In this study, the growth characteristics of
Campylobacter sputorum ss mucosalis were investigated
in twelve different types of cell cultures: primary
cultures of porcine kidney (PPK), chicken embryo
fibroblasts (CEF) and cell lines of porcine kidney (PK),
bovine kidney (BK), canine kidney (DK), monkey kidney
(Vero and LLCMK^)' baby hamster kidney (BHK-21), human
cervical carcinoma (HeLa) cells and lines of porcine
(PK ^), bovine (BK ^) and ovine (OK^) kidney cells
persistently infected with Newcastle disease virus.
As a prelude to infection of cell cultures prelim¬
inary studies on the morphology and ultrastructure of
C. sputorum ss mucosalis and C. coli were undertaken.
Negatively-stained preparations of serologically repre¬
sentative strains of mucosalis showed the presence of
comma, S-shaped and long filamentous forms. Organisms
were observed with three different surface coats:
i) rough and scaly types with deep transverse clefts,
ii) smooth types with longitudinal ridges and iii)
smooth types without clefts or ridges. The production
of these cell surface structures appeared to depend on
the age of the culture and the type of growth medium
used. Mucosalis organisms invariably possessed a single
polar flagellusnand the flagellar appendage showed the
presence of a 'collar-like® structure on either side
of the filament at the site of attachment to the
basal granule.
In thin-sections, the presence of a double layered
cell wall and cytoplasmic membrane were clearly visible,
the cytoplasmic substance was granular and contained
numerous polyphosphate crystals. By comparison, a
strain of _C. coli showed coccoid or comma-shaped organisms
with leathery cell surface with or without transverse
clefts, however on section this organism closely re¬
sembled C. sputorum ss mucosalis except for the presence
of a large cytoplasmic vacuole.
C. sputorum ss mucosalis requires a hydrogen microaerophilic
atmosphere for growth and maintenance,
without which it becomes non-viable after 10-12 hr.
Certain types of cell cultures can support 'parasitic
growth' of this organism in the absence of such a
hydrogen microaerophilic atmosphere. The evidence
suggests that this 'cell dependant' growth is due to
intracellular multiplication and release of bacteria
from the infected cell cultures. In infected BK cells
'parasitic growth' of mucosalis can persist for up to
7 weeks.
Investigations on the adhesive properties of
mucosalis have shown that bacterial attachment is specific and is 'transitory', lasting upto 10-12 hr postinoculation.
There appears to be a direct relationship
between the ability of cell cultures to support 'parasitic growth' and to attach mucosalis organisms. The
mechanisms involved in the adhesive process are complex
although bacterial motility seems to enhance the attachment of mucosalis organisms. Bacterial cell surface structures, but not the flagella, are involved in the
adhesive process to host-cell surface receptors.
Infection of suspensions of trypsinized cells or
preformed monolayers with C. sputorum ss mucosalis did
not appear to interfere with the initial attachment
and/or growth of infected cells, although characteristic
cytopathic changes are found during later stages of
infection. Electron microscopic studies show that
following attachment to cell surfaces bacteria are
engulfed by the infected cells and are later found
in the phagosomes or free in the cytoplasmic substance.
The intracellular fate of mucosalis organisms in the
infected cell cultures depends on the type of cell infected.
Pig kidney cells appear to destroy mucosalis organisms
rapidly and characteristic bacteria disappear from the
cytoplasm to be replaced by accumulations of granular
material. In contrast, bovine kidney cells infected
with mucosalis show, in addition to degenerate forms
(ghost cells), morphologically normal bacteria. Com¬
prehensive evidence has been obtained, by viable counts
of bacteria, light,immunofluorescent and electron
microscopy, which strongly suggests that mucosalis is
capable of intracellular multiplication, at least in
the BK cell line.
The response of different types of cell cultures
to infection with mucosalis differs and can be broadly
grouped as following:
i. cell cultures that show 'parasitic growth' of
mucosalis and rapid CPE with cell fusion and
destruction of the infected monolayers. These
include PPK, PK, and PK(pi) cells.
ii. cell cultures that are less readily destroyed
by mucosalis infection and give rise to the
production of markedly enlarged 'altered cells',
and limited cell fusion. Such cultures include
BK, BK(pi) ., OK(pi) ., BHK and HeLa cells.
iii. cell cultures including Vero and LLCNK₂ lines,
that are 'initially' refractory to 'parasitic
growth' and the production of CPE but which, on
re-infection, behave like those in group (ii).
iv. cell cultures that normally support 'parasitic
growth' but fail to show any CPE, of which DK
cells is an example.
v. cell cultures that fail completely to support 'parasitic growth' and which do not show a CPE, even aft
super-infection e.g. primary cultures of CEF.
The experimental evidence indicates that the cellular
abnormalities produced are associated with either the
intracellular growth, or the presence of intracellular
killed bacteria or bacterial products.
The significance and future application of the
findings of this work in relation to the pathogenesis of
porcine intestinal adenomatosis, the disease associated
with the presence of intracellular C. sputorum ss
mucosalis, are discussed.
