HighlightsAnalysis 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.

HighlightsIdentification 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.

ABSTRACT:Haemophilus parasuis is a common epiphyte of the upper respiratory tract of pigs. The factors of H. parasuis pathogenicity that enable some strains to be virulent and consequently cause a clinical disease have not been established yet. Fifteen serovars of H. parasuis have been described at present. Individual serovars differ in virulence, and considerable differences in virulence also exist within each serovar. Virulent strains can particularly participate as microorganisms secondary to pneumonia, cause septicaemia without polyserositis or Glässer's disease characterized by polyserositis, pericarditis, arthritis and meningitis. Clinical symptoms of this disease are highly variable. Therefore, culture detection of causative agent, particularly from the brain, joints and polyserositis is an essential diagnostic tool. The disease caused by H. parasuis can be treated with antibiotics; however, oral or parenteral administration of very high doses of antibiotics is necessary. The level of animal hygiene and animal husbandry are important factors for prevention of this disease. Commercial or autogenous vaccines can be used in the immunoprophylaxis of pre-parturient sows and their progeny after weaning. For the production of autogenous vaccines, it is most effective to use isolates from animals with lesions present in CNS. Isolates recovered from arthritic and systemic sites of infection are less suitable and isolates recovered from lungs are not suitable at all because of their heterogeneity.

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.

A total of 24 Pasteurella multocida rabbit isolates obtained from 24 rabbit flocks in the Czech Republic during the period of between 2001 and 2004 were analysed by capsular PCR typing. Apart from isolates identified as serogroups A (n = 14, 58.4%) and D (n = 2, 8.3%), eight isolates (33.3%) were identified as members of serogroup F. This serogroup had been predominantly associated with poultry infections so far. The rabbit serogroup F isolates were characterized in detail by ribotyping with restriction to endonuclease MspI revealing two distinct ribotypes. Seven serogroup F isolates were assigned to ribotype 1 and one isolate was assigned to ribotype 2.

Pasteurella multocida and Haemophilus parasuis pig isolates obtained in the Czech Republic were tested for their susceptibility against selected antimicrobial agents by broth microdilution method between 2008 and 2011. A low degree of resistance was observed for ampicillin, amoxicillin/clavulanic acid, ceftiofur, tulathromycin, tilmicosin, florfenicol and enrofloxacin in 20 (6.0%), 15 (4.5 %), 2 (0.6%), 8 (2.4%), 13 (3.9%), 5 (1.5%) and 5 (1.5%) P. multocida isolates as well as for tiamulin, gentamicin, tulathromycin, tilmicosin and ampicillin in 2 (2.4%), 2 (2.4%), 3 (3.6%), 3 (3.6%) and 6 (7.2%) H. parasuis isolates. In addition, moderate level of resistance to tiamulin was found in 60 (18.1%) P. multocida isolates and high level of resistance for tetracycline was detected in 107 (32.2 %) P. multocida isolates and in 23 (27.7 %) H. parasuis isolates. Differences between resistance rates of P. multocida and H. parasuis were significant (P ≤ 0.5) only for tiamulin. These data confirmed that antimicrobial resistance is not very widespread among current porcine P. multocida and H. parasuis isolates in the Czech Republic. Antimicrobial resistance, minimal inhibition concentration, respiratory diseases, pigsPasteurella multocida and Haemophilus parasuis are the causative agents of infectious diseases of porcine respiratory tract which cause major economic losses by their negative impact on the weight gain, feed conversion, and health status (Oliveira and Pijoan 2004;Shin et al. 2005). Antibiotic treatment is one of the commonly used measures for the control of pasteurellosis and H. parasuis infections but the use of antimicrobial agents leads to both selection and increase of resistance (Schwarz and Chaslus-Dancla 2001). Failure to treat infectious disease caused by resistant bacteria leads to increased morbidity and mortality (Kolar et al. 2010). Variations in the antimicrobial use for the control of bacterial pathogens in pigs from one country to another can contribute to evident differences in antimicrobial susceptibility of P. multocida and H. parasuis. In accordance with the recommendation of Schwarz et al. (2010) on the requirement of application of the same methodology and interpretive criteria (which would allow for re-evaluation of the original data if the interpretive criteria change over time), only a limited number of recent studies were conducted regarding the susceptibility of P. multocida and H. parasuis to antimicrobials with the use of microdilution method.Considering the lack of recent studies of antimicrobial susceptibility testing of P. multocida and H. parasuis within the European territory, the aim of this work was to examine a panel of various isolates obtained from different Czech locations to determine their susceptibility to selected antimicrobial agents by the broth microdilution method, in accordance with the guidelines issued by the Clinical and Laboratory Standards Institute (CLSI) in 2008. Materials and Methods SamplingAll P. multocida and H. parasuis isolates were obtained fro...

Streptococcus suis is an important pathogen of pigs but is also transmissible to humans, with potentially fatal consequences. Among 29 serotypes currently recognized, some are clinically and epidemiologically more important than others. This is particularly true for serotypes 2 and 14, which have a large impact on pig production and also on human health. Conventional PCR-based serotyping cannot distinguish between serotype 1/2 and serotype 2 or between serotype 1 and serotype 14. Although serotype 1/2 and serotype 2 have a very similar cps locus, they differ in a single-nucleotide substitution at nucleotide position 483 of the cpsK gene. Similarly, serotypes 1 and 14 have a very similar cps locus but also differ in the same nucleotide substitution of the cpsK gene. Fortunately, this cpsK 483G→C/T substitution can be detected by BstNI restriction endonuclease. A PCR-restriction fragment length polymorphism (RFLP) detection method amplifying a fragment of the cpsK gene digested by BstNI restriction endonuclease was developed and tested in reference strains of these serotypes and also in field isolates.

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