Serotype diversity and antimicrobial susceptibility profiles of Actinobacillus pleuropneumoniae isolated in Italian pig farms from 2015 to 2022.

Actinobacillus pleuropneumoniae (APP) is a bacterium frequently associated with porcine pleuropneumonia. The acute form of the disease is highly contagious and often fatal, resulting in significant economic losses for pig farmers. Serotype diversity and antimicrobial resistance (AMR) of APP strains circulating in north Italian farms from 2015 to 2022 were evaluated retrospectively to investigate APP epidemiology in the area. A total of 572 strains isolated from outbreaks occurring in 337 different swine farms were analysed. The majority of isolates belonged to serotypes 9/11 (39.2%) and 2 (28.1%) and serotype diversity increased during the study period, up to nine different serotypes isolated in 2022. The most common resistances were against tetracycline (53% of isolates) and ampicillin (33%), followed by enrofloxacin, florfenicol and trimethoprim/sulfamethoxazole (23% each). Multidrug resistance (MDR) was common, with a third of isolates showing resistance to more than three antimicrobial classes. Resistance to the different classes and MDR varied significantly depending on the serotype. In particular, the widespread serotype 9/11 was strongly associated with florfenicol and enrofloxacin resistance and showed the highest proportion of MDR isolates. Serotype 5, although less common, showed instead a concerning proportion of trimethoprim/sulfamethoxazole resistance. Our results highlight how the typing of circulating serotypes and the analysis of their antimicrobial susceptibility profile are crucial to effectively manage APP infection and improve antimicrobial stewardship.

Characterization of nonencapsulated Actinobacillus pleuropneumoniae serovar K12:O3 isolates.

Three Actinobacillus pleuropneumoniae isolates from clinical cases of porcine pleuropneumonia were positive by capsular serovar 12-specific PCR assay, but not reactive to antiserum prepared against serovar 12 using the rapid slide agglutination (RSA) test. The isolates were positive for apxIICA, apxIIICA, apxIBD, apxIIIBD, and apxIVA in the PCR toxin gene assay, which is the profile seen in serovars 2, 4, 6, 8, and 15, and reacted with antisera against serovars 3, 6, 8, 15, and 17. Nucleotide sequence analysis revealed that genes involved in the biosynthesis of capsular polysaccharide of the 3 isolates were identical or nearly identical to those of serovar 12. However, genes involved in the biosynthesis of O-polysaccharide of the 3 isolates were highly similar to those of reference strains of serovars 3, 6, 8, 15, 17, and 19. In agreement with results from the RSA test, transmission electron microscopic analysis confirmed the absence of detectable capsular material in the 3 isolates. The existence of nonencapsulated A. pleuropneumoniae serovar K12:O3 would hamper precise serodetection.

An improved multiplex PCR for Actinobacillus pleuropneumoniae, Glaesserella australis and Pasteurella multocida.

Glaesserella australis, a newly described bacterial species, has been isolated from pig lungs that displayed lesions very similar to those caused by Actinobacillus pleuropneumoniae, prompting the need for a validated diagnostic tool. In this work, we have altered a multiplex PCR used for the identification of cultures of G. australis, A. pleuropneumoniae and Pasteurella multocida to be more sensitive and then evaluated the use of the altered diagnostic tool on cultures and directly on tissues. The altered multiplex PCR was validated using 47 related species, both type/reference strains and field isolates. The sensitivity was assessed by serial dilutions and used a mixture of target bacteria in different concentrations. Further, 166 lung samples from 54 farms from four Australian States were used to validate the ability of the multiplex PCR to detect bacteria in lung swabs. The multiplex PCR was specific for the three target species. The assay could detect a minimum of 40 colony forming units (CFU) of G. australis, 786 CFU of A. pleuropneumoniae and 238 CFU of P. multocida. The multiplex PCR yielded more positives than coventional bacteriological examination. From a total of 166 lung samples, 51.9%, 51.9% and 5.6% of farms were PCR positive for P. multocida, A. pleuropneumoniae and G. australis, respectively. The results suggested that the new multiplex PCR was specific, sensitive and out performed traditional culture. The prevalence of G. australis was not very high, but it was the dominant pathogen in infected pigs.

Antibodies against Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae and influenza virus and their relationships with risk factors, clinical signs and lung lesions in pig farms with one-site production systems in Brazil.

A study was conducted on 21 pig herds using one-site production system in the southeast region of Brazil to assess the relationships among serological results for primary pathogens involved in respiratory diseases (Actinobacillus pleuropneumoniae, App; Mycoplasma hyopneumoniae, Mhyo; and swine influenza virus, SIV), cough index, pneumonia index, pleuritis and herd characteristics. The prevalence of antibodies against Mhyo and SIV increased throughout the raising phases, with the highest prevalence in slaughtered pigs (> 40%), while pigs in 65% (14/21) of nurseries demonstrated marked seroprevalence of App that decreased until the day of slaughter. Pleuritis and pulmonary consolidations were recorded in 9.0 and 72.4%, respectively, of the 908 evaluated lungs. Histopathological analysis of the lung lesions revealed suppurative bronchopneumonia in almost half of the lungs (48.9%). Regression analyses were conducted to identify risk factors associated with the cough index; pleuritis; pulmonary consolidation; and App, Mhyo and SIV serological results. All-in-all-out management in nursery buildings reduced the seroprevalence of Mhyo in herds. App seroprevalence was associated with pleuritis, and the presence of cough episodes in growing pigs was associated with SIV seropositivity in nursery pigs.

Actinobacillus pleuropneumoniae serotypes in Hungary.

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.

Proposal of serovars 17 and 18 of Actinobacillus pleuropneumoniae based on serological and genotypic analysis.

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.

New trends in innovative vaccine development against Actinobacillus pleuropneumoniae.

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.

Update on Actinobacillus pleuropneumoniae-knowledge, gaps and challenges.

Porcine pleuropneumonia, caused by the bacterial porcine respiratory tract pathogen Actinobacillus pleuropneumoniae, leads to high economic losses in affected swine herds in most countries of the world. Pigs affected by peracute and acute disease suffer from severe respiratory distress with high lethality. The agent was first described in 1957 and, since then, knowledge about the pathogen itself, and its interactions with the host, has increased continuously. This is, in part, due to the fact that experimental infections can be studied in the natural host. However, the fact that most commercial pigs are colonized by this pathogen has hampered the applicability of knowledge gained under experimental conditions. In addition, several factors are involved in development of disease, and these have often been studied individually. In a DISCONTOOLS initiative, members from science, industry and clinics exchanged their expertise and empirical observations and identified the major gaps in knowledge. This review sums up published results and expert opinions, within the fields of pathogenesis, epidemiology, transmission, immune response to infection, as well as the main means of prevention, detection and control. The gaps that still remain to be filled are highlighted, and present as well as future challenges in the control of this disease are addressed.

Isolation and molecular characterization of a urease-negative Actinobacillus pleuropneumoniae mutant.

An atypical urease-negative mutant of Actinobacillus pleuropneumoniae serovar 2 was isolated in Japan. Nucleotide sequence analysis of the urease gene cluster revealed that the insertion of a short DNA sequence into the cbiM gene was responsible for the urease-negative activity of the mutant. Veterinary diagnostic laboratories should be watchful for the presence of aberrant urease-negative A. pleuropneumoniae isolates.

Molecular serotyping and antimicrobial resistance profiles of Actinobacillus pleuropneumoniae isolated from pigs in South Korea.

BACKGROUND: Actinobacillus pleuropneumoniae (APP) causes porcine pleuropneumonia (PP). OBJECTIVE: Serotypes and antimicrobial resistance patterns in APP isolates from pigs in Korea were examined. METHODS: Sixty-five APP isolates were genetically serotyped using standard and multiplex PCR (polymerase chain reaction). Antimicrobial susceptibilities were tested using the standardized disk-agar method. PCR was used to detect ß-lactam, gentamicin and tetracycline-resistance genes. The random amplified polymorphic DNA (RAPD) patterns were determined by PCR. RESULTS: Korean pigs predominantly carried APP serotypes 1 and 5. Among 65 isolates, one isolate was sensitive to all 12 antimicrobials tested in this study. Sixty-two isolates was resistant to tetracycline and 53 isolates carried one or five genes including tet(B), tet(A), tet(H), tet(M)/tet(O), tet(C), tet(G) and/or tet(L)-1 markers. Among 64 strains, 9% and 26.6% were resistance to 10 and three or more antimicrobials, respectively. Thirteen different antimicrobial resistance patterns were observed and RAPD analysis revealed a separation of the isolates into two clusters: cluster II (6 strains resistant to 10 antimicrobials) and cluster I (the other 59 strains). CONCLUSION: Results show that APP serotypes 1 and 5 are the most common in Korea, and multi-drug resistant strains are prevalent. RAPD analysis demonstrated that six isolates resistant to 10 antimicrobials belonged to the same cluster.

Exposure of extensively farmed wild boars (Sus scrofa scrofa) to selected pig pathogens in Greece.

BACKGROUND: Increased density and distribution of wild boar populations are likely to promote interactions and transmission of certain pathogens, not only among wild boar but also from wild boar to livestock or humans and vice versa. OBJECTIVE: The purpose of this study was to determine seroprevalence against seven selected pathogens in wild boar living in four different areas in Greece. ANIMALS AND METHODS: In total, 359 serum samples were collected from extensively farmed wild boar (Sus scrofa scrofa) originating from four distinct geographical areas throughout Greece from April 2012 to August 2013. Samples were tested for antibodies to Actinobacillus pleuropneumoniae, African swine fever virus (ASFV), Aujeszky's disease virus (ADV), classical swine fever virus (CSFV), Erysipelothrix rhusiopathiae, Mycoplasma hyopneumoniae and porcine reproductive and respiratory syndrome virus (PRRSV). Prevalence was compared among the four regions using Fisher's exact test. RESULTS: Low overall seropositivities of 2.4% and 5.6% were detected for E. rhusiopathiae and PRRSV, respectively, higher ones for ADV (32.0%) and the highest (72.5% and 90.5%) for M. hyopneumoniae and A. pleuropneumoniae, respectively. All sera tested were found negative for antibodies directed against CSFV and ASFV. CONCLUSIONS: This is the first report of exposure of wild boars to selected pig pathogens in Greece. These results are indicative of the circulation of these pathogens in Greece with the exception of CSFV and ASFV and suggestive of the potential role of wild boars on their maintenance and transmission to their domestic counterparts and vice versa.

Transmission of Actinobacillus pleuropneumoniae among weaned piglets on endemically infected farms.

Clinical outbreaks due to Actinobacillus pleuropneumoniae occur recurrently, despite the wide-scale use of antimicrobials or vaccination. Therefore, new approaches for the prevention and control of these outbreaks are necessary. For the development of alternative measures, more insight into the transmission of the bacterium on farms is necessary. The aim of this cohort study was to quantify transmission of A. pleuropneumoniae amongst weaned piglets on farms. We investigated three possible transmission routes: (i) indirect transmission by infected piglets within the same compartment, (ii) transmission by infected pigs in adjacent pens and (iii) transmission by direct contact within pens. Additionally, we evaluated the effect of independent litter characteristics on the probability of infection. Two farms participated in our study. Serum and tonsil brush samples were collected from sows pre-farrowing. Serum was analysed for antibodies against Apx toxins and Omp. Subsequently, tonsil brush samples were collected from all piglets from these dams (N=542) in three cohorts, 3 days before weaning and 6 weeks later. Tonsil samples were analysed by qPCR for the presence of the apxIVA gene of A. pleuropneumoniae. Before weaning, 25% of the piglets tested positive; 6 weeks later 47% tested positive. Regression and stochastic transmission models were used to assess the contribution of each of the three transmission routes and to estimate transmission rates. Transmission between piglets in adjacent pens did not differ significantly from that between non-adjacent pens. The transmission rate across pens was estimated to be 0.0058 day(-1) (95% CI: 0.0030-0.010), whereas the transmission rate within pens was ten times higher 0.059 day(-1) (95% CI: 0.048-0.072). Subsequently, the effects of parity and serological response of the dam and litter age at weaning on the probability of infection of pigs were evaluated by including these into the regression model. A higher dam ApxII antibody level was associated with a lower probability of infection of the pig after weaning; age at weaning was associated with a higher probability of infection of the pig after weaning. Finally, transmission rate estimates were used in a scenario study in which the litters within a compartment were mixed across pens at weaning instead of raising litter mates together in a pen. The results showed that the proportion of infected piglets increased to 69% if litters were mixed at weaning, indicating that farm management measures may affect spread of A. pleuropneumoniae.

Simulation study of the mechanisms underlying outbreaks of clinical disease caused by Actinobacillus pleuropneumoniae in finishing pigs.

Actinobacillus pleuropneumoniae is a major cause of respiratory disease in pigs. Many farms are endemically infected without apparent disease, but occasionally severe outbreaks of pleuropneumonia occur. To prevent and control these outbreaks without antibiotics, the underlying mechanisms of these outbreaks need to be understood. Outbreaks are probably initiated by a trigger (common risk factor) changing the host-pathogen interaction, but it is unclear whether this trigger causes all cases directly (trigger mechanism), or whether the first case starts a transmission chain inducing disease in the infected contacts (transmission mechanism). The aim of this study was to identify conditions under which these mechanisms could cause A. pleuropneumoniae outbreaks, and to assess means for prevention and control. Outbreaks were first characterised by data from a literature review, defining an average outbreak at 12 weeks of age, affecting 50% of animals within 4 days. Simple mathematical models describing the two mechanisms can reproduce average outbreaks, with two observations supporting the trigger mechanism: (1) disease should be transmitted 50 times faster than supported by literature if there is a transmission chain; and (2) the trigger mechanism is consistent with the absence of reported outbreaks in young pigs as they have not yet been colonised by the bacterium. In conclusion, outbreaks of A. pleuropneumoniae on endemic farms are most likely caused by a trigger inducing pneumonia in already infected pigs, but more evidence is needed to identify optimum preventive interventions.

Efficacy of vaccination against Actinobacillus pleuropneumoniae in two Belgian farrow-to-finish pig herds with a history of chronic pleurisy.

The efficacy of an Actinobacillus pleuropneumoniae subunit vaccine based on ApxIA, ApxIIA, ApxIIIA and OMP-2 (Porcilis App, MSD) was investigated in two farrow-to-finish pig herds (A and B) affected by chronic pleurisy. In total, 1161 pigs were included. At three weeks of age, the pigs were randomly allocated to non-vaccinated control (NV; n=580) and vaccinated (V; n=581) groups. At 6 and 10 weeks of age, pigs were injected with Porcilis-APP (V group) or adjuvant (NV group). At slaughter (26 weeks), pleurisy and pneumonia lesions were assessed. All pigs were weighed individually at 6 and 26 weeks of age, and average daily weight gain (ADG; g/pig/day) was calculated. Mortality and days of additional treatment (DAT) were registered during the whole experiment. Data were analysed using binary logistic regression or analysis of variance for proportions or continuous variables, respectively. The prevalence of pleurisy and pneumonia was (NV-A=19.3, V-A=7.9, (P=0.000); NV-B=17.9, V-B=0.7, (P=0.000)) and (NV-A=42.4, V-A=21.2, (P=0.000); NV-B=46.7, V-B=19.0, (P=0.000)), respectively. The ADG was NV-A=632±157, V-A=647±91, (P=0.162); NV-B=660±115, V-B=670±82, (P=0.232). The mortality during the experiment was NV-A=5.7, V-A=1.8, (P=0.015); NV-B=2.3, V-B=1.0, (P=0.170) per cent. The DAT was: NV-A=15.04±1.41, V-A=14.95±0.67, (P=0.010); NV-B=21.68±2.43, V-B=16.99±0.62, (P=0.000). The present study showed a significant reduction of the prevalence of pleurisy and pneumonia, and antimicrobial use in V pigs from both herds, and in mortality in V pigs from one herd.

Actinobacillus pleuropneumoniae seroprevalence in farmed wild boars in Finland.

The aim of this study was to estimate the prevalence of serum antibodies to Actinobacillus pleuropneumoniae (APP) in Finnish farmed wild boars and to evaluate basic risk factors for seropositivity of the agent. A sampling frame was compiled based on the national record of wild boar farmers (n = 117), and 26 herds were included in the study. Serum samples (n = 206) were obtained from all animals slaughtered from these herds during the 2007-2008 period. Indirect enzyme-linked immunosorbent assay techniques were used to detect the possible presence of antibodies toward 12 serotypes of APP. Altogether, 1,442 analyses were performed, out of which 35 (2.4%) exceeded the positive cut-off value. Correspondingly, 26 of the pigs (12.6%) were found to be seropositive for any serotype of APP. The apparent animal prevalence of the cross-reacting APP serotypes 1, 9, and 11 was 3.9% (95% confidence interval [CI] 1-7%); it was 0.5% (CI 0-1%) for serotype 2; 3.4% (CI 1-6%) for serotypes 3, 6, and 8; 7.3% (CI 4-11%) for serotypes 4 and 7; 0.5% (CI 0-1%) for serotype 10; and 1.5% (CI 0-3%) for serotype 12. No antibodies against serotype 5s were detected. The apparent farm prevalence of APP serotypes 1,9, and 11 was 15.4% (CI 2-29%); that of serotype 2 was 3.8% (CI 0-11%); that of serotypes 3, 6, and 8 was 11.5% (CI 0-24%); that of serotypes 4 and 7 was 34.6% (CI 16-53%); that of serotype 10 was 3.8% (CI 0-11%); and that of serotype 12 was 7.7% (CI 0-18%). In conclusion, the prevalence of serum antibodies to different APP serotypes was low in farmed wild boar. Risk factor analysis did not reveal any significant animal- or herd-level risk factors for seropositivity of the agent.

Infectious agents associated with respiratory diseases in 125 farrow-to-finish pig herds: a cross-sectional study.

A study was carried out in 125 farrow-to-finish pig herds to assess the relationships between pathogens involved in respiratory disorders and to relate these findings to clinical signs of respiratory diseases and pneumonia and pleuritis at slaughter. Clinical examination and sampling were carried out on four different batches in each herd (pigs aged 4, 10, 16 and 22 weeks). Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, swine influenza viruses (SIV), porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) were detected by serological or PCR tests. Pneumonia-like gross lesions and pleuritis were scored at the slaughterhouse. The results indicate that the percentage of pigs PCR-positive for PCV2 at 4, 10 and 16 weeks old was associated with the percentage of pigs PCR-positive for M. hyopneumoniae at these ages. On the other hand, the percentage of pigs with antibodies against PRRSV at 10, 16 and 22 weeks was positively correlated with the percentage of pigs seropositive for M. hyopneumoniae at 22 weeks, with the percentage of pigs with antibodies against SIV H1N1 and SIV H1N2 and the percentage of pigs sero-positive for A. pleuropneumoniae serotype 2. The findings also indicate that, within the five studied pathogens, M. hyopneumoniae, PRRSV and SIV H1N1 are the major pathogens involved in pneumonia-like gross lesions even though PCV2 may play a role. A. pleuropneumoniae serotype 2, in association with PRRSV, is significantly associated with extensive pleuritis. Respiratory diseases could be significantly reduced by implementing measures including appropriate management practices to control these pathogens.