Molecular characterization of Streptococcus suis isolates recovered from diseased pigs in Europe.

Streptococcus suis is a major swine pathogen and zoonotic agent, causing important economic losses to the porcine industry. Here, we used genomics approaches to characterize 251 S. suis isolates recovered from diseased pigs across Belgium, France, Germany, Hungary, the Netherlands, Spain, and the United Kingdom. We identified 13 serotypes, being serotypes 9 and 2 the most prevalent, and 34 sequence types (STs), including 16 novel STs, although ST16 and ST1 dominated the strain population. Phylogenetic analysis revealed complex genetic relationships, notable geographic clustering, and potential differential capacity for capsular switching among serotype 9 isolates. We found antimicrobial resistance (AMR) genes in 85.3% of the isolates, with high frequencies of genes conferring resistance to tetracyclines and macrolides. Specifically, 49.4% of the isolates harbored the tetO gene, and 64.9% possessed the ermB gene. Additionally, we observed a diverse array of virulence-associated genes (VAGs), including the classical VAGs mrp, epf, and sly, with variable presence across different genotypes. The high genetic diversity among European S. suis isolates highlights the importance of targeted antimicrobial use and flexible vaccine strategies. Rapid strain characterization is crucial for optimizing swine health management, enabling tailored interventions like the development of autovaccines to mitigate S. suis infections.

Assessment of real-time PCR test in oral fluid samples for screening the serotypes of Actinobacillus pleuropneumoniae circulating in swine herds.

Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, causing remarkable economic losses in the global swine industry. The diversity of A. pleuropneumoniae is generally determined through serotype identification, which is commonly employed for control strategies and surveillance. However, serological methods currently in use still have significant limitations. This study explores the use of real-time polymerase chain reaction (qPCR) to detect circulating serotypes of A. pleuropneumoniae in non-diseased swine herds through testing of oral fluids. The study included three A. pleuropneumoniae-positive and three A. pleuropneumoniae-negative farms located in Quebec, Canada. Tonsil brushings, microbiological growths, and oral fluids were analyzed using qPCR to detect A. pleuropneumoniae and its distinct serotypes. Serological tests were performed using the LPS ELISA available at that time. In negative farms the absence of A. pleuropneumoniae and any serotype confirmed the specificity of the method. Positive farms, on the other hand, confirmed also the sensitivity of the analysis, with oral fluid samples consistently yielding positive results for the serotypes identified by ELISA. The qPCR test conducted on oral fluids offers a noninvasive and cost-effective method for monitoring, complementing traditional serological techniques. It provides qualitative information about serotype distribution, facilitating proactive surveillance and control strategies.

The granulocyte colony-stimulating factor produced during Streptococcus suis infection controls neutrophil recruitment in the blood without affecting bacterial clearance.

Streptococcus suis causes diseases in pigs and has emerged as a zoonotic agent. When infected, the host develops an exacerbated inflammation that can lead to septic shock and meningitis. Although neutrophils greatly infiltrate the lesions, their dynamics during S. suis infection remain poorly described. Moreover, very few studies reported on the production and role of a key factor in the regulation of neutrophils: the colony-stimulating granulocyte factor (G-CSF). In this study, we characterized the G-CSF-neutrophil axis in the pathogenesis of S. suis induced disease. Using a mouse model of S. suis infection, we first evaluated the recruitment of neutrophils and their activation profile by flow cytometry. We found that infection provokes a massive neutrophil recruitment from the bone marrow to the blood and spleen. In both compartments, neutrophils displayed multiple activation markers. In parallel, we observed high systemic levels of G-CSF, with a peak of production coinciding with that of neutrophil recruitment. We then neutralized the effects of G-CSF and highlighted its role in the release of neutrophils from the bone marrow to the blood. However, it did not affect bacteremia nor the cytokine storm induced by S. suis. In conclusion, systemic G-CSF induces the release of neutrophils from the bone marrow to the blood, but its role in inflammation or bacterial clearance seems to be compensated by unknown factors. A better understanding of the role of neutrophils and inflammatory mediators could lead to better strategies for controlling the infection caused by S. suis.

Techniques for Genetic Manipulation.

In the last few decades, molecular techniques and genetic modification have been used in genotype and phenotype studies of S. suis. Genomic modification of S. suis requires DNA acquisition and its stable insertion into the chromosome by allelic exchange. In this chapter, we described two techniques for the preparation of genomic constructs (cloning and overlapping extension PCR) and for DNA uptake (electroporation and transformation). The protocols are accompanied with examples. All described protocols were successful on our hands with the reference S. suis strain P1/7.

Immune response induced by a Streptococcus suis multi-serotype autogenous vaccine used in sows to protect post-weaned piglets.

Streptococcus suis is a bacterial pathogen that causes important economic losses to the swine industry worldwide. Since there are no current commercial vaccines, the use of autogenous vaccines applied to gilts/sows to enhance transfer of passive immunity is an attractive alternative to protect weaned piglets. However, there is no universal standardization in the production of autogenous vaccines and the vaccine formulation may be highly different among licenced manufacturing laboratories. In the present study, an autogenous vaccine that included S. suis serotypes 2, 1/2, 5, 7 and 14 was prepared by a licensed laboratory and administrated to gilts using a three-dose program prior to farrowing. The antibody response in gilts as well as the passive transfer of antibodies to piglets was then evaluated. In divergence with previously published data with an autogenous vaccine produced by a different company, the increased response seen in gilts was sufficient to improve maternal antibody transfer to piglets up to 5 weeks of age. However, piglets would still remain susceptible to S. suis disease which often appears during the second part of the nursery period. Vaccination did not affect the shedding of S. suis (as well as that of the specific S. suis serotypes included in the vaccine) by either gilts or piglets. Although all antibiotic treatments were absent during the trial, the clinical protective effect of the vaccination program with the autogenous vaccine could not be evaluated, since limited S. suis cases were present during the trial, confirming the need for a complete evaluation of the clinical protection that must include laboratory confirmation of the aetiological agent involved in the presence of S. suis-associated clinical signs. Further studies to evaluate the usefulness of gilt/sow vaccination with autogenous vaccines to protect nursery piglets should be done.

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.

Investigation of genomic and pathogenicity characteristics of Streptococcus suis ST1 human strains from Guangxi Zhuang Autonomous Region (GX) between 2005 and 2020 in China.

Streptococcus suis is a significant and emerging zoonotic pathogen. ST1 and ST7 strains are the primary agents responsible for S. suis human infections in China, including the Guangxi Zhuang Autonomous Region (GX). To enhance our understanding of S. suis ST1 population characteristics, we conducted an investigation into the phylogenetic structure, genomic features, and virulence levels of 73 S. suis ST1 human strains from GX between 2005 and 2020. The ST1 GX strains were categorized into three lineages in phylogenetic analysis. Sub-lineage 3-1a exhibited a closer phylogenetic relationship with the ST7 epidemic strain SC84. The strains from lineage 3 predominantly harboured 89K-like pathogenicity islands (PAIs) which were categorized into four clades based on sequence alignment. The acquirement of 89K-like PAIs increased the antibiotic resistance and pathogenicity of corresponding transconjugants. We observed significant diversity in virulence levels among the 37 representative ST1 GX strains, that were classified as follows: epidemic (E)/highly virulent (HV) (32.4%, 12/37), virulent plus (V+) (29.7%, 11/37), virulent (V) (18.9%, 7/37), and lowly virulent (LV) (18.9%, 7/37) strains based on survival curves and mortality rates at different time points in C57BL/6 mice following infection. The E/HV strains were characterized by the overproduction of tumour necrosis factor (TNF)-α in serum and promptly established infection at the early phase of infection. Our research offers novel insights into the population structure, evolution, genomic features, and pathogenicity of ST1 strains. Our data also indicates the importance of establishing a scheme for characterizing and subtyping the virulence levels of S. suis strains.

Characterization of Actinobacillus pleuropneumoniae biovar 2 isolates reportedly reacted with the serovar 4 antiserum, and development of a multiplex PCR for O-antigen typing.

We have analyzed the capsule (CPS) and the lipopolysaccharide O-Antigen (O-Ag) biosynthesis loci of twelve Spanish field isolates of Actinobacillus pleuropneumoniae biovar 2, eleven of them previously typed serologically as serovar 4 and one non-typable (NT) (Maldonado et al., 2009, 2011). These isolates have the common core genes of the type I CPS locus, sharing >98% identity with those of serovar 2. However, the former possesses the O-Ag locus as serovar 4, and the latter possesses the O-Ag locus as serovar 7. The main difference found between the CPS loci of the 11 isolates and that of serovar 2 reference strain S1536 are two deletions, one of an 8 bp sequence upstream of the coding sequence and one of 111 bp sequence at the 5' end of the cps2G gene. The deletion mutations mentioned lead to a defect in the production of CPS in these isolates, which contributed to their previous mis-identification. In order to complement the serotyping of A. pleuropneumoniae in diagnostics and epidemiology, we have developed a multiplex PCR for the comprehensive O-Ag typing of all A. pleuropneumoniae isolates.

Lipoteichoic acids influence cell shape and bacterial division of Streptococcus suis serotype 2, but play a limited role in the pathogenesis of the infection.

Streptococcus suis serotype 2 is a major swine pathogen and a zoonotic agent, causing meningitis in both swine and humans, responsible for substantial economic losses to the swine industry worldwide. The pathogenesis of infection and the role of bacterial cell wall components in virulence have not been fully elucidated. Lipoproteins, peptidoglycan, as well as lipoteichoic acids (LTA) have all been proposed to contribute to virulence. In the present study, the role of the LTA in the pathogenesis of the infection was evaluated through the characterisation of a mutant of the S. suis serotype 2 strain P1/7 lacking the LtaS enzyme, which mediates the polymerization of the LTA poly-glycerolphosphate chain. The ltaS mutant was confirmed to completely lack LTA and displayed significant morphological defects. Although the bacterial growth of this mutant was not affected, further results showed that LTA is involved in maintaining S. suis bacterial fitness. However, its role in the pathogenesis of the infection appears limited. Indeed, LTA presence reduces self-agglutination, biofilm formation and even dendritic cell activation, which are important aspects of the pathogenesis of the infection caused by S. suis. In addition, it does not seem to play a critical role in virulence using a systemic mouse model of infection.

Genomic epidemiology in Streptococcus suis: Moving beyond traditional typing techniques.

Streptococcus suis is a bacterial gram-positive pathogen that causes invasive infections in swine and is also a zoonotic disease agent. Traditional molecular typing techniques such as ribotyping, multilocus sequence typing, pulse-field gel electrophoresis, or randomly amplified polymorphic DNA have been used to investigate S. suis population structure, evolution, and genetic relationships and support epidemiological and virulence investigations. However, these traditional typing techniques do not fully reveal the genetically heterogeneous nature of S. suis strains. The high-resolution provided by whole-genome sequencing (WGS), which is now more affordable and more commonly available in research and clinical settings, has unlocked the exploration of S. suis genetics at full resolution, permitting the determination of population structure, genetic diversity, identification of virulent clades, genetic markers, and other bacterial features of interest. This approach will likely become the new gold standard for S. suis strain typing as WGS instruments become more widely available and traditional typing techniques are gradually replaced.

Genomic and phenotypic analysis of invasive Streptococcus suis isolated in Spain reveals genetic diversification and associated virulence traits.

Streptococcus suis is a zoonotic pathogen that causes a major health problem in the pig production industry worldwide. Spain is one of the largest pig producers in the world. This work aimed to investigate the genetic and phenotypic features of invasive S. suis isolates recovered in Spain. A panel of 156 clinical isolates recovered from 13 Autonomous Communities, representing the major pig producers, were analysed. MLST and serotyping analysis revealed that most isolates (61.6%) were assigned to ST1 (26.3%), ST123 (18.6%), ST29 (9.6%), and ST3 (7.1%). Interestingly, 34 new STs were identified, indicating the emergence of novel genetic lineages. Serotypes 9 (27.6%) and 1 (21.8%) prevailed, followed by serotypes 7 (12.8%) and 2 (12.2%). Analysis of 13 virulence-associated genes showed significant associations between ST, serotype, virulence patterns, and clinical features, evidencing particular virulence traits associated with genetic clusters. The pangenome was generated, and the core genome was distributed in 7 Bayesian groups where each group included a variable set of over- and under-represented genes of different categories. The study provides comprehensive data and knowledge to improve the design of new vaccines, antimicrobial treatments, and bacterial typing approaches.

Invasive Bacterial Infections of the Musculoskeletal and Central Nervous System during Pig Rearing: Detection Frequencies of Different Pathogens and Specific Streptococcus suis Genotypes.

Locomotor and central nervous system disorders occur during pig rearing, but there is no systematic recording of the different causative agents in Germany. Joint and meningeal swabs, kidneys, lungs, and eight different lymph nodes per pig were cultured, and isolated pathogens were identified using polymerase chain reactions (PCRs). The cps and pathotype of Streptococcus suis (S. suis) isolates were determined using multiplex-PCR. S. suis was the most important pathogen in the infected joints (70.8%) and meningeal swabs (85.4%) and was most frequently detected in both sites in suckling and weaning piglets. To elucidate the possible portal of entry of S. suis, eight different lymph nodes from 201 pigs were examined in a prospective study. S. suis was detected in all examined lymph nodes (n = 1569), including the mesenteric lymph nodes (15.8%; n = 121/765), with cps 9 (37.2%; n = 147) and cps 2 (24.3%; n = 96) being the most dominating cps types. In piglets with a systemic S. suis infection, different lymph nodes are frequently infected with the invasive S. suis strain, which does not help clarify the portal of entry for S. suis.

Encapsulated Streptococcus suis impairs optimal neutrophil functions which are not rescued by priming with colony-stimulating factors.

The porcine pathogen and zoonotic agent Streptococcus suis induces an exacerbated inflammation in the infected hosts that leads to sepsis, meningitis, and sudden death. Several virulence factors were described for S. suis of which the capsular polysaccharide (CPS) conceals it from the immune system, and the suilysin exhibits cytotoxic activity. Although neutrophils are recruited rapidly upon S. suis infection, their microbicidal functions appear to be poorly activated against the bacteria. However, during disease, the inflammatory environment could promote neutrophil activation as mediators such as the granulocyte colony-stimulating factor granulocyte (G-CSF) and the granulocyte-macrophages colony-stimulating factor (GM-CSF) prime neutrophils and enhance their responsiveness to bacterial detection. Thus, we hypothesized that CPS and suilysin prevent an efficient activation of neutrophils by S. suis, but that G-CSF and GM-CSF rescue neutrophil activation, leading to S. suis elimination. We evaluated the functions of porcine neutrophils in vitro in response to S. suis and investigated the role of the CPS and suilysin on cell activation using isogenic mutants of the bacteria. We also studied the influence of G-CSF and GM-CSF on neutrophil response to S. suis by priming the cells with recombinant proteins. Our study confirmed that CPS prevents S. suis-induced activation of most neutrophil functions but participates in the release of neutrophil-extracellular traps (NETs). Priming with G-CSF did not influence cell activation, but GM-CSF strongly promote IL-8 release, indicating its involvement in immunomodulation. However, priming did not enhance microbicidal functions. Studying the interaction between S. suis and neutrophils-first responders in host defense-remains fundamental to understand the immunopathogenesis of the infection and to develop therapeutical strategies related to neutrophils' defense against this bacterium.

Comparative genome analysis of Streptococcus suis serotype 5 strains from humans and pigs revealed pathogenic potential of virulent, antimicrobial resistance, and genetic relationship.

Streptococcus suis is a causative agent of swine and human infections. Genomic analysis indicated that eight S. suis serotype 5 strains recovered from human patients and pigs carried many virulence-associated genes and markers defining pathogenic pathotypes. The strains were sequence types diverse and clustered within either minimum core genome group 3 (MCG-3) or MCG-7-3. Almost all the serotype 5 strains were non-susceptible to penicillin, ceftriaxone, erythromycin, and levofloxacin. Resistance to tetracycline and clindamycin was observed in all strains. The antimicrobial resistance genes tet(O), tet(O/W/32/O), tet(W), tet(44), erm(B), ant(6)-Ia, lsaE, and lnuB were found in these strains. Moderate-to-large numbers of substitutions were observed in three penicillin-binding proteins (PBP)-PBP1A, PBP2B, and PBP2X-in the penicillin-non-susceptible serotype 5 isolates that were involved in ß-lactam-non-susceptibility. Comparative genomics between the serotype 5 and 2 strains revealed that only 15 genes absent from the serotype 2 strains were shared by all the serotype 5 strains. However, some additional genes were present only in some of the serotype 5 strains. This study highlighted the pathogenic potential of virulent serotype 5 strains in humans and pigs and the need for increased monitoring of penicillin-non-susceptibility in S. suis serotypes other than for serotype 2.

The emergence and diversification of a zoonotic pathogen from within the microbiota of intensively farmed pigs.

The expansion and intensification of livestock production is predicted to promote the emergence of pathogens. As pathogens sometimes jump between species, this can affect the health of humans as well as livestock. Here, we investigate how livestock microbiota can act as a source of these emerging pathogens through analysis of Streptococcus suis, a ubiquitous component of the respiratory microbiota of pigs that is also a major cause of disease on pig farms and an important zoonotic pathogen. Combining molecular dating, phylogeography, and comparative genomic analyses of a large collection of isolates, we find that several pathogenic lineages of S. suis emerged in the 19th and 20th centuries, during an early period of growth in pig farming. These lineages have since spread between countries and continents, mirroring trade in live pigs. They are distinguished by the presence of three genomic islands with putative roles in metabolism and cell adhesion, and an ongoing reduction in genome size, which may reflect their recent shift to a more pathogenic ecology. Reconstructions of the evolutionary histories of these islands reveal constraints on pathogen emergence that could inform control strategies, with pathogenic lineages consistently emerging from one subpopulation of S. suis and acquiring genes through horizontal transfer from other pathogenic lineages. These results shed light on the capacity of the microbiota to rapidly evolve to exploit changes in their host population and suggest that the impact of changes in farming on the pathogenicity and zoonotic potential of S. suis is yet to be fully realized.

Study of the Role of Lipoprotein Maturation Enzymes in the Pathogenesis of the Infection Caused by the Streptococcus suis Serotype 2 Sequence Type 25 North American Prototype Strain.

Streptococcus suis serotype 2 is an important swine bacterial pathogen causing sudden death, septic shock, and meningitis. However, serotype 2 strains are phenotypically and genotypically heterogeneous and composed of a multitude of sequence types (STs) whose distributions greatly vary worldwide. It has been previously shown that the lipoprotein (LPP) maturation enzymes diacylglyceryl transferase (Lgt) and signal peptidase (Lsp) significantly modulate the inflammatory host response and play a differential role in virulence depending on the genetic background of the strain. Differently from Eurasian ST1/ST7 strains, the capsular polysaccharide of a North American S. suis serotype 2 ST25 representative strain only partially masks sub-capsular domains and bacterial wall components. Thus, our hypothesis is that since LPPs would be more surface exposed in ST25 strains than in their ST1 or ST7 counterparts, the maturation enzymes would play a more important role in the pathogenesis of the infection caused by the North American strain. Using isogenic Δlgt and Δlsp mutants derived from the wild-type ST25 strain, our studies suggest that these enzymes do not seem to play a role in the interaction between S. suis and epithelial and endothelial cells, regardless of the genetics background of the strain used. However, a role in the formation of biofilms (also independently of the STs) has been demonstrated. Moreover, the involvement of LPP dendritic cell activation in vitro seems to be somehow more pronounced with the ST25 strain. Finally, the Lgt enzyme seems to play a more important role in the virulence of the ST25 strain. Although some differences between STs could be observed, our original hypothesis that LPPs would be significantly more important in ST25 strains due to a better bacterial surface exposition could not be confirmed.

Metabolic insights and background from naturally affected pigs during Streptococcus suis outbreaks.

Streptococcus suis (S. suis) is an endemic zoonotic pathogen still lacking adequate prevention in pigs. The present case study looked back to the occurrence and consequences of S. suis outbreaks in our swine research facilities in search of new metabolic and physiological insight. From a series of outbreaks, a dataset was created including 56 pigs sampled during disease detection based on clinical signs. Pigs suspected with S. suis infection were defined as diseased (n = 28) and included pigs defined as neurologically diseased (n = 20) when severe neurological signs (central nervous system dysfunctions, i.e., opisthotonos, ataxia, and generalized tremor) were observed. Another set of 28 pigs included respective pen mates from each case and were defined as control. Representative deaths were confirmed to be caused by S. suis. Tonsillar swabs were collected and analyzed by quantitative polymerase chain reaction (qPCR) for total bacteria, total S. suis, and S. suis serotypes (SS) 2 (and/or 1/2) and 9. Blood and sera were analyzed to quantify blood gases, minerals, and S. suis reactive immunoglobulins against current isolates. Data collected included litter sibling associations, birth and weaning body weight (BW), and average daily gain (ADG) 7 d after the disease detection. In general, the disease increased pH, sO2 and the incidence of alkalosis, but reduced pCO2, glucose, Ca, P, Mg, K, and Na in blood/serum compared to control. The SS2 (and/or SS1/2) prevalence was significantly (P < 0.05) increased in neurologically diseased pigs and its relative abundance tended (P < 0.10) to increase in tonsils. In contrast, the relative abundance of total S. suis was lower (P > 0.05) in diseased pigs than control pigs. Levels of S. suis reactive IgG2 were lower, but IgM were higher (P < 0.03) in neurologically affected pigs compared to control. Furthermore, there was an increased proportion of sibling pigs that were diseased compared to control. In conclusion, our results evidence that naturally affected pigs were associated to average performing pigs without any predisease trait to highlight but a sow/litter effect. Besides, neurologically affected pigs had increased S. suis (SS2 and/or 1/2) prevalence and relative abundance, a respiratory alkalosis profile, and mineral loss.

Evaluation of the Immunoprotective Capacity of Five Vaccine Candidate Proteins against Avian Necrotic Enteritis and Impact on the Caecal Microbiota of Vaccinated Birds.

Avian necrotic enteritis is an enteric disease of broiler chickens caused by certain pathogenic strains of Clostridium perfringens in combination with predisposing factors. A vaccine offering complete protection against the disease has not yet been commercialized. In a previous study, we produced five recombinant proteins predicted to be surface-exposed and unique to necrotic enteritis-causing C. perfringens and the immunogenicity of these potential vaccine candidates was assessed in broiler chickens. In the current work, the relative contribution of the antibodies raised by these putative antigens to protect broiler chickens was evaluated using an experimental necrotic enteritis induction model. Additionally, the link between the immune response elicited and the gut microbiota profiles in immunized birds subjected to infection with virulent C. perfringens was studied. The ELISA results showed that the IgY antibody titers in vaccinated birds on days 21 and 33 were significantly higher than those on days 7 and 14 and those in birds receiving the adjuvant alone, while the relative contribution of the specific immunity attributed to these antibodies could not be precisely determined using this experimental necrotic enteritis induction model. In addition, 16S rRNA gene amplicon sequencing showed that immunization of birds with recombinant proteins had a low impact on the chicken caecal microbiota.

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.