LUCRĂRI ŞTIINłIFICE MEDICINĂ VETERINARĂ VOL. XLI, 2008, TIMIŞOARA
SPECIFIC YOLK ANTIBODIES GROWTH INHIBITION TEST ON A
FIMBRIATED K88 E.COLI STRAIN
M. SĂRĂNDAN, R. TRIF, E. TÎRZIU
Faculty of Veterinary Medicine Timisoara
Summary
An aqueous solution containing IgY antibodies was tested for growth inhibitory
effect of an E. coli K88 positive strain compared to a control solution, with no specific
antibodies. Specific yolk immunoglobulins reduce the “in vitro” growth rate of an E. coli K88
positive strain
Key words: IgY, growth inhibition, E. coli, K88,
Yolk immunoglobulins have been demonstrated to have an „in vivo”
therapeutic effect on piglet colibacillosis caused by fimbriated strains of E. coli (2).
“In vitro” growth inhibition tests were previously performed on E. coli and
Salmonella strains (1, 5).The present experiment is meant to test yolk
immunoglobulins „in vitro” activity against a fimbriated E. coli strain.
Materials and methods
The E. coli strain used (NCTC 10650 serotype O149:K91, K88ac:H10) (4)
is a fimbriated, enterotoxigenic natural incident strain in the pig farms.
The strain was taken from the Microbiology laboratory collection. Prior to
the experiment it was repeatedly cultured on blood agar for 5 times.
The growth speed for the pure culture was assessed by inoculating the
strain on nutritive broth followed by incubation at 37°C. At the time of inoculation
and every hour for 5 hours afterwards 0.1 ml samples were taken and bacteria
were enumerated by plate count.
The antibodies were extracted from a specific anti E. coli K88 IgY product
(“Globigen® Pig Doser” supplied by EW Nutrition GmbH Germany) that has proven
to have therapeutic effect. Chloroform delipidation method (3) was preferred
because of the higher antibody concentration in the aqueous solution. The IgY
product does not contain any anti-bacterial substances
10 ml of IgY product were mixed with equal volumes of PBS, well
homogenized and kept cold (4°C) for 1 hour. 20ml of chloroform are added to the
mixture and homogenized. The mixture is centrifuged for 30min at 2500 rpm. 3
layers are formed. The supernatant, the aqueous antibody containing part, was
taken with a syringe and filter sterilized through a 0.2 µm sterile syringe filter
(Millipore).
The control solution was made using the same procedure from the yolk of
eggs laid from unvaccinated hens.
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LUCRĂRI ŞTIINłIFICE MEDICINĂ VETERINARĂ VOL. XLI, 2008, TIMIŞOARA
4 ml of the test solution are mixed with 7 ml of nutrient broth. 50 µl of the
E.coli strain are added and the mixture is incubated in 100 ml Erlenmeyer flasks at
37°C. Every 30 minutes the broth is homogenized. The mixture ratios were chosen
arbitrary.
At the time of inoculation and every hour for 5 hours afterwards 0.1 ml
samples were taken and bacteria were enumerated by plate count.
Results and discussions
After 24 hours at 37°C the UFC from the pure culture were count and a
growth curve was plotted. There is a classic growth curve, with the lag, exponential
growth and decline phases. The growth is very fast, after 5 hours the culture gets
into the decline phase. This curve was made to assess the strain’s growth speed in
order to use the data for the experimental inhibition test.
The total plate count for the experimental and control probes are shown in
table 1.
Table 1
The total plate count for the experimental and control probes
Hours
Control
Experimental
0
100000
100000
1
300000
250000
2
2500000
1500000
3
35000000
18000000
4
400000000
200000000
5
550000000
1000000000
The pH of the aqueous solutions was 6.12 for the control and 7.3 for the
experimental sample before they were added to culture media. As the bacteria
release metabolites in the medium, the pH lowers. Even if the pH conditions were
more suitable in the experimental sample, growth rate in the experimental sample
is lower than in the control and it continues to decrease. The number of bacteria in
the experimental sample is increasing but not as fast as in the control sample. We
presume this decrease is due to the antibody content of the sample. After 4 hours
the UFC in the experimental sample are half of those in the control. At 5 hours, the
antibodies are probably depleted and the UFC count from the experimental sample
compared to the control almost doubles.
Figure 1 is a graphical representation of the bacterial growth in the
experimental sample represented as percent form the control sample.
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LUCRĂRI ŞTIINłIFICE MEDICINĂ VETERINARĂ VOL. XLI, 2008, TIMIŞOARA
%
200
182
180
160
140
120
100
100
83
80
60
60
51
50
3 hours
4 hours
40
20
0
initial
one hour
2 hours
5 hours
Fig. 1. Total UFC from the experimental sample as percent from the control sample
The immunoglobulin is probably not killing the bacteria, as the loss of the
fimbriae is compatible to bacteria’s survivor, but its antibacterial potential may
result from the impairment of certain bacterial functions by the interaction of the
antibodies with the bacterial surface antigens.
Conclusion
An aqueous solution of specific yolk immunoglobulin reduces the “in vitro”
growth rate of an E. coli K88 positive strain.
References
1. Lee E. N., H. H. Sunwoo, K. Menninen, J. S. Sim In Vitro Studies of
Chicken Egg Yolk Antibody (IgY) Against Salmonella enteritidis and
Salmonella typhimurium 2002 Poultry Science 81:632–641
2. Marquardt Ronald R (2000) Control of Intestinal diseases in Pigs by
Feeding Specific Chicken Egg Antibodies CAB International. Egg Nutrition
and Biotechnology (eds J.S. Sim, S Nakai and W. Guenter).
3. Polson A. Isolation of IgY from the yolks of eggs by a chloroform
polyethylene glycol procedure. Immunol Invest. 1990 Jun;19(3):253-8.
4. Orskov, I. et al. (1969) Acta path. microbiol. scand. 75, 491
5. Sunwoo H.H., E.N. Lee, K. Menninen, M.R. Suresh, J.S. Sim Growth
Inhibitory Effect of Chicken Egg Yolk Antibody (IgY) on Escherichia coli
O157:H7 Journal of Food Science Volume 67 Issue 4 Page 1486-1494, May
2002
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