Rajasekhar, Malleshappa.

2018-05-22T12:47:18Z

2018-05-22T12:47:18Z

1981

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.

http://hdl.handle.net/1842/30667

The University of Edinburgh

Annexe Thesis Digitisation Project 2018 Block 19

Already catalogued

Growth characteristics of Campylobacter sputorum subspecies mucosalis in cell cultures

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy