2. ISOLATION, IDENTIFICATION, PATHOGENICITY AND
ANTIBIOTIC SENSITIVITY PATTERN OF ESCHERICHIA
COLI ISOLATED FROM SUSPECTED COLIBACILLOSIS
OF COMMERCIAL POULTRY
Advisory Committee
3. • Chitwan, one of the major pockets in Nepal, share
around 30% of the broiler and 80% of the total layer
chicks produced throughout the country (Thapa, 2005).
• Central region alone produced more than 51% of
chicken meat and egg in country (Prajuli, 2005).
4. • Colibacillosis is considered one of the leading causes of economic loss
in the poultry industry worldwide (Zanella et. al., 2000).
• Some of the strains of E. coli are zoonotically important (Cheville and
Arp 1978).
• Escherichia coli are present in the normal intestinal flora of birds.
5. • Only some strains with specific virulence attributes,
designated as avian pathogenic E. coli (APEC), are
able to cause disease such as acute colisepticaemia,
fibrinopurulent polyserositis, airsacculitis,
pericarditis, salpingitis, synovitis, omphalitis, yolk
sac infection, swollen head syndrome, coligranuloma,
and cellulitis (Vidotto et al., 1990; Dozois et al., 1994; Gomis et al.,
1997; Pourbakhsh et al., 1997;)
• Avian pathogenic Escherichia coli (APEC) is causative agent
of avian Colibacillosis .Respiratory tract infection with avian
pathogenic Escherichia coli result in depression and in birds of
4 to 9 weeks of age and may result in extensive economic loss
up to 20 % Mortality (Dho-Moulin and Fairbrother, 1999).
6. APEC is also associated with cellulitis of the lower
abdomen and thigh that is not associated with clinical
illness.
Gross lesions are typically 3 to 6 cm in diameter, in the
skin of the post ventral region and tend to the unilateral
with moderate to marked thickening of the skin (Messier et.
al., 1993).
7. The general objective of this study was to isolate, identify,
pathogenicity and antibiotic sensitivity pattern of
Escherichia coli isolates
•To isolate presence Escherichia coli in Colibacillosis
suspected chicken of Chitwan.
•To find out antibiotic sensitivity pattern of isolated E. coli
•To determine pathogenicity of E. coli in poultry by in-vitro
and in-vivo tests
•To determine the PCV, Hb, TRBC in experimentally tested in
control birds
9. Study site and sample collection
• The tissue samples were collected based on the clinical findings and
pathogonomic lesions observed during detail post-mortem
examination of poultry at Veterinary Hospital Research and Training
Center (VHRTC).
• A total of 440 tissues samples from the Colibacillosis suspected birds
including 240 dead and 200 diseased birds of broilers and layers were
collected in sterile containers following aseptic precautions and
transported to laboratory.
• Only tissue samples of liver and heart were obtained.
• Out of total samples, 300 samples were from broilers and 140 from
layers.
• The study was performed from July 2008 to June 2010.
11. Sample Type Perihepatitis Pericarditis Egg peritonitis Air Sacculitis
Broiler 184 (61.33) 54 (18.0) 12 (4.0) 156 (52.0)
Layers 116 (82.85) 48 (34.28) 34 (24.28) 51 (36.42)
Total 300 (68.18) 102 (23.18) 46 (10.45) 207 (47.04)
Figures in the parenthesis indicate percentages
Gross lesions recording of poultry
12. The study was conducted utilizing the
conventional methods for the detection of E.coli
following the standard guide lines from
Modified
Bacteriological Analytical
Manual of the Food and Drug
Administration (Updated December, 2007).
13. BAM)
Plates incubated at 370C for 24 hours.
Sample collection
Streaking on Mac-Konkey Agar
Positive colonies on MCA, Streaked on
EMB Agar
EMB +veMCA + ve was done Gram Staining
Gram’s staining, Pathogenicity test, Biochemical test and Antimicrobial
analysis
14. Preparation of media Plating of sample
Growth of colonies on MCA
Pink colonies on MCA Metallic growth on EMB Agar
15. • The positive colonies using the biochemical test was inoculated into
the nutrient broth (NB) (HiMedia Ref M002, LOT 0000012042) having the
volume 1.5 ml.
• It was incubated for six hour at 370C.
• Forty percent glycerin was already prepared in distill water by
autoclaving.
• The 0.5 ml of NB with colony suspension was transferred into the
three sterile serum screw vials.
• The 40% glycerin 0.5 ml of volume was also added by pasture pipette
to make 1ml of volume.
• Then it was preserved at the temperature -700C for further
investigation as well as confirmation (Bacteriological Analytical Manual 2007).
18. Preparation of sheep blood
Collection of sterile blood
Inoculating the isolates E. coli
Incubate the plates at 370C
for 24 hrs.
zone of lysis under bacterial
colony identified.
19.
20.
21. • A drop of broth culture was dropped in the
centre of clean cover slip
• Inverting it over a middle concave slide and
observed under microscopic.
22. • Antimicrobial resistance patterns of E. coli were
determined by the disk diffusion method using
Mueller Hinton Agar (HiMedia).
• Zone interpretations were based on the National
Committee on Clinical Laboratory Standards
(NCCLS, 2007)
• Inoculums were prepared in Nutrient Broth and
incubated for 4-6 hours resembling 0.5 Mc Farland
turbidity.
• Antimicrobial sensitivities were evaluated from the
diameter of the zone of inhibition of growth around
the disc.
23. • One hundred and twenty one day chicks (Cobb-100)
were obtained from M/S Kalyan Poultry Breeding
and Research Farm, Kalyanpur, Chitwan.
• The birds were fed with chick mash (From
Panchratan feed industries, Narayangarh, Chitwan)
and water provided ad libitum.
• The chicks were assigned into experimental group of
12 containing 10 birds in each group.
In vivo Pathogenicity Test
24. Numbers of the birds and inoculums allocated in each group
No. of birds:10
Inoculation:
105CFU of E.
coli/bird
(G1)
No. of birds:10
Inoculation:
104CFU of E.
coli/bird
(G2)
No. of birds:10
Inoculation:
103CFU of E.
coli/bird
(G3)
No. of birds:10
Inoculation: 102
CFU of E.
coli/bird
(G4)
No. of birds:10
Inoculation: 101
CFU of E. coli/bird
(G5)
No. of birds:10
Inoculation: 100µl
BHI/bird
(G6)
No. of birds:10
Inoculation:
105CFU of E.
coli/bird plus
Gentamicin (G7)
No. of birds:10
Inoculation:
105CFU of E.
coli/bird plus
Amoxicillin (G8)
No. of birds:10
Inoculation:
105CFU of E.
coli/bird plus
Enrofloxacin (G9)
No. of birds:10
Inoculation:
105CFU of E.
coli/bird plus
Norfloxacin (G10)
No. of birds:10
Inoculation:
105CFU of E.
coli/bird plus
Doxy+Clostin
(G11)
No. of birds:10
Inoculation:
105CFU of E.
coli/bird plus
Chloramphenicol
(G12)
26. • Blood sample was collected from CFU of E.coli
105, 104 fed group, and control group which
has no given CFU of E.coli on day 1,3 1nd 6
days.
• Blood sample were collected from jugular vein
in 2 ML EDTA vial.
• Sample processing was done in Automatic
Blood analyzer.
Haematology
27. Data entry, management and analysis were
done using the program Excel, (Microsoft® Office Excel
2007).
Descriptive statistics was used to describe the
result of prevalence analysis.
Chi-square analysis was performed by Fisher's
exact test using commercial software (Minitab V-
13.2) to compare proportion where required.
29. Broilers (n=300) Layers (n=140) Total (N=440)
67 (78.82)a 18 (21.17)a 85 (19.31)
Presence of Escherichia coli
Figures in the parenthesis indicate percentages
30. • In this study, out of 440 sample analyzed a total of 19.31% (85/440) of the
samples were affected with E. coli. The prevalence rate was higher in the
broiler 22.33% ( 67/300) than the layer 12.85% (18/140) .
• According to the annual technical report of National Avian
Lab,Bharatpur,Chitwan (2006), out of 525 samples obtained from post
mortem examination of clinically affected or dead birds 55.81% were
found E. coli positive . (Annual technical report of NAL 2006).Which is
comparatively higher than present study.
• Similarly, in a study at veterinary teaching hospital, IAAS, Rampur about 12%
(11/90) of the samples taken from postmortem cases were found positive for E. coli
(Sapkota et al., 2006) which is comparatively lower than in this present study.
• A higher prevalence rate in overall indicates poor hygienic condition of
chick production in the hatcheries and post death contamination to some
extent.
Discussion
31. Sample Type Perihepatitis Pericarditis
Broiler (n=67) 44 (65.67) 23 (34.32)
Layers (n=18) 13 (72.22) 5 (27.77)
Total (n=85) 57 (67.05) 28 (32.94)
Figures in the parenthesis indicate percentages
Total Isolation of Escherichia coli from gross lesion sample
32. Pathogenicity test Broiler (n=67) Layers (n=18)
Congo red dye 49 (73.13) 12 (66.66)
Haemolysis 38 (56.71) 8 (44.44)
Motility 67 (100.00) 18 (100.00)
Figures in the parenthesis indicate percentages
In- vitro pathogenicity studies on E. coli isolates
33. Discussion continue
● In this study, out of 67 isolates E.coli from broiler, 49 (73.13%) and
38 (56.71%) were positive for Congo red dye and haemolysis test
respectively. Likewise out of 18 isolated from layers, 12 (66.66%)
and 8 (44.44%) were positive for Congo red dye and haemolysis
test respectively, whereas all the isolates were motility positive for
both in broilers and layers isolates.
●Congo red binding has been used as a potential virulence marker
(Berkhoff et.al.1986) which indicated that the isolates were pathogenic.
●Berkhoff et al., (1986) also reported that generally, most CR positive E.
coli isolates will lose the ability to bind CR if sub cultured on complex
media (such as blood agar).
34. ● The expression of phenotypic marker of virulence such as CR
binding ability, iron-limited growth, and aerobactin bioassay
were strongly correlated to the day old chick lethality
(Swaminathan et. al., 2004).
● But it has been reported that hemolysis of the 5% sheep blood
agar can not always be taken as an in vitro measure of virulence
because virulence strain are found with negative hemolysis and
non-virulent with positive hemolysis (Swaminathan et. al., 2004).
● But Ragi et. al., (2003) had showed that CR medium also not
reliable method to test virulence character of E. coli.
Discussion continue
35. ● A clear distinction between pathogenic and non-pathogenic E.
coli could not be established based on haemolytic activity
(Kulshreshtha et. al., 1977).
● However, involvement of O9 and O88 serotypes of E. coli has
been reported to cause cellulitis and other colibacillosis lesions
in the broiler chickens (Susantha et. al., 2001).
● In this study also Congo red positive E. coli was found lethal to
the chick. This showed that in vitro Congo red uptake test was
consistent with in vivo day old chick lethality test. So it can be
concluded that ability to uptake Congo red dye will be a simple
method of testing virulence of E. coli.
Discussion continue
36. Antimicrobial disks were taken as
Amoxicillin (10mcg),
Chloramphenical (30mcg),
Norfloxacin (5mcg),
Colistin + Doxicyline (10mcg),
Enrofloxacin (5mcg),
Gentamycin (10mcg),
39. Discussion continue
● From this study it was found that, In Gentamicin (81.17%), Enrofloxacin
(63.52%), Amoxicillin (61.17%), Chloramphenicol, (61.16%),
Norfloxacin (56.74%) and Doxycycline + clostinsulphate (37.64%)
showed sensitive.
● The analysis of resistant pattern showed that Norfloxacin (38.82%),
Doxyxycline plus Clostin Sulphate (32.94%), Enrofloxacin (29.21%),
while Amoxocillin and Gentamicin had no resistant pattern to E. coli.
● In contrary to this result, Neupane et. al., (2005) reported that the most
sensitive antimicrobials were Gentamicin (81.81%) and Enrofloxacin
(72.72%) followed by Tetracycline and Chloramphenicol (63.63%).This
result is almost similar to present study.
40. ●Baral (2005) has reported that sensitivity of E. coli isolates from poultry
with Chloramphenicol and Neomycin was 81% and 46.87% respectively.
● One study conducted in Iran reported that high resistance of antimicrobials
(Tetracycline-96.57%, Enrofloxacine-78.11%, Chloramphenicol-63.09%)
was observed in E. coli from chickens (Salehi, 2005).
● In this study analysis of resistant pattern showed that Norfloxacin
(38.82%), Doxyxycline plus Clostin Sulphate (32.94%), Enrofloxacin
(29.21%), while Amoxocillin and Gentamicin had no resistant pattern to E.
coli
Discussion continue
41. Discussion continue
● The result is in contrary to the study at Michigan where a
quite low percentage of multi-drug resistance E. coli were
isolated (Sayah et. al., 2004); in which the percentage of E. coli
isolates resistance to agent were Norfloxacin38.82%, Doxyplus
colistin sulphate 32.94% and Enrofloxacin 29.21%.
● The use of fluroquinolones has been restricted since
1990s, after the rapid emergence of resistance to
fluoroquinolones after the introduction of Enrofloxacin into
chicken production in Europe (Gaskin et. al., 2002).
42. Discussion continue
Therefore, the present study suggests an alarming situation of
the presence of multi-drug resistance E. coli in Chitwan and its
adjoining Districts.
43. Group 1-4 5 6 7 8 9 Total
G1 105 cfu E. coli/bird 4 2 1 7
G2 1104 cfu E. coli/bird 2 2 4
G3 103 cfu E. coli/bird 1 1
G4 102 cfu E. coli/bird 2 2
G5 101 cfu E. coli/bird 2 1 1 4
G6 BHI broth100µl/bird
In Vivo pathogenicity Test( day old Chicks)
Day
44. Group 1-4 5 6 7 8 9 total
G7 105CFU of E. coli/bird plus Gentamicin
G8 105CFU of E. coli/bird plusAmoxic illin
G9105CFU of E. coli/bird plus Enrofloxacin 1 1
G10 105CFUofE.coli/bird plus Norfloxacin 2 2
G11 105CFUofE.coli/bird plus Doxy+Clostin 1 1 2
G12 105CFU of E. coli/bird plus Chloramph
Day
45. • In this study 70% mortality was recorded in the birds fed with
105 CFU/bird of the E. coli, 40% mortality in 104 CFU/bird.
This might be due to difference in generation of E. coli
isolates, no mortality were recorded in control group.
• In groups 7, 8 and 12, antibiotic Gentamicin, Amoxocillin and
Chloramphenicol were fed respectively together with 105 CFU
E. coli isolate there was no mortality. There was mortality in
group G9, G10 and G11 where antibiotic Enrofloxacin,
Norfloxacin and Doxy + Clostinsulphate were fed respectively
together with105 CFU E. coli isolate. E
Discussion continue
46. This might be due to difference in
generation of E. coli isolates. Berkhoff et.
al., 1986 also reported that generally when
E. coli isolate is sub cultured to produce
This might be due to difference in generation of E. coli
isolates. Berkhoff et. al., 1986 also reported that
generally when E. coli isolate is sub cultured to produce
successive generations on simple and complex media, it
loses its virulence. This result is also favored by
seasonal variation, breed of poultry health status and
probiotic use in feed.
Pathogenicity of the E. coli not only depends on the
virulence of the organism but depends largely on the
dose of exposure, route of infection, health status of the
bird and the normal flora of the intestinal tract (Vegad,
2004).
47. Pathological lesion associated with vivo pathogenicity
test
Chicks (n=120) Perihepatitis Pericarditis Air Sacculitis
E. coli CFU fed 11 (9.16 ) 8 (6.66) 21 (17.5)
E. coli CFU +
Antibiotic fed
7 (5.53 ) 3 (2.50) 12 (10.0)
Total 18 (15.0) 11 (9.16) 33 (27.5)
Figures in the parenthesis indicate percentages
49. Hematology
Mean hematological values of E. coli CFU fed and control birds
Group PVC
%
Hb
(gm/100ml)
RBC
million/cu.m
m
MCH
pg
MCHC
(gm/dl)
E. Coli
Fed (n-27)
0.2340a
(±0.0239)
6.95
(±0.67)
1.73a
(±0.23)
40.4b
(±4.25)
30.3b
(±2.60)
Controls
(n-9)
0.2563b
(±1.55)
7.22
(±0.42)
2.18b
(±0.10)
33.2a
(±1.97)
28.3a
(±1.9)
Figures in the parenthesis indicate standard deviation, were PCV = Packed Cell
Volume, RBC = Red Blood Cell, MCH = Mean Cell Hemoglobin and MCHT = Mean
Cell Haemoglobin Concentration
50. Daily PCV percentages of E.coli fed group and control (No E. coli fed) birds
19
20
21
22
23
24
25
26
27
1 2 3 4 5
PVC%
Daily PCV of E.coli infected and control birds
Infected
Control
51. The haematological values determined on the infected and
non infected birds .The experimental survivor group was
significantly lower for packed cell volume (P<0.05) and red cell
count (p<0.05) and significantly higher for mean cell
haemoglobin (P< 0.05) and mean cell haemoglobin
concentration (p < 0.05).
52. • The investigations demonstrated that E. coli septicaemia
causes a fall in packed cell volume, haemoglobin level and red
cell count 24 hours after inoculation and that these continue to
fall until the fifth day of infection.
• The results therefore suggest that this is not a true anaemia and
the occurrence of haemodilution in circumstances perhaps
favouring haemo-concentration must be considered. This
would require more detailed studies of blood volume and red
cell mass changes following infection.
53. CONCLUSION
• Prevalence of E. coli in both broiler and layer hatcheries were
high in Chitwan which indicate poor hygienic condition of chick
production. There is no difference (p>0.05) in prevalence of E.
coli in broilers and layers samples in Chitwan.
• The in-vitro pathogenicity tests showed high CR uptake (70-
100%) and motility (100% for all) which reflect the isolates
were pathogenic.
• All the isolates were highly sensitive to Gentamicin and
Enrofloxacin. Higher percentage of resistance of E. coli against
commonly used antimicrobials would be due to more use of
these Antimicrobials in treatment and prevention proposes in
birds.
Conclusion
54. Recommendations
• Before the treatment, drug sensitivity should be conducted to
know the resistance patterns.
• Antimicrobials resistance profiles of E. coli should be
investigated from domestic and wild animals, chicken, human
and their environment from all parts of the country.
• People awareness programs regarding the antimicrobial use
and additionally crowding and poor sanitation, hygienic food
production with careful handling should be conducted.
Recommendations
