Figure 4

Differentiated OTECs support the colonisation of a wide variety of M. haemolytica isolates including pathogenic and non-pathogenic strains. Differentiated OTECs were infected with the indicated M. haemolytica isolates for 2 h, 24 h and 72 h. Non-adherent bacteria were removed by washing and tissues were processed as follows. (A) Tissues were lysed by addition of 1% (v/v) Triton X-100 and bacteria were enumerated by serial dilution and spot-plating. The numbers of bacteria in the lysate were expressed as a percentage of the inoculum (colonisation efficiency [%]). Columns display means of three experiments +/− standard error, with colonisation efficiency being enumerated from triplicate inserts in each experiment. Bovine isolates are indicated by red columns, while ovine isolates are indicated by green columns. Solid colour indicates pathogenic isolates while dashed columns indicate non-pathogenic isolates. Statistical significance of differences between strains at each independent time-point was assessed by Welch’s unequal variance t test with multiple comparisons. Significance values of * and ** represent p < 0.05 and 0.01, respectively. (B) Tissues were fixed in paraformaldehyde. Following permeabilisation, bacteria were immunostained with an anti-M. haemolytica whole-cell antibody (green). Cilia and cytoskeletal microtubules were stained with anti-β-tubulin (magenta), host cell actin was stained with phalloidin Alexa-568 (red) and nuclei were stained with DAPI (blue). Samples were mounted and viewed by wide-field microscopy. In healthy regions of tissue, β-tubulin is concentrated in the apical cell surface-localised cilia while F-actin staining allows visualisation of the underlying epithelium. Upon formation of invasive foci of infection with pathogenic strains, this localisation and normal cellular morphology becomes disrupted. By comparison, with non-pathogenic strains and PH372 only sporadic aggregates of cilia-attached bacteria were observed.