Adherence to and invasion of mammalian cell lines by Pasteurella multocida B:2

Haemorrhagic septicemia (HS), caused by the Gram-negative bacterium Pasteurella multocida serotype B:2, is an economically important disease responsible for morbidity and mortality of bovines, especially buffaloes, in countries of South or Southeast Asia and Africa. A feature of this disease is the rapid spread of infecting bacteria from the respiratory tract to the blood and lymph to cause a fatal septicemia. To pass into the blood stream, the bacteria must migrate through the epithelial layer into the pulmonary interstitium. Avian serogroup A strains of P. multocida have been reported to invade cultured mammalian cells, but the behaviour of other of serogroups has not been reported. The main object of the work was to confirm that HS strains of P. multocida B:2 have the capacity to invade and survive within cultured mammalian cells, such as J774.2 cells (mouse macrophage-like cell lines) and BL-3 cells (bovine lymphoma cell line). Invasion, defined as adhesion to, followed by uptake by, or entry into, J774.2 macrophage cells or BL-3 cells was determined by: (I) counting of viable intracellular bacteria after killing extracellular bacteria with polymyxin and gentamicin, (II) Transmission electronic microscopy. Comparison of the invasiveness of a B:2 HS strain and its aroA derivative JRMT12 with that of P. multocida A:3 and E. coli XL1-Blue, showed that both P. multocida B:2 strains invaded both types of mammalian cells more readily than P. multocida A:3 and that E. coli XL1-Blue was essentially non-invasive. Both strains of P. multocida B:2 could survive within J774.2 macrophage and BL-3 cells for at least 2 h. A longer-term survival experiment (up to 6 h incubation) indicated that the numbers of intracellular bacteria declined between 4 to 6 h post-infection. It was shown by TEM that a significant proportion of the P. multocida B:2 bacteria were found within vacuoles in the cytoplasm of the mammalian cells with some free in the cytoplasm. A much reduced invasion capacity of P. multocida A:3 and E. coli XL1-Blue was detected. Different effects on the appearance and viability of J774.2 and BL-3 cells were observed by the trypan blue method and TEM when exposed to the P. multocida B:2 strains. Evaluation of cytotoxicity of P. multocida B:2 stains with J774.2 cells by the MTT assay produced unsatisfactory results