Intra-cellular and extra-cellular growth of L. monocytogenes in chick embryo fibroblast cell culture.

The growth of L. monocytogenes in isolated chick embryo fibroblast cell culture was studied. Hanks balanced salt solution and Eagles minimal essential medium were shown to support a limited growth of L. monocytogenes. Extra-cellular growth on the maintenance medium occurs for 48 h prior to the establishment of intra-cellular organisms. As the uptake of the parasite by the cell culture takes place, intra-cellular replication begins with subsequent release of the organisms into the surrounding medium. The organism continues to replicate intra-cellularly until all the cell culture is destroyed.

The effect of progesterone on the growth and virulence of Listeria monocytogenes.

Progesterone was examined for action on the virulence of Listeria monocytogenes and the toxicity of its haemolysin. Progesterone at concentrations between 5 and 20 micrograms/ml reduced the numbers of L. monocytogenes over the first two hours of growth. Virulence and haemolysin toxicity were assessed using the allantoic sac route of inoculation into embryonated hens eggs. Increasing the concentrations of progesterone in the culture medium resulted in a decrease in the mortality of chick embryos inoculated with either organisms, or cell-free extracts or purified haemolysin. Progesterone had no effect on the lethality of pre-formed haemolysin.

Role of haemolysin and temperature in the pathogenesis of Listeria monocytogenes in fertile hens' eggs.

The haemolysin produced by Listeria monocytogenes at 37 degrees C and 4 degrees C was examined in fertile hens' eggs. Organisms, cell free extracts and purified haemolysin derived from broth cultures grown at the lower temperature were more pathogenic for chick embryos, induced higher mortality with toxic changes in the embryos. These effects were most pronounced with the purified haemolysin as shown by LD50 determinations and following inoculation of constant haemolytic doses. Pathological changes induced by the haemolysin included sub-cutaneous haemorrhage due to endothelial damage, hepatosplenomegaly with macroscopic and histological lesions in heart, spleen and liver in the absence of an inflammatory response. At the cellular level, the myocardial tissue, and hepatocyte structure were destroyed with intravascular haemolysis, fatty degeneration of mitochondria, dilation of endoplasmic reticulum and distortion of liver cell nuclear membranes evident. The mortality and morphological data showed an increase in virulence for Listeria after culture at 4 degrees C compared with 37 degrees C and suggested a more cytotoxic component of the haemolysin which was activated at lower temperatures.

Pathogenesis and growth of Listeria monocytogenes in fertile hens' eggs.

The LD50 for 15-day old chicken embryos inoculated with Listeria monocytogenes into the allantoic sac was determined. The growth cycle of the organism was investigated in different tissues and fluids derived from embryonated eggs following inoculation with a suspension of L. monocytogenes equivalent to the LD50. Eggs receiving doses of 100 and 1000 times the LD50 were used to examine the effect of high doses on the pathogenesis and growth of Listeria in ovo. The pattern of growth of the organism in embryonic blood showed two distinct peaks and correlated with these was the development of large and small pock lesions on the chorioallantoic membrane. Bacterial growth in the internal organs exhibited a single peak. Histological and electron microscopic evidence indicated that the primary cellular damage was due to a soluble haemolysin present prior to the establishment of the organism within the tissues.

Pathogenicity of natural and experimental listeriosis in newly hatched chicks.

The pathogenicity of Listeria monocytogenes for newly hatched chickens exposed to natural infection was examined. Organisms entered through the alimentary tract and dissemination followed bacteraemia. Among a number of symptoms recorded were unilateral and bilateral toe paralysis. In addition to gross abnormalities in the following tissues, histological lesions were seen in the liver, spleen, heart and kidneys of all infected chicks but brain lesions were observed only in birds with central nervous system involvement. The organism was recovered from some tissues derived from apparently healthy chicks as well as those with listeriosis. The use of trypsin in the isolation process increased the probability of a positive result from tissues, reduced the storage time needed and had no adverse effect on the rate of organism growth.

Infection, haemorrhagia and death of chick embryos experimentally inoculated with Listeria monocytogenes by the intra-allantoic route.

When allantoic sacs of embryonated (SPF) chicken eggs were inoculated with different doses to investigate the pathogenicity of two strains of Listeria monocytogens (4379 and NCTC 5214), infection resulted which spread rapidly throughout the embryonated eggs. When low doses were used small pock lesions on the chorio-allantoic membrane (CAM), generalized haemorrhage (especially on the head region) and deaths of the embryos with necrotic foci on the liver and heart were observed. Neither the pock lesions nor the haemorrhage were detected in embryos dying from high doses of the bacterium. Bacteria were recovered from the CAM's, allantoic fluids, amniotic fluids and selected organs of the dead embryos. The pathogenicity was shown to be strain dependent, strain 4379 being more pathogenic than strain NCTC 5214. In vitro studies indicated that brain homogenates of uninoculated chicken embryos are not inhibitory to Listeria monocytogenes at 37 degrees C and will increase the viable count.