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J. Phy8iol. (1975), 253, pp.

223-232
With 5 text-figure8
Printed in Great Britain

223

AVIAN FEBRILE RESPONSE

BY LOUIS G. D'ALECY AND MATTHEW J. KLUGER


From the Department of Physiology, University of Michigan
Medical School, Ann Arbor, Michigan 48104, U.S.A.

(Received 13 May 1975)


SUMMARY

1. The febrile response of the pigeon (Columba livia) was characterized


for comparison with our present day understanding of mammalian
fever.
2. When injected with live Pasteurella multocida the birds became
febrile and died.
3. Varying doses of dead bacteria produced a complex dose-dependent
febrile response.
4. Antipyretics effectively attenuated the febrile response to dead
bacteria.
5. The similarities of reptilian, avian and mammalian fever suggest
a common origin and perhaps a similar adaptive role of fever in increasing
host survival during bacterial infection.
INTRODUCTION

A careful review of the literature reveals little information on the


evolution of fever. Until recently it was generally assumed that the
ability to generate a fever in response to infection was limited to endothermic organisms, but Vaughn, Bernheim & Kluger (1974) have now
demonstrated that a reptile (Dipsosaurus dorsalis) will develop a fever
by behavioural means in response to bacterial infection. Whether the
febrile responses of this reptile and of the mammals to infection are of
common origin or have evolved independently is unestablished. However, as the point of common ancestry of the mammals and birds was
in the early anapsid reptiles, the demonstration of a similar febrile
response to infection in birds would greatly strengthen the possibility
of a genetic link between the febrile responses of reptiles, birds and
mammals.
While many texts on avian biology refer to fever as a clinical sign in
many bird diseases, little information exists on the characterization of
the febrile state in birds. In order to demonstrate similarities between
8

PHY 253

L. G. D'ALECY AND M. J. KLUGER


bird and mammalian fever, we have studied the pigeon, Columba livia,
and have attempted to answer the following questions about avian
fever: (1) when injected with live pathogenic bacteria, will birds develop
a fever; (2) when injected with dead pathogenic bacteria (containing
endotoxin), will birds still respond, as do mammals, by generating a fever;
(3) is the magnitude of the febrile response dose-dependent; (4) can
fever in birds be attenuated or abolished by antipyretic drugs such as
sodium salicylate?
224

METHODS
Male white carneaux pigeons (Columba livia) weighing between 450 and 600 g
were used. Each pigeon was individually housed and given food (Washtenaw's
Check-R-Mix scratch bird feed, Ann Arbor, Michigan) and water ad libitum. The
daily photoperiod was 12 hr and ambient temperature was maintained at 22-23 C.
Room temperature and the body temperature of each bird were recorded about
every 30 sec on a multipoint recorder (Honeywell Electronik 112). Copperconstantan thermocouples (36 s.w.G.) were placed inside polyethylene tubing
(P.E. 205) which was sealed at the end and placed about 20 mm into the cloaca
of each bird. The thermocouple lead wire was then taped to the tail feathers and
allowed to drape freely from the cage. This allowed the birds unrestrained movement
in their cages during the course of the experiment. Each group of birds was allowed
one day to adjust to the handling and placement of the cloacal probes. The second
day generally served as a control period and the experimental manipulations were
carried out on the third day.
The bacterium Pa8teurella multocida was chosen because it is a gram-negative
bacterium that is known to cause fowl cholera (Heddleston, 1972). The stock
cultures of bacteria were supplied by Dr G. R. Carter of thq Department of
Microbiology at Michigan State University. The bacteria were grown on sheep
blood agar at 370 C, washed twice with sterile pyrogen-free saline, centrifuged
and re-suspended in pyrogen-free saline. The appropriate concentrations were
made by diluting this suspension until they matched the reference concentration
of McFarland barium sulphate standards. Dead bacteria were obtained by initially
washing the agar plates with 70 % ethyl alcohol. Confirmation that the bacteria
were dead was made by plating the final suspension of bacteria on sheep blood
agar plates and observing no growth. The various concentrations of dead or live
bacteria as well as sterile pyrogen-free saline (controls) were all injected i.P. For
each experimental situation the injections and administration of sodium salicylate
(as well as the handling of the birds) was controlled for by injection of pyrogen-free
saline or the administration of water by mouth. To study the effect of antipyretics
on the fever, we administered 0-67 ml. tap water or sodium salicylate (300 mg/ml.)
orally by inserting 20-30 mm polyethylene P.E. 160 tubing, connected by an
18 s.W.G. needle (1-2 mm o.d.) to a syringe, into the mouth of each bird.
RESULTS

Normal body temperature variations


As reported for other avian species (Dawson & Hudson, 1970) the
pigeons used in this study displayed a pronounced circadian pattern in
body temperature. The average body temperature of twenty-two birds

225
EVOLUTION OF FEVER
ranged from 40-72 + 0.040 C s.E. of mean during the day (08.00-18.00 hr)
to 39 70 + 0.09 C s.E. of mean at night (18.00-04.00 hr). (See Table 1 and
Fig. 1.)
TABLE 1. Febrile responses in pigeons
Time
1st lOhr
(08.00-18.00)
A, control body temperature 40-72 + 004
B, 5 x 107 P. multocida
+0-150-07
C, 5 x 108 P. multocida
+0-45 0*11
D, 5 x 109 P. multocida
- 0.01 + 0-10
E, 5 x 109 P. multocida
-0-080 11
F, 5 x 1010 P. multocida
+ 0 19 0-13
- 0 45 + 0-10
G, 5 x 108 P. multocida
+ sodium salicylate
H, sodium salicylate
-0-500-10

2nd 10 hr
(18.00-04.00)
39-70 0 09
+ 0-06 0-02

24 hr
(08.00-08.00)
40-21 + 0-11

+ 178+0-12
-0-23+0-10

+1-060-17
-0-320-06

N
22
5
5
7
4
5
8

-0-21 0-08

- 0-32 + 0-12

+0-09+0-03
+0-800-10
+0-50+ 011

+1X210-07
+1-01+0-04
+1-030-08

+ 0-48 + 0-12

A, average body temperature of pigeons injected with 1 ml. sterile pyrogenfree saline.
B-F, average changes in body temperature in response to varying concentrations
of dead Pasteurella multocida (1 ml., i.P.).
E, pigeons were re-inoculated with the same dose (5 x 109) dead P. multocida
after 6 days.
G, average changes in body temperature in response to 5 x 108 P. multocida and
sodium salicylate (0-67 ml. 300 mg/ml. at 08.00 and 16.00 hr).
H, average changes in body temperature in response to sodium salicylate (0-67 ml.
300 mg/ml. at 08.00 and 16.00 hr).
+ s.E. of mean, n = number of pigeons.

41-0

. .40.01

CL

E
0

390
39-0

12.00

I,,| | i

18.00

24.00

II

06.00

Hours

Fig. 1. Mean body temperature ( s.E. of mean) of twenty-two pigeons


injected with 1 ml. sterile pyrogen-free saline at the beginning of a 24 hr
control period.
8-2

226

L. a. D'ALECY AND M. J. KLUGER

Response to live Pasteurella multocida


After a control period of 24 hr, seven pigeons were injected (i.P.) with
live Pasteurella multocida (1 ml. of 1 x 107 organisms/ml.). All birds developed a fever and the response of a representative animal is shown in
Fig. 2. The fever begins to develop after 7-8 hr and remains elevated
above the control even during the normal nocturnal decline in body
45

44

0_1%~ ~ ~~1

U 43_

39~~~~~~~~~~~~~~I

40 _
12.00

a
18.00

24.00
Hours

06.00 10.00

Frig. 2. Body temperature of a representative pigeon during a 24 hr control


period (open circles) and after injection at 10.00 hr (filled circles) with
I ml. I x 107 organism/ml. live Pasteurelld mnultocida.

temperature.After 18 hr the bird's body temperature rises sharply and at a


temperature of 45 C it dies. The other birds in this group lived for varying lengths of time but in each case there was a sharp rise in body
temperature just preceding death. This terminal hyperpyrexia was only
observed in the birds given live bacteria.
Dose-dependent response to Pasteurella multocida
In mammals it has been demonstrated that dead bacteria, which
contain endotoxin, are sufficient to induce fever (Seibert, 1925). In order
to determine whether avian fever is similar to mammalian fever, dead
bacteria were injected at varying concentrations into birds that had not
previously been exposed to Padteurella multocida. For each concentration
of bacteria injected, the protocol was the same. On day 1 a copperconstantan thermocouple was inserted in the cloaca and taped to the

227
EVOLUTION OF FEVER
tail of each bird. On day 2, at 08.00 hr, 1 ml. sterile pryrogen-free saline
was injected (i.r.) into each bird. On day 3, at 08.00 hr, 1 ml. Pasteurella
multocida in saline vehicle was injected (i.P.). The concentrations of
bacteria used were 5 x 107 (five birds), 5 x 108 (five birds), 5 x 109 (seven
birds) and 5 x 1010 (five birds) organisms/ml. The results of a typical
experiment in which 5 x 109 organisms/ml. were injected on the 3rd day
are compared to the saline control injection on the 2nd day in Fig. 3.
The average data for each concentration of bacteria are presented as the
differences in body temperature at each hour between the control and
the experimental observations (Fig. 4A and B). A concentration of
5 x 107 organisms/ml. led to a minimal change in body temperature when

42

U 41:14

%- I

~40

391.

J lS
12.00

Lghts off rb

I I
18.00
24.00
Hours

1.

06.00

Fig. 3. Body temperature of a representative pigeon injected with sterile


pyrogen-free saline on day 1 (open circles) and 5 x 109 dead Pasteurella
multocida at 08.00 hr on day 2 (filled circles). Note the initial decline in
body temperature occurring about 2 hr after the injection of the bacteria.

compared to the control day. A concentration of 5 x 108 organisms/ml.


induced a fever that began about 3 hr after injection and produced
a mean increase in body temperature of 1-210 C for a 10 hr period
(18.00-04.00 hr). Table 1 gives the average increase in body temperature
during the lights-on interval, the lights-off interval and the total experimental period.
The concentration of 5 x 109 organisms/ml. led to an initial fall (P < 0-01)
in body temperature followed by several oscillations about control body

L. G. D'ALECY AND M. J. KLUGER

228

20 r A

, 10

e
M

0.CL

E0
0
.0

.C
0
C

-1.0
2
1800
Hours

P. multocida
5 x 10' (n=5)
onto 5 x 10' (n=7)
-.

0IS..
0

'a
0

0.

E0
0
.0
C

0
C

12.00

18.00

24.00

06.00

Hours

Fig. 4A and B. Average changes in pigeon body temperature in response


to injection of dead Paateurella multocida at 08.00 hr (1 ml. 5 x 107-5 x 1010/
ml.). Injection of 5 x 107 organisms led to little elevation in body
temperature. 5 x 108 led to an average fever of 0-80 0- 10 S.E. of mean.
Injection of 5 x 109 or 5 x 1010 led to an initial decline in body temperature
followed by a series of oscillations and then ultimately to fevers averaging
0-50 0-11 and 1-06 + 0-17 s.E. of mean, respectively (see Table 1)
(n = number of pigeons).

229
EVOLUTION OF FEVER
temperature (Fig. 4B). After this initial drop and oscillation, a steady
fever developed with a magnitude similar to that in the group of animals
who were injected with 5 x 108 organisms/ml. The pattern of response to
5 x 1010 organisms/ml. showed a similar initial drop and oscillation before
the full development of the fever. The possible significance of these
oscillations will be developed in the discussion.
To determine whether the febrile response in birds was reduced in
response to a subsequent injection of dead Pa8teurella multocida, four
birds were injected a second time with 1 ml. 5 x 109 organisms/ml. Six
days were allowed for recovery between the first and second experimental
sessions. The protocol for this tolerance test was that used for the initial
2-0

1-5
01W

L-

1*0

0.

E
V

0-5

.0
C

80
C

-0.5

-1 *0

12.00

24.00

18.00

06.00

Hours

Fig. 5. Average changes in pigeon body temperature ( +.E. of mean) in


response to injection of 1 ml. 5 x 108 Pa8teurella multocida at 08.00 hr plus
oral administration of water at 08.00 and 16.00 hr (filled circles), 5 x 108
P. multocida at 08.00 hr plus oral administration of sodium salicylate at
08.00 and 16.00 hr (open circles), and to sodium salicylate alone at 08.00
and 16.00 hr (triangles). Injection of P. multocida alone leads to responses
similar to that shown in Figure 4A. No differences were observed between
the birds receiving sodium salicylate and the birds receiving P. multocida
and sodium salicylate (n = numbers of pigeons).

L. . D'ALECY AND M. J. KLUGER


230
injections. The magnitude (day, night, or 24 hr) of the fever in these
birds was not different from that on initial exposure to Pasteurella
multocida (see Table 1).
Antipyresis of bird fever
A group of fifteen birds were injected with 5 x 108 dead Pasteurella
muitocida. Eight of these birds were given 200 mg sodium salicylate orally
at 08.00 and 16.00 hr. The seven other birds (controls) were given tap
water at 08.00 and 16.00 hr. Effective antipyresis is illustrated in Fig. 5.
Four additional birds were given the same dose of sodium salicylate at
08.00 and 16.00 hr but received no Pasteurella multocida. Fig. 5 and
Table 1 show that body temperature was lowered to the same extent by
sodium salicylate whether or not Pasteurella multocida was also given.
DISCUSSION

These results demonstrate that birds will develop a fever in response


to injection of live or dead bacteria and that the febrile response is
dose-dependent. These characteristics of avian fever, as well as the
attenuation of the fever by a common antipyretic, clearly establish the
similarity between avian and mammalian fever.
The circadian pattern of body temperature in these pigeons appears
to accentuate the febrile response to injected pyrogens. Although the
fever can be demonstrated during daytime hours, the difference in body
temperatures between control and infected birds is much greater during
the lights-off period. This is due, however, not only to an increase in the
infected animal's body temperature during the night but also to the
absence of the normal decline in the animal's temperature during this
lights-off period. It is nevertheless clear that the birds can become
febrile during the lights-on period (see Fig. 4A-B).
A curious fall in body temperature is observed in response to the
higher concentrations of dead bacteria (5 x 109 and 5 x 1010 organisms/ml.).
In Fig. 4B a dip in body temperature is observed which reaches statistical
significance (P < 0 01) at about 3 hr following injection. This dip and
the subsequent oscillations of body temperature about control values
(with 5 x 109) and at about + 0.50 C (with 5 x 1010) suggests that there
might be competing mechanisms at work during this initial stage of
fever development. The dip could be due to some cryogenic response,
perhaps some circulating factor analogous to, but opposite in effect of,
endogenous pyrogen (endogenous cryogen?). An alternative to this could
be a simple vasodilation leading to excessive heat loss. A vasodilation
with a similar time course in response to local inflammation in birds
has been reported previously (Glatt, Peskar & Brune, 1974). The second

231
EVOLUTION OF FEVER
mechanism, with a somewhat longer latency, could be the development
of granulocytic endogenous pyrogen which would then produce the
observed elevation in body temperature. Regardless of the mechanism,
this cryogenic response at higher doses obscures the initiation of the
daytime fever at the higher doses of bacteria.
TABLE 2. Febrile responses in the terrestrial vertebrates

Fever produced with live bacteria


Fever produced with dead bacteria
Sodium salicylate attenuates fever
Fever is beneficial

Reptiles

Birds

+*

+
+
+
?

+
+*

Mammals
+
+
+
?

+, response positive; -, response negative; ?, response unknown.


* Unpublished observations of H. Bernheim.

The evolution of fever


In an earlier study it was demonstrated that the lizard DipsosaurU8
dorsalis develops a fever in response to infection with dead Aeromoras
hydrophila, gram-negative bacteria pathogenic to lizards (Vaughn et al.
1974). The lizard actively seeks a warmer microclimate and this results
in it raising its deep-body temperature. In a subsequent study, it was
shown that this elevation in body temperature results in a significant
increase in the ability of the lizard to withstand an infection with the
live bacteria (Kluger, Ringler & Anver, 1975). It is not known at present
whether the febrile responses of mammals also decrease their mortality
from bacterial infection. If the febrile responses of the mammalian, avian
and reptilian classes had a common origin, then it is likely that the
function of fever has not greatly changed and that therefore sub-lethal
intensity fever might be beneficial in mammals. An understanding of
the phylogeny of fever might thus provide clues as to whether fever is
beneficial or harmful. A comparison of what is presently known about
the febrile responses in the terrestrial vertebrates is shown in Table 2.
The similarities between reptilian, avian and mammalian fever are
striking. While data like these do not prove that the febrile mechanism
had a common origin (fever might have independently evolved in all
three groups of animals) they are highly suggestive of this premise.
Characterization of amphibian and fish thermal responses to bacterial
infection would greatly enhance our understanding of the evolution of
fever and might add to this phylogenetic interpretation. If fever had
a common origin in the terrestrial vertebrates, then it might be expected
that the function of fever is similar in these classes. Since it was recently
demonstrated that the normal fever in lizards leads to a significant

L. G. D'ALECY AND M. J. KLUGER


increase in host survival, one might expect to find the same results in
birds and mammals. Clearly this is speculative, and remains to be
determined experimentally.
232

This work was supported by NSF research Grant GB 42749X. We are most
grateful to G. Carter for providing us with the stock culture of Pa8teurella multocida.
We thank H. Bernheim, W. Dawson, D. Mouw and L. Vaughn for their critical
review of this manuscript.
Special thanks goes to M. Foster for her technical assistance in the microbiological
aspects of the study and to C. Rose for her assistance in data reduction and
analysis.

REFERENCES
DAwsoN, W. R. & HUDSON, J. W. (1970). Birds. In Comparative Physiology of
Thermoregulation, vol. 1, Invertebrates and Nonmammalian Vertebrate8, ed.
WHITTOW, G. C., pp. 223-310. New York and London: Academic Press.
GLATT, M., PESKAR, B. & BRUNE, K. (1974). Leucocytes and prostaglandins in
acute inflammation. Experientia 30, 1257-1259.
HEDDLESTON, K. L. (1972). Avian pasteurellosis. In Diseawes of Poultry. 6th edn.,
ed. HOFSTAD, M. S., pp. 219-251. Ames: Iowa State University Press.
KLUGER, M. J., RINGLER, D. H. & ANVER, M. R. (1975). Fever and survival. Science,
N.Y. 188, 166-168.
SEIBERT, F. B. (1925). The cause of many febrile reactions following intravenous
injections. I. Am. J. Phy8iol. 71, 621-651.
VAUGHN, L. K., BERNIHEIN, H. A. & KLUGER, M. J. (1974). Fever in the lizard.
Dipeo8aurue dormali. Nature, Lond. 252, 473-474.

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