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Highlights • Analysis of complete capsule loci in all 18 serovars of A. pleuropneumoniae. • Novel insights into evolution of capsule loci in A. pleuropneumoniae. • Development of two mPCRs for comprehensive capsule typing., 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.

Highlights • Identification of two new serovars of Actinobacillus pleuropneumoniae. • Serological confirmation of specific reactivity with homologous antisera. • Characterization of the capsule loci of serovars 17 and 18. • Development of PCRs for molecular diagnostics., 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.

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.

Summary Background: Pleuropneumonia with high economic losses caused by Actino bacillus pleuropneumoniae is common throughout the world. A. pleuropneumo niae has two biotypes and 16 serotypes. Serotype 16 was described recently in Hungary and it has not been isolated from other countries yet. Genes for the production (apxIA) and secretion (apxIB) of ApxI, the gene for the expression of ApxII and apxIV gene were detected in it. Objectives: Serotype 16 of A. pleuropneumoniae was diagnosed in a large scale pig herd with 1350 sows in the Eastern part of Hungary in acute cases of pleuro pneumonia. On the basis of the bacteriological examinations the formerly used marketed, registered vaccine produced from inactivated A. pleuropneumoniae strains was replaced with a marketed and registered vaccine containing inacti vated toxins of A. pleuropneumoniae and outer membrane protein in standardi zed amount. The objective was to evaluate its protective effect. Materials and methods: Four lungs of dead pigs and four lungs collected at the slaughterhouse, showing lesions of acute haemorrhagic-necrotic pneumonia and fibrinous pleuritis were sent to the laboratory. After changing the vaccine, a few months later 14 lungs with chronic pleuropneumonia were examined. The isolated A. pleuropneumoniae strains were serotyped using the indirect haem agglutination test. Results: Serotype 16 of A. pleuropneumoniae was isolated from all the 8 acute and 14 chronic forms. In that part of the herd where the animals were inject ed with the toxoid vaccine (only healthy animals were vaccinated), the acute respiratory signs have not been seen any more, however A. pleuropneumoniae serotype 16 strains could be still isolated from lung samples collected at the slaughterhouse from animals having chronic lesions.

SUMMARY Background: Actinobacillus pleuropneumoniae is an important pathogen of swine, it can cause respiratory disease in piglets sometimes from 6 weeks of age, but typically 12-16 week-old feeder pigs are susceptible. It has two biotypes and 16 serotypes, and due to the different pattern of toxin production there are great differences in the virulence of the strains. Objectives: Besides reaching etiologic diagnosis, the objective of the examina tion was to prove that in case of special circumstances sometimes young pig lets around weaning age can show the clinical signs and post mortem lesions caused by Actinobacillus pleuropneumoniae. Materials and methods: Lungs of six 28 and 31-day-old piglets showing lesions of acute haemorrhagic-necrotic pneumonia and fibrinous pleuritis were sent from a large scale farm with 3000 sows in the Eastern part of Hungary. According to our previous examinations A. pleuropneumoniae was present in the herd; serotype 16 strains were isolated earlier but the pigs in the farm were not vaccinated against A. pleuropneumoniae. The lung samples were inoculated on blood agar plates; they were cross-inoculated with Staphylococcus aureus and incubated at 37 ºC for 24 hours in the presence of 5% carbon dioxide. The isolated A. pleuropneumoniae strains were serotyped using the indirect haemagglutination test. Results and Discussion: Actinobacillus pleuropneumoniae biotype 1 serotype 11 strains were isolated from the lungs; this serotype was not detected in this farm earlier. The examinations confirmed that A. pleuropneumoniae can cause disease not only in grower and feeder pigs, but a newly introduced serotype can also result severe clinical signs and lesions of the disease around weaning in the absence of maternal protection. The age of the diseased animals can be informative in setting up the diagnosis of an infectious disease; however the circumstances have to be always carefully evaluated.