This Btudy utilised cell cultures in an attempt to elucidate the
aetiological cause of the bacterial associated porcine proliferative enteropathies.
Initial work evaluated the attachment of one possible agent Campylobacter
sputorum subspecies mucosalis (CSM) to cells, CSM did not adhere to PK15 cells
during the first 8 hours of cellular development whereas, after 20—24 hours of
growth, the majority of cells in the monolayer showed maximum bacterial attachment.
Adhesion was influenced by the number of viable organisms in the inoculum, by the
degree of cell to cell contact in the monolayer and by the stage of development of
the cells when exposed to infection, but, in all instances, a small number of cells
were refractory to bacterial attachment. Adhesion to PK15 cells was completely
inhibited by rabbit anti-CSM serum, partially inhibited by wheat germ agglutinin
(WGA) and soya bean agglutinin (SBA) but was not affected by any of the metallic
salts tested. Attachment of CSM to in vitro preparations of pig intestinal brush
borders was scanty compared with that obtained with PK15 cells and did not vary
between cells from different pig genotypes.
Following attachment to PK15 cell surfaces, the bacteria were engulfed
and later appeared within phagosomes or lying freely in the cytoplasm. Cytopathic
effects were induced by CSM infecting doses of at least 81og^Q organisms per ml
but not by 51og1Q organisms per ml. Bacterial counts of supernatant fluids and
lysed cells from infected PK15 cultures, supported by immunoflourescence staining,
indicated cell-associated growth of CSM but limited survival in the extracellular
Infection of tissue culture cells by CSM grown in cell-free culture (CSM-C)
was compared with that of bacteria derived directly from the tissues of the
proliferative enteropathies. Three types of bacteria were isolated from filtered
homogenised PIA tissues. A purified inoculum of the filtrate prepared by dilution
and further filtration contained organisms that stained as the intracellular
organisms, grew in cell-free culture and were identified as CSM (CSM-T). Such
CSM-T derived by filtration produced cytopathic changes in PK15 cells similar to
those obtained with cultured CSM (CSM-C). Progressive infection of cell cultures
was initiated with only 3»481og1_ per ml of CSM-T, but not with 5»26log..Q per ml of
CSM-C. Irrespective of source, whether from lesions, tissue culture or cell-free
cultures, it was not possible to passage CSM organisms serially in PK15 cells.
Campylobacter-like forms in filtrates of PHE tissues did not multiply in PK15 cells
nor produce cytopathic changes. In contrast, Campylobacters and other bacteria
isolated from PIA or PHE by filters of larger pore diameter multiplied in the
extracellular fluids and caused rapid destruction of PK15 cells.
The novel intracellular, campylobacter-like antigen (CJ) of proliferative
porcine enteropathies and hamster ileitis, which differs from the surface antigen
of known Campylobacters, was also detected in PK15 cells following infection with
CSM-C or CSM-T organisms.
A line of PK15 cells, once-infected with CSM and subsequently self-cured,
was developed but the cells showed only minor differences in characteristics from
those of the parent line.
The significance and future applications of the findings of this present
investigation in relation to the pathogenesis of porcine proliferative enteropathies,
the diseases associated with intracellular Campylobacters are discussed.