillus pleuropneumoniae has been established in all suspected cases of pleuropneumonia at several farms that have been included in the research. Equal incidence of pleuropneumonia has been found both among piglets for breeding and for fattening. The health monitoring of herds is extremely important, firstly because of the need for the adequate strategy to be chosen for controlling the Actinobacillus-caused pleuropneumonia and, at the same time, in order to prevent enormous economic losses that this disease may cause. The morphological, physiological and biochemical characteristics of isolated strains of Actinobacillus pleuropneumoniae were completely identical to those of referent strains. It has been found that Actinobacillus pleuropneumoniae grows most abundantly on chocolate agar when PolyVitex (bioMerieux) is used as the substrate. Out of 237 samples of altered parts of swine lungs, 13 bacterial species have been found in 193 (81%), and the incidence of Actinobacillus pleuropneumoniae within this percentage was 33%. Of all bacterial species isolated in pure culture from all investigated specimen the most dominant species were Pasteurella multocida with the incidence of 32.64% and Actinobacillus pleuropneumoniae with 29.01%. Their common incidence in all positive findings was 61.65%. If one adds to this their participation in mixed infections (2.59%), this percentage is higher (64.24%). The high percentage of these two bacterial species shows that they are at the same time the most common bacterial pathogens causing pneumonia in pigs. The incidence of other species of bacteria isolated from the lungs of diseased pigs ranged from 0.51 to 10.88%. The sensitivity of isolated strains of Actinobacillus pleuropneumoniae to the selected range of antibiotics used in clinical veterinary medicine (penicillin, ampicillin, amoxicillin, cephalosporin (III gen.) gentamycin, streptomycin, neomycin, thylosine, enrofloxacin, linkomycin-spectinomycin, tetracycline, florphenycol, trimethoprimsulphomethoxazol and tulatromycin) was tested by the disc-diffusion method, with the implementation of antibiogram tabletes (Torlak) and antibiogram discs (Oxoid), on chocolate agar and on Chaemophilus test medium (HTM, Biomedics). All tested strains of Actinobacillus pleuropneumoniae were sensitive to thulatromycin, while resistance of same strains was the highest to tetracycline (53%) and trimethoprim/sulphomethoxazole (56%).

Isolation of Actinobacillus pleuropneumoniae Serovar 15-Like Strain from a Field Case of Porcine Pleuropneumonia in Japan

An Actinobacillus pleuropneumoniae strain isolated from a field case of porcine pleuropneumonia in Japan, was closely related to a reference strain of serovar 15, which is a newly proposed serovar according to an analysis of field isolates originating from Australia. The isolate had biological and biochemical properties consistent with A. pleuropneumoniae biovar 1, and reacted strongly to a rabbit antiserum raised against a reference strain of serovar 15 in an agar gel precipitation test. The nucleotide sequence of a hyper variable region in the 16S RNA gene of the isolate was identical to that of the reference strain of serovar 15. The isolate possessed A. pleuropneumoniae-RTX toxin (Apx) II, III, and IV genes, consistent with serovar 15. Its virulence in mice was lower than that of ApxI-bearing strains but higher than that of other ApxIII-bearing strains. This is the first report describing the isolation of A. pleuropneumoniae serovar 15-like strain from a country or region other than Australia.

Cloning and characterization of the groE locus from Actinobacillus pleuropneumoniae.

A 4.4-kb DNA fragment was cloned from Actinobacillus pleuropneumoniae (strain 4074, serotype 1) by genetic complementation with Escherichia coli groES-groEL mutant strains. Sequence analysis of this fragment revealed a purine nucleoside phosphorylase (DeoD)-encoding gene homolog (deoD), heat-shock response-encoding genes for the small (groES) and large subunits (groEL) and a partial open reading frame encoding an alcohol dehydrogenase homolog (adhE). The predicted amino-acid sequence of groES and groEL genes showed extensive sequence identity (80-95%) with other Pasteurellaceae. The gene organization surrounding the groE locus was different from that of Haemophilus infuenzae. When expressed in E. coli, groES-groEL genes were capable of complementing the growth of a lambda lytic phage, indicating a structural as well as functional conservation.

Systemic and Local Antibody Responses after Experimental Infection withActinobacillus pleuropneumoniaein Piglets with Passive or Active Immunity

The objectives of the present study was to describe different dynamics of humoral immune responses to experimental infection in piglets of different stages of infection and immunity. Two groups of piglets originating from non-immune (group 1) and immune (group 2) sows at the age of 3 weeks were subdivided as follows: a half of each group of piglets was exposed to a low-dose infection with Actinobacillus pleuropneumoniae (APP) strain 9. At the age of 8 weeks, all four groups of piglets were challenged with a high infection dose of APP of the same strain. Isotype characterization of the specific antibodies in sera and in bronchoalveolar lavage fluids (BALF) to a lipopolysaccharide was carried out, besides monitoring clinical signs and post-mortem examinations. A typical primary immune response was observed in specific antibody-free piglets infected with a challenge infection. Colostrum-derived immunoglobulin-G (IgG) antibodies persisted in sera and BALF of piglets up to the age of 8 weeks. However, they did not prevent induction of specific-primary antibody response, either in 8 or 4 weeks of age, when levels of specific colostrum-derived antibodies were still high. It was demonstrated by the increase of specific IgM antibodies in sera. The infection induced an increase in the levels of IgA antibodies in BALF regardless the severity of infection and presence of specific colostrum-derived antibodies. The specific antibodies of IgG isotype increased only in BALF from piglets without colostrum-derived antibodies.