Avian Pathology

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Haematological and biochemical aspects of an E.

coli septicaemia in brown leghorn chickens

G. Christie & W.G. Halliday

To cite this article: G. Christie & W.G. Halliday (1979) Haematological and biochemical
aspects of an E. coli septicaemia in brown leghorn chickens, Avian Pathology, 8:1, 45-55, DOI:
10.1080/03079457908418326

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Avian Pathology, 8: 45-55, 1979

HAEMATOLOGICAL AND BIOCHEMICAL ASPECTS

OF AN E. COLI SEPTICAEMIA IN BROWN LEGHORN
CHICKENS

G. CHRISTIE and W.G. HALLIDAY

Ministry of Agriculture, Fisheries and Food
Veterinary Laboratory, Eskgrove, Lasswade
Midlothian EH18 1HU, Scotland

SUMMARY
A series of experiments is described in which E. coli septicaemia was pro-
duced in 6-week-old Brown Leghorn male chickens by air sac inoculation
of a pathogenic strain of E. coli. Groups of birds were slaughtered for ex-
amination on days 1 to 5 post infection and haematological and biochemical
examinations performed on blood samples collected just prior to slaughter.
Changes in erythrocyte values and serum chemistry were observed and
the clinical pathology is discussed. Consideration is given to the role of
incidental haemodilution in the haematological changes observed.

INTRODUCTION
Numerous serotypes of E. coli are potentially pathogenic for poultry; however, only
a few commonly cause disease (Gross, 1956; Sojka and Carnaghan, 1961; Harry,
1964 and Sojka, 1965). These serotypes are widely distributed and cause various
disease conditions of economic importance. The most important of these is Coli sep-
ticaemia in broilers which frequently follows respiratory infection associated with
viruses or mycoplasmas acting singly or together (Sojka, 1965).
Many aspects of Coli septicaemia have been studied by several workers (Piercy and
West, 1976). These studies have included clinical pathology. Cole and Boyd (1965a
and b) studied the effects of Coli septicaemia on blood glucose, creatine, urea nitro-
gen and amino acids. Panigraphy etal. (1969) studied changes in chickens due to
E. coli septicaemia in combination with inanition and varying intake of dietary pro-
tein and energy sources and demonstrated that alterations of plasma cholesterol,
alkaline phosphatase and protein levels were independent of inanition. The effects of
E. coli endotoxin on poultry have been studied and a fall in serum zinc and serum
iron, raised caeruloplasmin levels and changes in the mineral element concentrations
have been observed (Butler and Curtis, 1973a and b, and 1975).

Received 23 February 1978

Accepted 1 September 1978
46 G. Christie and W.G. Halliday

The present investigation was undertaken to examine further the haematological and
biochemical changes associated with E. coli septicaemia over a period of 5 days after
inoculating a strain of E. coli into the left abdominal air sac with the aim of providing
additional information on the clinical pathology of E. coli infection.

MATERIALS AND METHODS

The birds used (110 experimental and 50 controls) were Brown Leghorn males approx-
imately 6 weeks old from the laboratory's specific pathogen free flock. They had been
hatched and reared in isolation. The birds were wing banded, caged in pairs and provi-
ded with food and water ad libitum.
The experimental design is shown in Table 1. In experiments 1, 2 and 3 the birds were
bled only on the day they were killed. Two blood samples were taken from each bird
at about the same time each day. The birds were bled from a wing vein using a triangu-
lar bayonet pointed needle and an unconstricted blcod flow allowed to accumulate in
chemically clean glass tubes for biochemical studies and into plastic tubes containing
an anticoagulant (KEDTA) for haematological studies. About 1 ml was taken for
haematology and about 6 ml for biochemistry. In experiment 4 a different procedure
was followed: initially the 40 birds were bled and packed cell volume determined;
the birds were then randomised and allocated into an experimental group of 30
birds and a control group of 10 birds; the two groups were bled daily to obtain a small
amount of blood for monitoring the PCV values; on the day of killing larger blood
samples were taken for fuller haematological and biochemical examination.

Table 1. Experimental design.

Number of Number of
survivors Day when bled
Experiment Inoculum birds Day killed
inoculated with marked
lesions
1 E. Coli 50 33 1 1

2 E. Coli 15 10 2 2

3 E. Coli 15 6 3 3
Combined
Controls Sterile broth 40 - Day 1, 2 or 3 Day 1, 2 or 3
1, 2 & 3
4 E. Coli 30 13 13 on days 1 8 on day 4
to 4 5 on day 5
5 on day 5
Controls Sterile broth 10 — 1 to 5 5
inclusive

An 02 strain of E. coli (225), isolated at Lasswade from a typical case of coli-pericar-

ditis and serotyped at the Central Veterinary Laboratory, Weybridge was used. The
organism, which was a smooth strain, had been repeatedly sub-cultured following iso-
lation. Six-hour broth cultures were prepared and 0.5 ml inoculated into the left ab-
dominal air sac of the experimental birds using a W fine bore needle. To do this the
birds were held in an extended position, lying on the right side. Control birds were
given 0.5 ml of sterile broth in a similar manner. All birds were examined post-mortem.
 E. coli septicaemia in the fowl 47

Birds which did not die from the infection were killed by an intra-peritoneal injection
of sodium pentabarbitone solution.

The haematological examination carried out on the unclotted blood sample included
estimations of the packed cell volume (PCV), the haemoglobin concentration (Hb)
and red blood cell count (RBC). From these were calculated the mean cell volume
(MVC), the mean cell haemoglobin (MCH) and the mean cell haemoglobin concen-
tration (MCHC). The blood chemistry examination included estimations on the
serum protein, albumin, globulin, inorganic phosphate, uric acid, blood glucose, chol-
esterol, magnesium, calcium, sodium and potassium. Weighing, biochemical and
haematological procedures were carried out according to the methods described by
Rossera/. (1976).

RESULTS
Post-mortem examination
Of the 110 birds injected with E. coli, 11 exhibited the post-mortem features of Coli
septicaemia and the organism was successfully recovered in pure culture from pericar-
dial lesions when isolations were made from a sample of these birds.
Clinical observations
The control birds in all four experiments remained well and free fromiT. coli infection.
Seventy-five per cent of the 110 birds injected with E. coli developed clinical signs
which varied from slight depression to recumbency. Many of the birds which became
ill were affected with a watery, stained, diarrhoea and many were not inclined to eat
or drink. In all 15 birds died: two in experiment 1, three in experiment 3 and 10 in
experiment 4.
The effect of E. coli infection on body weight was recorded in experiment 4. The
mean weight of the survivors is compared with that of the controls in Text-fig. 1. The
control birds gained weight steadily compared to the affected birds whose weight
dropped significantly within 24 hours (P < 0.001).
Haematology
The haematological values determined on the birds from experiments 1, 2 and 3 are
shown in Table 2. There was a significant drop in haemoglobin concentration
(/> < 0.01) and in the mean cell haemoglobin concentration (P <0.02) in experiment
1. At 48 hours post-inoculation the birds from experiment 2 had highly significantly
reduced (f < 0.001) packed cell volume, haemoglobin concentration and mean cell
volume and significantly reduced red blood cell count (p < 0.05) and mean cell
haemoglobin values (P < 0.02).
In experiment 4 the mean packed cell volume of the survivors was significantly lower
(P < 0.01) than their controls at 24 hours post-infection. This difference persisted
throughout the course of the experiment (Text-fig.2). The packed cell volume of the
controls also showed a marked fall over the 5-day period indicating that the results ob-
tained with the infected birds were affected by the repeated bleeding involved during
sampling. Packed cell volume, haemoglobin concentration, red blood cell count and
indices were estimated on blood collected just prior to slaughter from eight survivors
4 days after infection, and five remaining survivors 5 days after infection, as well as
on blood collected from the 10 control birds slaughtered by day 5. The data of the
survivors on days 4 and 5 were combined and compared to the data for the controls
bled on day 5 (Table 3). The experimental survivor group was significantly lower for
 Table 2. Mean haematological values obtained in experiments 1, 2 and 3.

Number of Hb Rbc MCV MCH MCHC

Experiment samples PCV (g/100 ml) (x 1 0 1 2 / l ) (g/100 ml)
(fl) (Pg)
0.3221 11.74*** 2.45 129.14 47.50 36.69**
1 31 (0.0297) 3 (1.02) (0.26) (7.34) (2.95) (1.58) 9
0.2775**** 10.65**** 2.12**** 131.42 50.88 38.71 S-.
2 10 (0.0463) (1.2) (0.31) (12.4) (6.5) (3.3
0.2450**** 9.86**** 2.26* 108.03**** 43.04** 40.41
3 6 (0.0351) (2.1) (0.15) (11.7) (9.3) (11.0) a
Combined 0.3233 12.42 2.53 129.06 49.60 38.52
Controls 40 (0.0200) (1.1) (0.3) (11.5) (5.4) (3.8)

a Standard deviation
* S% level of significance of difference from controls
** 2% level of significance of difference from controls
*** 1% level of significance of difference from controls
**** 0.1% level of significance of difference from controls
 E. coli septicaemia in the fowl 49

• 13, SURVIVORS

650- • 5, DAY 5 SURVIVORS

O CONTROLS

400

Text-fig. 1. Daily mean weight ± one standard deviation of affected

survivors and controls in experiment 4.
Table 3. Mean haematological values of days 4 and 5 of experiment 4.

Group PCV
Hb Rbc MCV MCH MCHC
(g/100 ml) ( x l O 1 2 / D (fl) (pg) (g/100 ml)
E. Coli 0.2300** 6.95 1.73**** 134** 40.4**** 30.3**
infected (0.0239) 3 (0.67) (0.23) (15.42) (4.25) (2.60)
Controls 0.2563 7.22 2.18 118 33.2 28.3
(1.55) (0.42) (0.10) (5.78) (1.97) (1.9)
a Standard deviation
** 7% level of significance of difference from controls
• • • • 0.1% level of significance of difference from controls

packed cell volume (P < 0.02) and red cell count (/> < 0.001) and significantly higher
for mean cell volume (P < 0.02), mean cell haemoglobin (P < 0.001) and mean cell
haemoglobin concentration (P < 0.02).
Blood chemistry
The following differences were observed between the infected and control groups of
birds. There were highly significant (p < 0.001) increase in serum total protein and
globulin (p < 0.001), significantly reduced albumin (/» < 0.05) and highly significant
decreases in blood glucose and inorganic phosphate in experiment 1 due to infection.
In experiment 2 serum potassium was highly significantly reduced (P < 0.001) and
cholesterol significantly reduced (P < 0.05). In experiment 3 serum total protein was
raised significantly (p < 0.05) (Table 4).
 301

PCV
2
L ^
• 13, SURVIVORS

• 5, DAY 5 SURVIVORS

O CONTROLS

20

DAYS
Text-fig. 2. Daily mean packed cell volume ± one standard deviation of affected survivors and controls in experiment 4.
 Table 4. Mean biochemical values for experiments 1, 2 and 3.
Total Albumin Globulin Inorganic Magnesium
Glucose Cholesterol Phosphorus Calcium Sodium Potassium
Experiment protein
(g/100 ml) (g/100 ml) (g/100 ml) (mg/100 ml) (mg/100 ml) (mg/100 ml) mg/100 ml) mg/100 ml) (mEq/1) (mEq/1)
3.62**** 1.09* 2.51**** 122.1**** 98.2 4.70**** 1.89 10.21 140.1 7.73
1 (0.32)a (0.24) (0.29) (26) (28.8) (1.53) (0.23) (0.83) (3.63) (0.57)
i
3.27 •1.62 1.66 159.3 88.6* 7.33 2.09 7.80 137.8 5.80**** •a
2 (0.43) (0.11) (0.32) (59.39) (18.5) (1.15) (0.13) (2.44) (5.91) (0.84) S

3.51* 1.60 1.92 174 90.3 9.33 1.93 8.73 139.6 7.70
3 (0.37) (0.18) (0.24) (70.4) (18.2) (1-53) (0.96) (2.02) (5.22) (1.41)
Combined 3.19 1.34 1.85 177.5 110.1 7.04 1.94 10.63 139.5 7.20
Control (0.27) (0.44) (1.73) (0.34) (3.89) (5.17) (1.0)
Values (0.41) (19.5) (25.49)

a Standard deviation
* 5% level of significance of difference from controls
**** 0.1% level of significance of difference from controls
 Table 5. Mean biochemical values for days 4 and 5 of experiment 4.
P
Group
Total
protein Albumin Globulin Cholesterol Inorganic
phosphate Magnesium Calcium Sodium Potassium g
(g/100ml) (g/100 ml) (g/100 ml) (mg/100 ml) (mg/100 ml) (mg/100 ml) (mg/100 ml (mEq/1) (mEq/1)
6
E. Coli 3.88 1.71 2.17* 100.8 4.55 1.60 7.44 129.7* 6.95
infected a
(0.89)a (0.22) (0.75) (29.9) (0.78) (0.42) (1.21) (5.78) (1.15)
Controls 3.31 1.72 1.59 101.3 4.86 1.45 6.29 125.5 6.38
(0.52) (0.16) (0.38) (15.37) (0.38) (0.35) (2.53) (6.21) (1.53)

Standard deviation
5% level of significance of difference from controls
 E. coli septicaemia in the fowl 53

In experiment 4 on the day of slaughter, the infected survivor birds had significantly
higher (P < 0.05) serum globulin and sodium concentrations (P < 0.05) (Table 5).
A further statistical analysis was undertaken to compare the haematological and bio-
chemical changes in the 62 survivors with "classical E. coli septicaemia" at post
mortem examination with the 18 infected survivors which had less severe signs. This
showed that in the less severely affected birds statistically significant changes were
limited to reductions in packed cell volume (P < 0.002), haemoglobin concentration
(P < 0.02), serum inorganic phosphate (P < 0.01) and blood glucose (P < 0.05).

DISCUSSION
The investigations demonstrated that£". 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. When compared with uninfec-
ted control birds the erythrocytes were microcytic in the infected birds bled on one
occasion, and macrocytic in those infected birds bled on a number of occasions. The
macrocytosis in the repeatedly bled infected birds probably reflects a response due
to the haemorrhagic anaemia induced by the blood letting and it therefore seems that
E. coli septicaemia produces microcytosis. The apparent anaemia is generally normo-
chromic although there is some indication of hypochromia on the 3rd day.
The rapid onset of the anaemia does not suggest an aplastic anaemia, while the ab-
sence of haemorrhages at post-mortem examination and the presence of microcytosis
would not support a diagnosis of haemorrhagic anaemia. The absence of an increased
serum potassium level, which has been demonstrated to be a feature of haemolytic
anaemia in poultry, would suggest that this is not a haemolytic anaemia (Christie, in
preparation). The results therefore suggest that this is not a true anaemia and the oc-
currence of haemodilution in circumstances perhaps favouring haemoconcentration
must be considered. This would require more detailed studies of blood volume and
red cell mass changes following infection. A consideration of the serum chemistry
results does suggest that haemodilution may have occurred.
The investigations demonstrated that E. coli infection results in a rise in serum globu-
lin, falls in serum albumin, glucose and inorganic phosphate, a variable fall in serum
cholesterol. With the exception of the fall in serum glucose these observations confirm
earlier studies of Panigraphy et al. (1969). The increased total protein and serum glo-
bulin values agree with similar observations made by Cole and Boyd (1965b) and
Panigraphy et al (1969) who attributed this to an increased production of globulins
resulting directly from infection. Falls in serum albumin, cholesterol and inorganic
phosphate can be attributed directly to the infection or to anorexia while a fall in
serum glucose would be consistent with fasting or anorexia (Hazelwood and Lorenz,
1959). The reduction in serum potassium and these other values may perhaps indicate
the occurrence of haemodilution in this condition. The fall in serum potassium dis-
cussed earlier does not substantiate the presence of a haemolytic anaemia and the
other indications perhaps suggest that haemodilution is a feature of infection rather
than anaemia.
Acknowledgements
The authors wish to thank Dr. J.G. Ross for helpful advice and encouragement and
G. Arthur, Miss I. Innes, S. Jess and Miss J. Andrew for excellent technical assistance.
54 G. Christie and W.G. Halliday

REFERENCES
Butler, E.J., Curtis, M.J. and Watford, M. (1973a). The effect of Escherichia coli endotoxin on
the plasma iron concentration in the domestic fowl. Research in Veterinary Science, 15:
267-269.
Butler, E.J. and Curtis, M.J. (1973b). Effects of E. coli endotoxin and ACTH on the plasma zinc
concentration in the domestic fowl. Research in Veterinary Science, 15: 363-367.
Butler, E.J. and Curtis, M.J. (1975). The effects of Escherichia coli endotoxin on the concentra-
tion of mineral elements in the plasma of the domestic fowl. Research in Veterinary
Science, 18: 36-40.
Cole, J.R. Jr. and Boyd, F.M. (1965a). Chemical analyses of the blood of chicks infected or endo-
intoxicated with Escherichia coli. Poultry Science, 44: 1551-1558.
Cole, J.R. Jr. and Boyd, F.M. (1965b). Serum protein and amino acid levels of Escherichia coli —
infected chicks on high and low levels of dietary protein. Poultry Science, 44: 1580-1582.
Gross, W.B. (1956). Escherichia coli as a complicating factor in chronic respiratory disease of
chickens and infectious sinusitis of turkeys. Poultry Science, 35: 765-771.
Harry, E.G. (1964). A study of 119 outbreaks of coli septicaemia in broiler flocks. Veterinary
Record, 76: 443-449.
Hazelwood, R.L. and Lorenz, F. W. (1959). Effects of fasting and insulin on carbohydrate meta-
bolism of the domestic fowl. American Journal of Physiology, 197: 47-51.
Panigraphy, B., Waibel, P.E. and Pomeroy, B.S. (1969). Influence of E. coli septicaemia and
nutrition on growth and tissue and fluid changes of the chick. Poultry Science, 48:
1695-1703.
Piercy, D.W.T. and West, B. (1976). Experimental Escherichia coli infection in broiler chickens:
cause of the disease induced by inoculation via the air sac route. Journal of Comparative
Pathology, 86: 203-210.
Ross, J.G., Christie, G., Halliday, W.G. and Jones, R.M. (1976). Determination of haematology
and blood chemistry values in healthy six-week-old broiler hybrids. Avian Pathology, 5:
273-281.
Sojka, W.J. (1965). Escherichia coli in domestic animals and poultry. Commonwealth Agricultural
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RESUME
Aspects hématologiques et biochimiques d'une septicémie
a E. coli chez des poulets Leghorn bruns
Une série d'expériences est décrite dans lesquelles une septicémie iE. coli a été
produite, chez des poulets mâles Leghorn bruns de 6 semaines, par inoculation dans
le sac aérien d'une souche pathogène d'E. coli. Les différents groupes d'oiseaux ont
été sacrifiés pour examen entre le 1er et le 5ème jour suivant l'infection et il a été
procédé à des contrôles hématologiques et biochimiques sur les échantillons de sang
prélevés juste avant l'abattage.
Des modifications dans les valeurs érythrocytaires et sériques ont été observées; la
pathologie clinique est discutée.
Des commentaires sont donnés sur le rôle de l'hémodilution dans les modifications
hématologiques observées.

ZUSAMMENFASSUNG
Haematologische und biochemische Aspekte bei einer
E. coli-Septicaemie von braunen Leghornhühnern
Es wird eine Reihe von Experimenten beschrieben, bei denen eine E. coli-Septicaemie
bei 6 Wochen alten Hähnchen der braunen Leghornrasse mittels Inokulation eines
pathogenen E. coli-Stammes in die Luftsäcke ausgelöst wurde. Kükengruppen wurden
 E. coli septicaemia in the fowl 55
1-5 Tage nach der Infektion geschlachtet und die kurz vor der Schlachtung entnom-
menen Blutproben haematologisch und biochemisch untersucht.
Es wurden Veränderungen der Erythrozytenwerte und der chemischen Zusammen-
setzung des Serums beobachtet. Die klinische Pathologie wird erörtert. Die Rolle
einer zufälligen Haemodilution bei den veränderten Blutwerten wird diskutiert.