an epidemiological study of listeriosis in dairy cattle

mx =
nx + 10 1 nx+1 + 10 2 nx+2 .+10 3 nx+3..
where cx and nx refer to the counts and number of plates at lowest dilution and cx+1 and
cx+1 etc. refer to successively higher dilution. The best estimate of the mean count in the
starting inoculum was then calculated as below
M= mx x 10 x x y
where y is the number of drops per ml of the dropping pipette.
The values calculated for the bacterial concentration in the original inoculum were
then used to calculate the limit of detection.
1g of known Listeria - positive faeces sample was inoculated into 9ml of LSEB
and incubated at 37 0 C for 48 hours and processed as above. The detection limit was
determined in a similar manner using the same method of calculation.
5. 2. 3. Serology
a) Antigen Preparation :
Serum antibody to L. monocytogenes was detected using an ELISA. The antigen
used in this test was cholesterol precipitated Listeriolysin O (CP-LLO).
This was prepared according to the method of Vazquaz-Boland and others
(1989b) and Jagger (1993) and is described below.
L. monocytogenes serotype 4b was streaked out onto a Horse (Sheep) Blood
Agar plate and incubated at 37ºC for 24 hours. A colony from the plate was inoculated
into 10ml Brain Heart Infusion Broth (BHIB) and incubated at 37ºC for 24 hours. The
growth was harvested by centrifuging the BHIB at 2000g for 20 minutes. The
supernatant was poured off and the pellet was washed twice in sterile phosphate
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