Generation and Evaluation of a Glaesserella (Haemophilus) parasuis Capsular Mutant

Abstract

Glaesserella (Haemophilus) parasuis is a commensal bacterium of the upper respiratory tract in pigs and also the causative agent of Glässer's disease, which causes significant morbidity and mortality in pigs worldwide. Isolates are characterized into 15 serovars by their capsular polysaccharide, which has shown a correlation with isolate pathogenicity. To investigate the role the capsule plays in G. parasuis virulence and host interaction, a capsule mutant of the serovar 5 strain HS069 was generated (HS069Δcap) through allelic exchange following natural transformation. HS069Δcap was unable to cause signs of systemic disease during a pig challenge study and had increased sensitivity to complement killing and phagocytosis by alveolar macrophages. Compared with the parent strain, HS069Δcap produced more robust biofilm and adhered equivalently to 3D4/31 cells; however, it was unable to persistently colonize the nasal cavity of inoculated pigs, with all pigs clearing HS069Δcap by 5 days postchallenge. Our results indicate the importance of the capsular polysaccharide to G. parasuis virulence as well as nasal colonization in pigs.

Keywords: Glaesserella parasuis; Glässer’s disease; acapsular mutant; capsular polysaccharide.

FIG 1

Capsule locus deletion and verification. The entirety of the capsule locus was deleted from neuA_3 to etk as indicated (A). The loss of the capsule locus was verified using the Artemis compare tool (B). No sequence reads mapped to the region of the capsule locus.

FIG 2

Transmission electron microscopy visualization of capsule. The capsule layer of G. parasuis HS069 wild type (A) and HS069Δcap (B) were visualized using transmission electron microscopy to verify HS069Δcap was deficient in capsular polysaccharide production. The surface of HS069Δcap lacked a thickened, irregular surface associated with capsule production.

FIG 3

Biofilm production by HS069 wild type and HS069Δcap. The capacity of G. parasuis HS069 and HS069Δcap to produce biofilm under static growth conditions was quantified using microtiter assays. Results here represent data from replicates with a starting OD₆₀₀ of 0.05. The average absorbance at 538 nm is shown (column) with standard deviations indicated (error bars). Statistical significance is indicated by the asterisk (*) at a P value of <0.05.

FIG 4

Evaluation of sensitivity to complement killing. G. parasuis HS069 and HS069Δcap were screened for resistance to complement-mediated killing. Statistical analysis of the log₁₀ reduction in CFU/ml was analyzed, and statistical significance is indicated by the asterisk (*) at a P value of <0.05.

FIG 5

Adherence capacity of HS069 (A) and HS069Δcap (B) to 3D4/31 cells. The capacity for HS069 and HS069Δcap to adhere to porcine alveolar macrophages was evaluated using the 3D4/31 cell line. The interaction between G. parasuis and 3D4/31 cells was evaluated by confocal microscopy in chamber slides. G. parasuis strains were stained using a monoclonal antibody to the outer membrane protein P5. Bacterial aggregates were noted when evaluating HS069Δcap (B) but were not produced by the wild-type HS069 isolate.

FIG 6

Adherence capacity to porcine alveolar macrophages (3D4/31 cell line). The capacity of G. parasuis HS069 and HS069Δcap to adhere to porcine alveolar macrophages was evaluated using the 3D4/31 cell line. Due to the clusters of bacterial cells noted using confocal microscopy, adherence was evaluated both as bacterial cells detected per 3D4/31 cell (A) and fluorescent intensity per 3D4/31 cell (B). No statistical differences were noted in bacteria per cell or fluorescent intensity per cell.

FIG 7

Evaluation of the susceptibility to phagocytosis. G. parasuis HS069 and HS069Δcap were screened for susceptibility to phagocytosis using porcine alveolar macrophages. Log fold reduction in G. parasuis [log₁₀(CFU/ml)] is represented for both time points (1-hour and 2-hour incubation). The reduction of G. parasuis HS069 and HS069Δcap was not statistically different after 1 hour of incubation (P = 0.93); however, after 2 hours of incubation with PAMs, significantly more G. parasuis HS069Δcap was phagocytosed than HS069 wild type (P < 0.01).

FIG 8

Serum antibody titers for pigs inoculated with HS069Δcap. The serum antibody titer was detected using an ELISA to whole-cell sonicate. The data presented in this graph represent the animals in the second study investigating colonization with HS069Δcap.