- Email: [email protected]
The effect of bacteria infection on mean selected body temperature in the common agama,Agama agama: A dose-response study
0300-9629/93 $6.00 + 0.00
Camp. Biochem. Physiol.Vol. 105A, No. 3, pp. 479-484, 1993 0
Printed in Great Britain
1993 Pergamon
Press Ltd
THE EFFECT OF BACTERIA INFECTION ON MEAN SELECTED BODY TEMPERATURE IN THE COMMON AGAMA, AGAMA AGAMA: A DOSE-RESPONSE STUDY ANA B. RAMOS,MICHAELT. DON and ALAN E. MUCHLINSKI* Department of Biology, California State University, Los Angeles, Los Angeles, CA 90032, U.S.A. (Tel. 2 13-343-2050; Fax 213-343-2670) (Received 1 September 1992; accepted 30 September 1992)
Abstract-l. The fever response was studied in 43 common agamas using a self-pairing experiment in which animals received an intraperitoneal injection of sterile saline and an injection of one of six dosages of dead Aeromonas sobriu (1 x 106, 1 x lo’, 1 x IO*, 1 x 109, 1 x IO”, and 1 x 10” total organisms). 2. The results demonstrated a significant increase in T, (1.k3.l”C) above the mean selected body temperature (MSBT) of the saline injection animals over a bacteria infection range of three orders of magnitude. At 1 x lOa organisms, an increase was observed on bacteria day 1 while at dosages of 1 x lo9 and 1 x 1O’Oan increase was observed on bacteria days 1 and 2. 3. At dosages of 1 x lo6 and 1 x 10’ there was no difference between saline MSBT and bacteria MSBT. 4. At a dosage of 1 x IO”, MSBTs on bacteria days 1 and 2 were below saline MSBT. 5. The average duration of the fever response is related to the level of infection; however, the magnitude of the fever is relatively independent of the level of infection.
INTRODUCTION Febrile responses produced by injection of live or dead gram-negative bacteria such as Aeromonus hydrophiiu or A. sobria have been observed experimentally in several reptile species. The desert iguana, Dipsosaurus dorsalis (Vaughn et al., 1974), the green iguana, Iguana iguana (Kluger, 1978), the collared lizard, Crotaphytus colluris (Firth et al., 1980), the chuckwalla, Sauromalus obesus (Muchlinski et al., 1989), the spotted monitor tegu, Callopistes maculatus (Hallman et al., 1990), the common agama, spiny lizard, Agama agama, and the granite Sceloporus orcutti (Ortega et al., 1991) are all lizards that have displayed febrile responses. Non-lizard reptilian species such as the box turtle, Terrapene Carolina, and
(Monagas
the painted
turtle,
Chrysemys
and Gatten,
picta
1983), the gopher snake, Pituophis melanoleucus, and the glossy snake, Arizona eleguns (Burns, 1991), and the American alligator, Alligator mississippienis (Lang, 1986) have also been reported to exhibit a febrile response upon bacteria injection. Although a fever was reported in each of the above experiments, the level of infection (total number of organisms injected) was restricted to one dosage. Only a limited number of studies (Laburn et al., 1981; Zurovsky et al., 1987a; Zurovsky et al., 1987b) have been conducted on the possible relationship between the level of lipopolysaccharide (LPS) or bacteria infection and the magnitude and duration (or the presence or absence) of the fever *To whom all correspondence
should be addressed.
response in reptiles, and all of the studies have produced negative results. The armadillo lizard Cordylus catuphractus (Labum et al., 1981) did not demonstrate any fever response after intracardiac injection of Aeromonas hydrophila in dosages of 4 x 109, 4 x lo’, and 4 x 10’ total organisms. Lipopolysaccharide, extracted from Salmonella typhosu, at concentrations of 1 pg/kg and lOpg/kg failed to produce a fever in the leopard tortoise, Geochelone pardalis (Zurovsky et al., 1987b), and in the olive grass snake, Psammophis phillipsii (Zurovsky et al, 1987a). Hallman et al. (1990), using 1 x 10” A. sobriu, did not observe a statistically significant increase in MSBT in the tawny plated lizard, Gerrhosaurus major, or in the Savannah monitor lizard, Varanus exanthematicus. They did suggest, however, that detailed dose-response studies be conducted on species known to exhibit a fever response as well as on species known to be afebrile at one bacteria dosage, thereby gaining more information on possible dose-response relationships. Therefore, to further our understanding of the febrile response in reptiles as it relates to dosage we conducted a dose-response study on the common agama, Agama aguma, previously shown to exhibit a fever response at a dosage of 1 x 10” A. sobriu (Ortega et al., 1991). The results of our experiments show that Agama agama exhibits a fever response over a bacteria infection range of three orders of magnitude. We also show that while the duration of the fever response is related to the level of infection, the magnitude of the fever response is relatively independent of the level of bacteria infection. 479
480
ANA B. RAMOSet MATERIALS AND METHODS
The common agamas (Agama ugamu) used in this study were obtained from California Zoological Supply (Santa Ana, CA). A total of 43 animals were used in the experiments and the animals ranged in weight from 40.6 to 82.8 g. Prior to being used in an experiment, the animals were housed in groups in square arenas (125 x 125 cm) that contained a sand substrate, a rock for basking, and a 250 W heat lamp. The animals’ diet consisted of crickets and meal worms. At least 3 days prior to being used in an experiment, each lizard was allowed to acclimate individually to square arenas (125 x 125 cm) which contained a sand substrate, a 250 W heat lamp, and a rock for basking. The temperature gradient within the arenas ranged from approximately 25 to 50°C. Animals were maintained under a 12L : 12D cycle for both room lighting and heat lamps. For the determination of the fever response the animals were used in a self-pairing experiment in which each animal received a sterile pyrogen-free saline injection and one of six dosages of alcohol killed Aeromonas sobriu. Vials of freeze-dried A. sobria (ATCC9071) were obtained from the American Type Culture Collection (Rockville, MD). The bacteria solutions were prepared following the procedures in Ortega et al. (1991). Body temperature measurements Data were collected using an Apple II computer with a thermocouple data acquisition board and software (Omega Engineering Inc, Stamford, CT). Body temperatures (Tb) were measured using 30 gauge type K thermocouples inserted at least 3 cm into the animal’s cloaca and taped to the tail. Tb readings were collected during the light hours at 4min intervals. After each experimental run the thermocouples were removed and calibrated against a National Bureau of Standards calibated thermometer (Fisher Scientific, Pittsburgh, PA) with a resolution of +O.l”C. The type K thermocouples combined with the data acquisition board and software gave a resolution of +_O.1°C. On the first day of experimentation, animals were given an i.p. injection of sterile pyrogen-free saline in a volume of either 0.45 or 0.60 ml between 7.30 and 8 a.m. T,s were recorded over one 12 hr light period starting at 8.30 a.m. Following a 24 hr rest period, animals were given an i.p. injection of alcohol killed A. sobriu using the same volume as the saline injection. The bacteria injections were also administered between 7.30 and 8 a.m. The dosages of total number of bacteria organisms injected and the lizard’s average body weight per dosage were: 1 x lo6 (58.0 g), 1 x IO’ (53.9 g), 1 x lOa (72.1 g), 1 x lo9 (58.3 g). 1 x lOto (60.8 g), and 1 x 10” (51.2 g). Because the fever response can last through the second day, bacteria injection T,s were collected for 2 consecutive days. In an additional control experiment, six animals
al.
were first given a saline injection with T, monitored for 1 day, they they had 1 day of rest and were subsequently given another saline injection with Tb monitored for 2 days. This control experiment was conducted to ensure that the second injection itself did not cause an increase in T,,. Individual mean selected body temperatures (MSBTs) were calculated for saline injections by taking the mean of all Tb measurements from the first high T, set-point until the animal permanently left the heated area of the arena near the end of the 12 hr light period. MSBTs for bacteria injections in which a fever was evident were calculated by taking the mean of all Tb measurements from the first febrile high set-point until the animal returned to its saline MSBT. In those instances where a fever was not evident, the MSBTs were calculated in the same manner as saline MSBTs. A sudden rise in T, signals the onset of the fever response in Agama agamu so subjectivity in determining which data points to use and hence possible bias in data selection was kept to a minimum (Ortega et al., 1991). Statistical analysis Data analysis regarding a possible increase in MSBT after bacteria injection was conducted using the multivariate general linear hypothesis module of the SYSTAT statistical program (Version 5.0) Within this program univariate F tests were calculated for saline, bacteria day 1 and bacteria day 2 MSBTs. Individual hypothesis testing was used to compare pairs of selected between or within dosage saline, bacteria day 1 and bacteria day 2 MSBTs. The benefit of using a multivariate statistical test to analyse the data presented in this paper is that each test uses the pooled variance from all dosages and, therefore, the chances of a false significant result are decreased. Tests for differences in fever magnitude and duration were conducted using Wilcoxon’s two-sample rank test while the saline-saline control experiment was analysed using Wilcoxon’s signed rank test. All values in the text are expressed as mean + SE. RESULTS
Mean dosage MSBTs for the six levels of bacteria injection and controls are shown in Table 1. Analysis of the data using the SYSTAT Multivariate General Table 1. Average mean selected body temperatures in the common agama, Agama aganra, under saline injection and six different bacteria injection conditions Dosage
N
1x106 1x10’ 1x108 1x10’
8 8 7 8
; “, ;;::
%
Saline (“C) 36.8 * 37.5 * 36.5 + 36.3 + 36.8 f 37.1 f
0.3 0.2 0.3 0.2 0.3 0.5
Bacteria 1 (“C)
Bacteria 2 (“C)
31.4 * 37.9 f 39.4 f 39.4 * 39.5 f 30.7 f
36.9 f 36.9 f 36.8 f 37.9 f 39.1 f 31.7 f
0.4 0.3 0.3. 0.2. 0.2’ 0.5’
0.2 0.4 0.4 0.4. 0.4. 1.6.
An * indicate.8that the value is significantly different (P < 0.05) from the saline control value.
Bacteria infection in Agama agama
d
44
g
42
H
3::30
i
1 X ,08
BACTERIA DAY 1
I
13:30
18:30
TIME Fig. 1. Body temperature regulation patterns for one common agama, Agama agama, on the day of injection of 0.45 ml of sterile pyrogen-free saline and on day 1 after the injection of 1 x lo8 Aeromonas sobria. Linear Hypothesis program demonstrated a significant overall difference (P < 0.05). Univariate F tests demonstrated that there were no significant differences (P > 0.05) among saline MSBTs in the six different experiments but there were significant differences (P < 0.05) among bacteria day 1 MSBTs and among bacteria day 2 MSBTs. Individual hypothesis testing demonstrated a significant difference (P < 0.05) between saline MSBT and bacteria day 1 MSBT or bacteria day 2 MSBT at the following dosages and on the indicated days: (1) 1 x lo*; saline vs bacteria day 1, (2) 1 x 109; saline vs bacteria day 1, saline vs bacteria day 2, and bacteria day 1 vs bacteria day 2, (3) 1 x 10”; saline vs bacteria day 1, saline vs bacteria day 2, (4) 1 x 10”; saline vs bacteria day 1 and saline vs bacteria day 2. At bacteria injection dosages of 1 x lo*, 1 x 109, and 1 x 10’0, bacteria injection MSBTs compared to saline controls indicated the presence of a fever response. At a bacteria injection dosage of 1 x lo”, bacteria injection MSBTs on both day 1 and day 2 were depressed below saline MSBTs indicating the possibility of endotoxic shock at this high dosage. Mean selected body temperatures obtained in the saline-saline control experiment were not significantly different (P > 0.05). Differences between the first saline injection average MSBT and the first and second days average MSBT after the second saline injection were 0.1 and 0.4”C, respectively.
481
T, response after an injection of sterile saline and on days 1 and 2, respectively, after an injection of 1 x lo9 A. sobria. At this dosage of bacteria there was a significant (P < 0.05) increase in MSBT both on day 1 and day 2 after bacteria injection. The fever on day 1 averaged 3.1 f 0.4”C while the fever on day 2 averaged 1.6 f 0.3”C and the difference between these two values was significant (P < 0.05). The duration of the fever response on day 1 was 6.8 f 0.3 hr. After injection of bacteria on day 2 the mean fever duration was 6.8 f 1.4 hr (range 2.0-l 1.Ohr) for those animals exhibiting a fever (five of the eight animals exhibited a fever on day 2) and 4.25 f 1.6 hr when all individuals (febrile and non-febrile) are included. There was no significant difference (P > 0.05) between the mean day 1 fever duration or fever magnitude at 1 x lo* and 1 x lo9 A. sobria. The 1 x lo9 A. sobria dosage was the lowest dosage at which a significant number of the animals exhibited a fever on day 2 after bacteria injection. 1 x 10”. Figures 3A and B show an animal’s typical T,, response after an injection of sterile saline and on days 1 and 2, respectively, after an injection of 1 x 10” A. sobria. MSBTs on day 1 and day 2 after
TIME
(w
F 1 X 10’ BACTERIA DAY 2
Fever response dosages
1 x lo*. Figure 1 shows an animal’s typical T, response after an injection of sterile saline and on day 1 after an injection of 1 x IO* A. sobria. The mean increase in T, on day 1 of bacteria injection was 2.9 + 0.3”C (39.4 vs 36.5%) and the mean fever duration was 7.2 f 0.9 hr (range 2.25-9.0 hr). Only one of the seven animals exhibited an increase in T, on day 2 (magnitude = 2.6”C, duration = 5.5 hr). 1 x 109. Figures 2A and B show an animal’s typical
Fig. 2. Body temperature regulation patterns for one common agama, Agama agama, (A) on the day of injection of 0.6 ml of sterile pyrogen-free saline and on the day of injection of I x lo9 Aeromonas sobria and (B) on the day of injection of 0.6 ml of sterile pyrogen-free saline and on the day after the injection of 1 x IO9Aeromonas sobria.
ANA
B. l&ios er nl. duration
on day 2 at a dosage of 1 x 10” was greater (P < 0.05) than the average fever duration on day 2 at 1 x lo9 (when all animals, febrile and non-febrile, are included in the I x 10’ data set). The average durations on day 2 at 1 x IO9 and 1 x 10” were both greater (P < 0.05) than the average day 2 duration at 1 x lo8 where only one animal exhibited a fever response. significantly
Non -fever response dosages %30
13:30
MSBTs for saline injections and bacteria days 1 and 2 after 1 x lo6 and 1 x 10’ A. sobria injections
18:30
TIME
(B) 1 X IO’ ’ BACTERIA DAY 2
SALINE 3Oi---__ 8:30
13:30
were not significantly different (P > 0.05). These data are important, however, because they again demonstrate that no portion of the increase in MSBT during
the bacteria injection trials was due to the experimental design of having the animals injected first with sterile saline followed by a dose of A. sobria. All six animals showed a pronounced hypothermia on day 1 of 1 x 10” bacteria injection (mean MSBT 30.7 f O.YC) and three of these animals still exhibited a pronounced hypothermia on day 2 after bacteria injection. One of the six animals died on day 2 of bacteria injection and the other two animals become normothermic on day 2.
18:30 DISCUSSION
TIME Fig. 3. Body temperature regulation patterns for one common agama, Agnma aguma, (A) on the day of injection of 0.6mf of sterile pyrogen-free saline and on the day of injection of 1 x lOto Aeromonas sobria and (B) on the day of injection of 0.6 ml of sterile pyrogen-free saline and on the day after the injection of 1 x 10” Aeromonas sobria.
The data show that there is a positive relationship between the level of bacteria infection (from 1 x 10s
4.01
bacteria injection were sibilantly (P < 0.05) higher than the mean saline MSBT (2.7 + 0.4”C higher on day 1; 2.3 + 0.4”C higher on day 2). Fever duration on day 1 averaged 7.3 f 1.1 hr (range 3-10 hr) while the mean duration on day 2 was 9.3 zf:O.Shr (range 5.5-11.0 hr). All animals demonstrated a fever response on day 2. There were no significant differences (P > 0.05) between average MSBTs on day 1 of bacteria injection at dosages of 1 x 108, 1 x log, or 1 x 10” A. sobria and there were no significant differences between the mean fever magnitudes on day 1 of bacteria infection at dosages of I x IO’, 1 x IO’, and 1 x lOto A. sobriu (Fig. 4). There was no significant difference (P > 0.05) between average MSBTs on day 2 of bacteria injection at dosages of 1 x log and 1 x 10r”. The only significant difference (P c 0.05) in average fever MSBTs and in fever magnitude was at a dosage of 1 x lo9 where the average MSBT and fever magnitude on bacteria day 2 was lower than the average MSBT on bacteria day 1 at the same dosage (39.4 f 0.2”C bacteria day 1 vs 37.9 f 0.4”C bacteria day 2). In terms of fever duration (Fig. 4), the average
p
2.0
2 _
4.0
2 z
8.0
%
LEVEL OF INFECTION 1x108 1x109 lxlOl0 *
8.0
P
Fig. 4. Fever magnitude and duration in the common agama, Agama agama, on the day of injection and the day after injection of 1 x i08, 11 x 109, and 1 x 10” Aeromonas s&in. Vertical bars = f I SE and an * indicates a significant difference between these two values at P i 0.05. Bl = Bacteria day 1, B2 = Bacteria day 2. An averge fever duration value is not indicated for bacteria day 2 at 1 x IO* because only one of seven animals exhibited a fever response on this day and the average MSBT on bacteria day 2 was not significantly different from the average saline MSBT.
Bacteria infection in Agarna agama
to 1 x 10” A. sob&) and the average duration of the fever response in the common agama. The data also indicate that the magnitude of the fever response on the day of bacteria infection is independent of the level of bacteria infection. It is only on the day after bacteria infection at a dosage of 1 x lo9 A. sobria that any effect of dosage upon average fever magnitude is evident. Results from previous studies are contradictory regarding the effect of bacteria infection or LPS level upon fever magnitude. Various doses of live bacteria (range 0.002-0.2 mg bacteria), including ones high enough to drastically reduce survivorship, did not significantly effect the average magnitude of the fever in rats (Banet, 1981) and these results agree with our results in the common agama. On the other hand, a complex dose-dependent relationship between level of infection and fever magnitude was reported in pigeons (D’Alecy and Kluger, 1975); a significant difference in average fever magnitude during the first 24 hr was reported between two levels of bacteria infection (3 x 10” and 6 x 10” Pasteurella multocida) in the rabbit (Kluger and Vaughn, 1978) and a dose-dependent fever was demonstrated in the leech in response to LPS (Cabanac, 1989). Data from species such as the rat (Morimoto et af., 1989; Stitt, 1991), the rabbit (Morimoto et al., 1988) the cat (Milton and Wendlandt, 1970), and the crayfish (Casterlin and Reynolds, 1978) have shown a positive relationship between fever magnitude and level of injection of prostaglandin E (PGE). PGE is postulated to be a central nervous system modulator of the fever response and among vertebrate animals PGE probably effects hypothalamic neurons that regulate body temperature set-point. The general hypothesis regarding the elevation of T, set-point after bacteria infection includes release of interleukin1 (IL-l) from macrophages and other cells after phagocytosis of bacterial endotoxin with subsequent (although the mechanism is not clear) production and release of PGE in the thermosensitive preoptic-anterior hypothalamic (POAH) or organum vasculosum laminae terminali (OVLT) area (Stitt, 1991). Although it seems clear that increased levels of PGE increase T, set-point in a dose-dependent manner, there is no evidence available from reptiles or other animals which indicate that either increased levels of endotoxin result in a dose-dependent increase in circulating levels of IL-l or that increased levels of IL- 1 result in the dose-dependent production and release of PGE in the POAH or OVLT area. Kluger (1991) has stated that there is a poor correlation between plasma IL-l activity and body temperature in animals given injections of bacteria or LPS. The only study to show an increase in plasma IL-l (Hesse et al., 1988) used a very high dose of live E. coli in the baboon. However, this dose of E. coli also led to endotoxic shock and a decrease in T,, not a fever. A dose-dependent relationship between intravenous injection of endogenous pyrogen (derived
483
from human monocytes) and mean rise in T, has been demonstrated in rabbits and rats (Stitt et al., 1985); however, no significant difference in maximum change in T, was observed in rats after intravenous injection of 0.1 and 1.0 pg/rat IL-1B (Dascombe et al., 1988). The fever after injection of l.Opg/rat IL-1B did have a longer duration. If, as hypothesized, PGE release in the POAH or OVLT increases T, set-point, the data from Agama agama suggest that either the 1 x lo*, 1 x 109, and 1 x 10” doses of A. sobria produce the same amount of circulating IL-l or that different levels of circulating IL- 1, above some threshold level and to some maximum level, have the same effect upon PGE levels in the POAH or OVLT areas. Different levels of IL-l either in the plasma or in the brain could have similar effects on T, increase if IL-l receptors were saturated at the lowest fever producing dose. The dose-response data presented in this paper strongly suggest that the negative fever results published for some reptile species (Laburn et al., 1981; Zurovsky et al., 1987a; Zurovsky et al., 1987b; Mitchell et al., 1990) and possibly for species other than reptiles (Cabanac and Rossetti, 1987; Rossetti and Nagasaka, 1988) may be due to the use of an inappropriate (either too low or too high) dose of dead bacteria or LPS. Also, since some studies have used LPS from bacteria other than Aeromonas sobria or have used dead bacteria of other species, the dose-response system described here for Agama aguma may not be appropriate for lizards when either LPS or dead bacteria of another species are used. We would urge that dose-response studies using Aeromonas sobria be conducted on a number of different species in an attempt to describe various dose-response patterns that may exist. We also feel that dose-response studies using bacteria species other than A. sobria could provide data useful to the study of comparative fever biology. Acknowledgements-We wish to thank Helen Barath for assistance in preparing the bacteria cultures and Robert Deshamais for providing statistical advice. This study was supported by an NIH-Minority Biomedical Research Support grant (5S06RR08101) to California State University, Los Angeles, CA.
REFERENCES
Banet M. (1981) Fever and survival in the rat: metabolic versus temperature response. Experientia 37, 130221304. Burns G. A. (1991) Survey of the fever response in temperate North Amercan desert and grassland/forest snakes. M.Sc. Thesis, California State University, Los Angeles, CA. Cabanac M. (1989) Fever in the leech, Nepheiopsis obscura (Annelida). J. camp. Physiol. B. 159, 281-285. Cabanac M. and Rossetti Y. (1987) Fever in snails, reflection on a negative result. Camp. Biochem. Physiol. WA, 1017-1020. Casterlin M. E. and Reynolds W. W. (1978) Prostaglandin E, fever in the crayfish Cambarus bartoni. Pharmac. Biochem. Behav. 9, 593-595.
484
ANA B. RAMOSet al.
D’Alecy L. G. and Kluger M. J. (1975) Avian febrile response. J. Physiol. 253, 223-232. Dascombe M. J., Rothwell N. J., Sagay B. 0. and Stock M. J. (1988) Pyrogenic and thermogenic effects of interleukin 1B in the rat. Am. J. Physiol. 256 (Endocr. Metab. 19), E7-Ell. Firth B. I., Ralph C. L. and Boardman T. J. (1980) Independent effects of the pineal and a bacterial pyrogen on behavioral thermoregulation in lizards. Nature, Land. 285,399-400.
Hallman G. M., Ortega C. E., Towner M. C. and Muchlinski A. E. (1990) Effect of bacterial pyrogen on three lizard snecies. Como. Biochem. Phvsiol. %A, 383-386. H&se D. G., *Tracy K. J., Fond Y., Manogue K. R., Palladino M. A. Jr, Cerami A. G., Shitres T. and Lowry S. F. (1988) Cytokine appearance in human endotoxemia and primate endotoxemia. Surg. Gynecol. Obstet. 166, 147-153. Kluger M. J. (1978) The evolution and adaptive value of fever. Am. Sci. 66, 38-43. Kluger M. J. (1991) Fever: role of pyrogens and cryogens. Physiol. Rev. 71, 93-123. Kluger M. J. and Vaughn L. K. (1978) Fever and survival in rabbits infected with Pasteurella multocida. J. Physiol. 282, 243-25 1. Labum H. P., Mitchell K., Kenedi E. and Louw G. N. (1981) Pyrogens fail to produce fever in a cordylid lizard. Am. J. Physiol. 241 (Regulatory lo), R198R202.
Integrative camp. Physiol.
Lang J. W. (1986) Crocodilian thermal selection. In Wildlife Management Crocodiles and Alligators (Edited by Webb G. J. W., Manois S. C. and Whitehead P. J.), pp. 301-037. Surrey Beathy and Sons, Vancouver. Milton A. S. and Wendlandt S. (1970) A possible role for prostaglandin E, as a modulator for temperature regulation in the central nervous system of the cat. J. Physiol. 207, 7677.
Mitchell D., Labum H. P., Matter M. and McClain E. (1990) Fever in namib and other ectotherms. In Namib Ecology: 25 Years of Namib Research (Edited by Seely, M. K.), Monograph No. 7, pp. 179-192. Transvaal Museum, Pretoria, R.S.A.
Monagas W. R. and Gatten R. E. Jr (1983) Behavioral fever in the turtles Terrapens Carolina and Chrysemus picta. J. Therm. Biol. 8, 285-288.
Morimoto A., Murakami N., Makamori T., Sakata Y. and Watanade T. (1989) Possible involvement of prostaglandin E in development of ACTH response in rats induced by human recombinant interleukin-1. J. Physiol. 411, 245-256.
Morimoto A., Nakamori T., Watanabe T., Ono T. and Murakami N. (1988) Pattern differences in experimental fevers induced by endotoxin, endogenous pyrogen, and prostaglandins. Am J. Physiol. 294 (Regulatory Inregrative camp. Physiol. 23), R633-R640. Muchlinski A. E., Stoutenburgh R. J. and Hogan J. M. (1989) Fever response in laboratory-maintained and free ranging chuckwallas (Sauromalus obesus). Am. J. Physiol. 257
(Regulatory
Integrative
camp.
Physiol.
26)
R15GR155. Ortega C. E., Stranc D. S., Casal M. P., Hallman G. M. and Muchlinski A. E. (1991) A positive fever response in Agama agama and Sceloporus orcutri (Reptilia: Agamidae and Iguanidae). J. camp. Physiol. B. 161, 377-381. Rossetti Y. and Nagasaka T. (1988) Prostaglandin E,, prostaglandin E,, and endotoxin failure to produce fever in the Japanese fresh water snail Semisulcospira libertina. Jpn. J. Physiol. 38, 178-186.
Stitt J. T. (1991) Differential sensitivity in the sites of fever production by prostaglandin E, within the hypothalamus of the rat. J. Physiol. 432, 99-110. Stitt J. T., Shimada S. G. and Bemheim H. A. (1985) Comnarison of febrile responsiveness of rats and rabbits to enhogenous pyrogen. j. appl. Physiol. 59, 1121-1125. Vauahn L. K.. Bemheim H. A. and Kluaer M. J. (1974) F&er in the lizard Dipsosaurus dorsak. Nature 252, 413-474.
Zurovsky Y., Brain T., Labum H. and Mitchell D. (1987a) Pyrogens fail to produce fever in snakes Psammophis philipsii and Lamprophis fuliginosis. Comp. Biochem. Physiol. 87A, 911-914. Zurovsky Y., Mitchell D. and Labum H. (1987b) Pyrogens fail to produce fever in the leopard tortoise Geochelone pardalis.
Comp. Biochem. Physiol. WA, 467469.
Camp. Biochem. Physiol.Vol. 105A, No. 3, pp. 479-484, 1993 0
Printed in Great Britain
1993 Pergamon
Press Ltd
THE EFFECT OF BACTERIA INFECTION ON MEAN SELECTED BODY TEMPERATURE IN THE COMMON AGAMA, AGAMA AGAMA: A DOSE-RESPONSE STUDY ANA B. RAMOS,MICHAELT. DON and ALAN E. MUCHLINSKI* Department of Biology, California State University, Los Angeles, Los Angeles, CA 90032, U.S.A. (Tel. 2 13-343-2050; Fax 213-343-2670) (Received 1 September 1992; accepted 30 September 1992)
Abstract-l. The fever response was studied in 43 common agamas using a self-pairing experiment in which animals received an intraperitoneal injection of sterile saline and an injection of one of six dosages of dead Aeromonas sobriu (1 x 106, 1 x lo’, 1 x IO*, 1 x 109, 1 x IO”, and 1 x 10” total organisms). 2. The results demonstrated a significant increase in T, (1.k3.l”C) above the mean selected body temperature (MSBT) of the saline injection animals over a bacteria infection range of three orders of magnitude. At 1 x lOa organisms, an increase was observed on bacteria day 1 while at dosages of 1 x lo9 and 1 x 1O’Oan increase was observed on bacteria days 1 and 2. 3. At dosages of 1 x lo6 and 1 x 10’ there was no difference between saline MSBT and bacteria MSBT. 4. At a dosage of 1 x IO”, MSBTs on bacteria days 1 and 2 were below saline MSBT. 5. The average duration of the fever response is related to the level of infection; however, the magnitude of the fever is relatively independent of the level of infection.
INTRODUCTION Febrile responses produced by injection of live or dead gram-negative bacteria such as Aeromonus hydrophiiu or A. sobria have been observed experimentally in several reptile species. The desert iguana, Dipsosaurus dorsalis (Vaughn et al., 1974), the green iguana, Iguana iguana (Kluger, 1978), the collared lizard, Crotaphytus colluris (Firth et al., 1980), the chuckwalla, Sauromalus obesus (Muchlinski et al., 1989), the spotted monitor tegu, Callopistes maculatus (Hallman et al., 1990), the common agama, spiny lizard, Agama agama, and the granite Sceloporus orcutti (Ortega et al., 1991) are all lizards that have displayed febrile responses. Non-lizard reptilian species such as the box turtle, Terrapene Carolina, and
(Monagas
the painted
turtle,
Chrysemys
and Gatten,
picta
1983), the gopher snake, Pituophis melanoleucus, and the glossy snake, Arizona eleguns (Burns, 1991), and the American alligator, Alligator mississippienis (Lang, 1986) have also been reported to exhibit a febrile response upon bacteria injection. Although a fever was reported in each of the above experiments, the level of infection (total number of organisms injected) was restricted to one dosage. Only a limited number of studies (Laburn et al., 1981; Zurovsky et al., 1987a; Zurovsky et al., 1987b) have been conducted on the possible relationship between the level of lipopolysaccharide (LPS) or bacteria infection and the magnitude and duration (or the presence or absence) of the fever *To whom all correspondence
should be addressed.
response in reptiles, and all of the studies have produced negative results. The armadillo lizard Cordylus catuphractus (Labum et al., 1981) did not demonstrate any fever response after intracardiac injection of Aeromonas hydrophila in dosages of 4 x 109, 4 x lo’, and 4 x 10’ total organisms. Lipopolysaccharide, extracted from Salmonella typhosu, at concentrations of 1 pg/kg and lOpg/kg failed to produce a fever in the leopard tortoise, Geochelone pardalis (Zurovsky et al., 1987b), and in the olive grass snake, Psammophis phillipsii (Zurovsky et al, 1987a). Hallman et al. (1990), using 1 x 10” A. sobriu, did not observe a statistically significant increase in MSBT in the tawny plated lizard, Gerrhosaurus major, or in the Savannah monitor lizard, Varanus exanthematicus. They did suggest, however, that detailed dose-response studies be conducted on species known to exhibit a fever response as well as on species known to be afebrile at one bacteria dosage, thereby gaining more information on possible dose-response relationships. Therefore, to further our understanding of the febrile response in reptiles as it relates to dosage we conducted a dose-response study on the common agama, Agama aguma, previously shown to exhibit a fever response at a dosage of 1 x 10” A. sobriu (Ortega et al., 1991). The results of our experiments show that Agama agama exhibits a fever response over a bacteria infection range of three orders of magnitude. We also show that while the duration of the fever response is related to the level of infection, the magnitude of the fever response is relatively independent of the level of bacteria infection. 479
480
ANA B. RAMOSet MATERIALS AND METHODS
The common agamas (Agama ugamu) used in this study were obtained from California Zoological Supply (Santa Ana, CA). A total of 43 animals were used in the experiments and the animals ranged in weight from 40.6 to 82.8 g. Prior to being used in an experiment, the animals were housed in groups in square arenas (125 x 125 cm) that contained a sand substrate, a rock for basking, and a 250 W heat lamp. The animals’ diet consisted of crickets and meal worms. At least 3 days prior to being used in an experiment, each lizard was allowed to acclimate individually to square arenas (125 x 125 cm) which contained a sand substrate, a 250 W heat lamp, and a rock for basking. The temperature gradient within the arenas ranged from approximately 25 to 50°C. Animals were maintained under a 12L : 12D cycle for both room lighting and heat lamps. For the determination of the fever response the animals were used in a self-pairing experiment in which each animal received a sterile pyrogen-free saline injection and one of six dosages of alcohol killed Aeromonas sobriu. Vials of freeze-dried A. sobria (ATCC9071) were obtained from the American Type Culture Collection (Rockville, MD). The bacteria solutions were prepared following the procedures in Ortega et al. (1991). Body temperature measurements Data were collected using an Apple II computer with a thermocouple data acquisition board and software (Omega Engineering Inc, Stamford, CT). Body temperatures (Tb) were measured using 30 gauge type K thermocouples inserted at least 3 cm into the animal’s cloaca and taped to the tail. Tb readings were collected during the light hours at 4min intervals. After each experimental run the thermocouples were removed and calibrated against a National Bureau of Standards calibated thermometer (Fisher Scientific, Pittsburgh, PA) with a resolution of +O.l”C. The type K thermocouples combined with the data acquisition board and software gave a resolution of +_O.1°C. On the first day of experimentation, animals were given an i.p. injection of sterile pyrogen-free saline in a volume of either 0.45 or 0.60 ml between 7.30 and 8 a.m. T,s were recorded over one 12 hr light period starting at 8.30 a.m. Following a 24 hr rest period, animals were given an i.p. injection of alcohol killed A. sobriu using the same volume as the saline injection. The bacteria injections were also administered between 7.30 and 8 a.m. The dosages of total number of bacteria organisms injected and the lizard’s average body weight per dosage were: 1 x lo6 (58.0 g), 1 x IO’ (53.9 g), 1 x lOa (72.1 g), 1 x lo9 (58.3 g). 1 x lOto (60.8 g), and 1 x 10” (51.2 g). Because the fever response can last through the second day, bacteria injection T,s were collected for 2 consecutive days. In an additional control experiment, six animals
al.
were first given a saline injection with T, monitored for 1 day, they they had 1 day of rest and were subsequently given another saline injection with Tb monitored for 2 days. This control experiment was conducted to ensure that the second injection itself did not cause an increase in T,,. Individual mean selected body temperatures (MSBTs) were calculated for saline injections by taking the mean of all Tb measurements from the first high T, set-point until the animal permanently left the heated area of the arena near the end of the 12 hr light period. MSBTs for bacteria injections in which a fever was evident were calculated by taking the mean of all Tb measurements from the first febrile high set-point until the animal returned to its saline MSBT. In those instances where a fever was not evident, the MSBTs were calculated in the same manner as saline MSBTs. A sudden rise in T, signals the onset of the fever response in Agama agamu so subjectivity in determining which data points to use and hence possible bias in data selection was kept to a minimum (Ortega et al., 1991). Statistical analysis Data analysis regarding a possible increase in MSBT after bacteria injection was conducted using the multivariate general linear hypothesis module of the SYSTAT statistical program (Version 5.0) Within this program univariate F tests were calculated for saline, bacteria day 1 and bacteria day 2 MSBTs. Individual hypothesis testing was used to compare pairs of selected between or within dosage saline, bacteria day 1 and bacteria day 2 MSBTs. The benefit of using a multivariate statistical test to analyse the data presented in this paper is that each test uses the pooled variance from all dosages and, therefore, the chances of a false significant result are decreased. Tests for differences in fever magnitude and duration were conducted using Wilcoxon’s two-sample rank test while the saline-saline control experiment was analysed using Wilcoxon’s signed rank test. All values in the text are expressed as mean + SE. RESULTS
Mean dosage MSBTs for the six levels of bacteria injection and controls are shown in Table 1. Analysis of the data using the SYSTAT Multivariate General Table 1. Average mean selected body temperatures in the common agama, Agama aganra, under saline injection and six different bacteria injection conditions Dosage
N
1x106 1x10’ 1x108 1x10’
8 8 7 8
; “, ;;::
%
Saline (“C) 36.8 * 37.5 * 36.5 + 36.3 + 36.8 f 37.1 f
0.3 0.2 0.3 0.2 0.3 0.5
Bacteria 1 (“C)
Bacteria 2 (“C)
31.4 * 37.9 f 39.4 f 39.4 * 39.5 f 30.7 f
36.9 f 36.9 f 36.8 f 37.9 f 39.1 f 31.7 f
0.4 0.3 0.3. 0.2. 0.2’ 0.5’
0.2 0.4 0.4 0.4. 0.4. 1.6.
An * indicate.8that the value is significantly different (P < 0.05) from the saline control value.
Bacteria infection in Agama agama
d
44
g
42
H
3::30
i
1 X ,08
BACTERIA DAY 1
I
13:30
18:30
TIME Fig. 1. Body temperature regulation patterns for one common agama, Agama agama, on the day of injection of 0.45 ml of sterile pyrogen-free saline and on day 1 after the injection of 1 x lo8 Aeromonas sobria. Linear Hypothesis program demonstrated a significant overall difference (P < 0.05). Univariate F tests demonstrated that there were no significant differences (P > 0.05) among saline MSBTs in the six different experiments but there were significant differences (P < 0.05) among bacteria day 1 MSBTs and among bacteria day 2 MSBTs. Individual hypothesis testing demonstrated a significant difference (P < 0.05) between saline MSBT and bacteria day 1 MSBT or bacteria day 2 MSBT at the following dosages and on the indicated days: (1) 1 x lo*; saline vs bacteria day 1, (2) 1 x 109; saline vs bacteria day 1, saline vs bacteria day 2, and bacteria day 1 vs bacteria day 2, (3) 1 x 10”; saline vs bacteria day 1, saline vs bacteria day 2, (4) 1 x 10”; saline vs bacteria day 1 and saline vs bacteria day 2. At bacteria injection dosages of 1 x lo*, 1 x 109, and 1 x 10’0, bacteria injection MSBTs compared to saline controls indicated the presence of a fever response. At a bacteria injection dosage of 1 x lo”, bacteria injection MSBTs on both day 1 and day 2 were depressed below saline MSBTs indicating the possibility of endotoxic shock at this high dosage. Mean selected body temperatures obtained in the saline-saline control experiment were not significantly different (P > 0.05). Differences between the first saline injection average MSBT and the first and second days average MSBT after the second saline injection were 0.1 and 0.4”C, respectively.
481
T, response after an injection of sterile saline and on days 1 and 2, respectively, after an injection of 1 x lo9 A. sobria. At this dosage of bacteria there was a significant (P < 0.05) increase in MSBT both on day 1 and day 2 after bacteria injection. The fever on day 1 averaged 3.1 f 0.4”C while the fever on day 2 averaged 1.6 f 0.3”C and the difference between these two values was significant (P < 0.05). The duration of the fever response on day 1 was 6.8 f 0.3 hr. After injection of bacteria on day 2 the mean fever duration was 6.8 f 1.4 hr (range 2.0-l 1.Ohr) for those animals exhibiting a fever (five of the eight animals exhibited a fever on day 2) and 4.25 f 1.6 hr when all individuals (febrile and non-febrile) are included. There was no significant difference (P > 0.05) between the mean day 1 fever duration or fever magnitude at 1 x lo* and 1 x lo9 A. sobria. The 1 x lo9 A. sobria dosage was the lowest dosage at which a significant number of the animals exhibited a fever on day 2 after bacteria injection. 1 x 10”. Figures 3A and B show an animal’s typical T,, response after an injection of sterile saline and on days 1 and 2, respectively, after an injection of 1 x 10” A. sobria. MSBTs on day 1 and day 2 after
TIME
(w
F 1 X 10’ BACTERIA DAY 2
Fever response dosages
1 x lo*. Figure 1 shows an animal’s typical T, response after an injection of sterile saline and on day 1 after an injection of 1 x IO* A. sobria. The mean increase in T, on day 1 of bacteria injection was 2.9 + 0.3”C (39.4 vs 36.5%) and the mean fever duration was 7.2 f 0.9 hr (range 2.25-9.0 hr). Only one of the seven animals exhibited an increase in T, on day 2 (magnitude = 2.6”C, duration = 5.5 hr). 1 x 109. Figures 2A and B show an animal’s typical
Fig. 2. Body temperature regulation patterns for one common agama, Agama agama, (A) on the day of injection of 0.6 ml of sterile pyrogen-free saline and on the day of injection of I x lo9 Aeromonas sobria and (B) on the day of injection of 0.6 ml of sterile pyrogen-free saline and on the day after the injection of 1 x IO9Aeromonas sobria.
ANA
B. l&ios er nl. duration
on day 2 at a dosage of 1 x 10” was greater (P < 0.05) than the average fever duration on day 2 at 1 x lo9 (when all animals, febrile and non-febrile, are included in the I x 10’ data set). The average durations on day 2 at 1 x IO9 and 1 x 10” were both greater (P < 0.05) than the average day 2 duration at 1 x lo8 where only one animal exhibited a fever response. significantly
Non -fever response dosages %30
13:30
MSBTs for saline injections and bacteria days 1 and 2 after 1 x lo6 and 1 x 10’ A. sobria injections
18:30
TIME
(B) 1 X IO’ ’ BACTERIA DAY 2
SALINE 3Oi---__ 8:30
13:30
were not significantly different (P > 0.05). These data are important, however, because they again demonstrate that no portion of the increase in MSBT during
the bacteria injection trials was due to the experimental design of having the animals injected first with sterile saline followed by a dose of A. sobria. All six animals showed a pronounced hypothermia on day 1 of 1 x 10” bacteria injection (mean MSBT 30.7 f O.YC) and three of these animals still exhibited a pronounced hypothermia on day 2 after bacteria injection. One of the six animals died on day 2 of bacteria injection and the other two animals become normothermic on day 2.
18:30 DISCUSSION
TIME Fig. 3. Body temperature regulation patterns for one common agama, Agnma aguma, (A) on the day of injection of 0.6mf of sterile pyrogen-free saline and on the day of injection of 1 x lOto Aeromonas sobria and (B) on the day of injection of 0.6 ml of sterile pyrogen-free saline and on the day after the injection of 1 x 10” Aeromonas sobria.
The data show that there is a positive relationship between the level of bacteria infection (from 1 x 10s
4.01
bacteria injection were sibilantly (P < 0.05) higher than the mean saline MSBT (2.7 + 0.4”C higher on day 1; 2.3 + 0.4”C higher on day 2). Fever duration on day 1 averaged 7.3 f 1.1 hr (range 3-10 hr) while the mean duration on day 2 was 9.3 zf:O.Shr (range 5.5-11.0 hr). All animals demonstrated a fever response on day 2. There were no significant differences (P > 0.05) between average MSBTs on day 1 of bacteria injection at dosages of 1 x 108, 1 x log, or 1 x 10” A. sobria and there were no significant differences between the mean fever magnitudes on day 1 of bacteria infection at dosages of I x IO’, 1 x IO’, and 1 x lOto A. sobriu (Fig. 4). There was no significant difference (P > 0.05) between average MSBTs on day 2 of bacteria injection at dosages of 1 x log and 1 x 10r”. The only significant difference (P c 0.05) in average fever MSBTs and in fever magnitude was at a dosage of 1 x lo9 where the average MSBT and fever magnitude on bacteria day 2 was lower than the average MSBT on bacteria day 1 at the same dosage (39.4 f 0.2”C bacteria day 1 vs 37.9 f 0.4”C bacteria day 2). In terms of fever duration (Fig. 4), the average
p
2.0
2 _
4.0
2 z
8.0
%
LEVEL OF INFECTION 1x108 1x109 lxlOl0 *
8.0
P
Fig. 4. Fever magnitude and duration in the common agama, Agama agama, on the day of injection and the day after injection of 1 x i08, 11 x 109, and 1 x 10” Aeromonas s&in. Vertical bars = f I SE and an * indicates a significant difference between these two values at P i 0.05. Bl = Bacteria day 1, B2 = Bacteria day 2. An averge fever duration value is not indicated for bacteria day 2 at 1 x IO* because only one of seven animals exhibited a fever response on this day and the average MSBT on bacteria day 2 was not significantly different from the average saline MSBT.
Bacteria infection in Agarna agama
to 1 x 10” A. sob&) and the average duration of the fever response in the common agama. The data also indicate that the magnitude of the fever response on the day of bacteria infection is independent of the level of bacteria infection. It is only on the day after bacteria infection at a dosage of 1 x lo9 A. sobria that any effect of dosage upon average fever magnitude is evident. Results from previous studies are contradictory regarding the effect of bacteria infection or LPS level upon fever magnitude. Various doses of live bacteria (range 0.002-0.2 mg bacteria), including ones high enough to drastically reduce survivorship, did not significantly effect the average magnitude of the fever in rats (Banet, 1981) and these results agree with our results in the common agama. On the other hand, a complex dose-dependent relationship between level of infection and fever magnitude was reported in pigeons (D’Alecy and Kluger, 1975); a significant difference in average fever magnitude during the first 24 hr was reported between two levels of bacteria infection (3 x 10” and 6 x 10” Pasteurella multocida) in the rabbit (Kluger and Vaughn, 1978) and a dose-dependent fever was demonstrated in the leech in response to LPS (Cabanac, 1989). Data from species such as the rat (Morimoto et af., 1989; Stitt, 1991), the rabbit (Morimoto et al., 1988) the cat (Milton and Wendlandt, 1970), and the crayfish (Casterlin and Reynolds, 1978) have shown a positive relationship between fever magnitude and level of injection of prostaglandin E (PGE). PGE is postulated to be a central nervous system modulator of the fever response and among vertebrate animals PGE probably effects hypothalamic neurons that regulate body temperature set-point. The general hypothesis regarding the elevation of T, set-point after bacteria infection includes release of interleukin1 (IL-l) from macrophages and other cells after phagocytosis of bacterial endotoxin with subsequent (although the mechanism is not clear) production and release of PGE in the thermosensitive preoptic-anterior hypothalamic (POAH) or organum vasculosum laminae terminali (OVLT) area (Stitt, 1991). Although it seems clear that increased levels of PGE increase T, set-point in a dose-dependent manner, there is no evidence available from reptiles or other animals which indicate that either increased levels of endotoxin result in a dose-dependent increase in circulating levels of IL-l or that increased levels of IL- 1 result in the dose-dependent production and release of PGE in the POAH or OVLT area. Kluger (1991) has stated that there is a poor correlation between plasma IL-l activity and body temperature in animals given injections of bacteria or LPS. The only study to show an increase in plasma IL-l (Hesse et al., 1988) used a very high dose of live E. coli in the baboon. However, this dose of E. coli also led to endotoxic shock and a decrease in T,, not a fever. A dose-dependent relationship between intravenous injection of endogenous pyrogen (derived
483
from human monocytes) and mean rise in T, has been demonstrated in rabbits and rats (Stitt et al., 1985); however, no significant difference in maximum change in T, was observed in rats after intravenous injection of 0.1 and 1.0 pg/rat IL-1B (Dascombe et al., 1988). The fever after injection of l.Opg/rat IL-1B did have a longer duration. If, as hypothesized, PGE release in the POAH or OVLT increases T, set-point, the data from Agama agama suggest that either the 1 x lo*, 1 x 109, and 1 x 10” doses of A. sobria produce the same amount of circulating IL-l or that different levels of circulating IL- 1, above some threshold level and to some maximum level, have the same effect upon PGE levels in the POAH or OVLT areas. Different levels of IL-l either in the plasma or in the brain could have similar effects on T, increase if IL-l receptors were saturated at the lowest fever producing dose. The dose-response data presented in this paper strongly suggest that the negative fever results published for some reptile species (Laburn et al., 1981; Zurovsky et al., 1987a; Zurovsky et al., 1987b; Mitchell et al., 1990) and possibly for species other than reptiles (Cabanac and Rossetti, 1987; Rossetti and Nagasaka, 1988) may be due to the use of an inappropriate (either too low or too high) dose of dead bacteria or LPS. Also, since some studies have used LPS from bacteria other than Aeromonas sobria or have used dead bacteria of other species, the dose-response system described here for Agama aguma may not be appropriate for lizards when either LPS or dead bacteria of another species are used. We would urge that dose-response studies using Aeromonas sobria be conducted on a number of different species in an attempt to describe various dose-response patterns that may exist. We also feel that dose-response studies using bacteria species other than A. sobria could provide data useful to the study of comparative fever biology. Acknowledgements-We wish to thank Helen Barath for assistance in preparing the bacteria cultures and Robert Deshamais for providing statistical advice. This study was supported by an NIH-Minority Biomedical Research Support grant (5S06RR08101) to California State University, Los Angeles, CA.
REFERENCES
Banet M. (1981) Fever and survival in the rat: metabolic versus temperature response. Experientia 37, 130221304. Burns G. A. (1991) Survey of the fever response in temperate North Amercan desert and grassland/forest snakes. M.Sc. Thesis, California State University, Los Angeles, CA. Cabanac M. (1989) Fever in the leech, Nepheiopsis obscura (Annelida). J. camp. Physiol. B. 159, 281-285. Cabanac M. and Rossetti Y. (1987) Fever in snails, reflection on a negative result. Camp. Biochem. Physiol. WA, 1017-1020. Casterlin M. E. and Reynolds W. W. (1978) Prostaglandin E, fever in the crayfish Cambarus bartoni. Pharmac. Biochem. Behav. 9, 593-595.
484
ANA B. RAMOSet al.
D’Alecy L. G. and Kluger M. J. (1975) Avian febrile response. J. Physiol. 253, 223-232. Dascombe M. J., Rothwell N. J., Sagay B. 0. and Stock M. J. (1988) Pyrogenic and thermogenic effects of interleukin 1B in the rat. Am. J. Physiol. 256 (Endocr. Metab. 19), E7-Ell. Firth B. I., Ralph C. L. and Boardman T. J. (1980) Independent effects of the pineal and a bacterial pyrogen on behavioral thermoregulation in lizards. Nature, Land. 285,399-400.
Hallman G. M., Ortega C. E., Towner M. C. and Muchlinski A. E. (1990) Effect of bacterial pyrogen on three lizard snecies. Como. Biochem. Phvsiol. %A, 383-386. H&se D. G., *Tracy K. J., Fond Y., Manogue K. R., Palladino M. A. Jr, Cerami A. G., Shitres T. and Lowry S. F. (1988) Cytokine appearance in human endotoxemia and primate endotoxemia. Surg. Gynecol. Obstet. 166, 147-153. Kluger M. J. (1978) The evolution and adaptive value of fever. Am. Sci. 66, 38-43. Kluger M. J. (1991) Fever: role of pyrogens and cryogens. Physiol. Rev. 71, 93-123. Kluger M. J. and Vaughn L. K. (1978) Fever and survival in rabbits infected with Pasteurella multocida. J. Physiol. 282, 243-25 1. Labum H. P., Mitchell K., Kenedi E. and Louw G. N. (1981) Pyrogens fail to produce fever in a cordylid lizard. Am. J. Physiol. 241 (Regulatory lo), R198R202.
Integrative camp. Physiol.
Lang J. W. (1986) Crocodilian thermal selection. In Wildlife Management Crocodiles and Alligators (Edited by Webb G. J. W., Manois S. C. and Whitehead P. J.), pp. 301-037. Surrey Beathy and Sons, Vancouver. Milton A. S. and Wendlandt S. (1970) A possible role for prostaglandin E, as a modulator for temperature regulation in the central nervous system of the cat. J. Physiol. 207, 7677.
Mitchell D., Labum H. P., Matter M. and McClain E. (1990) Fever in namib and other ectotherms. In Namib Ecology: 25 Years of Namib Research (Edited by Seely, M. K.), Monograph No. 7, pp. 179-192. Transvaal Museum, Pretoria, R.S.A.
Monagas W. R. and Gatten R. E. Jr (1983) Behavioral fever in the turtles Terrapens Carolina and Chrysemus picta. J. Therm. Biol. 8, 285-288.
Morimoto A., Murakami N., Makamori T., Sakata Y. and Watanade T. (1989) Possible involvement of prostaglandin E in development of ACTH response in rats induced by human recombinant interleukin-1. J. Physiol. 411, 245-256.
Morimoto A., Nakamori T., Watanabe T., Ono T. and Murakami N. (1988) Pattern differences in experimental fevers induced by endotoxin, endogenous pyrogen, and prostaglandins. Am J. Physiol. 294 (Regulatory Inregrative camp. Physiol. 23), R633-R640. Muchlinski A. E., Stoutenburgh R. J. and Hogan J. M. (1989) Fever response in laboratory-maintained and free ranging chuckwallas (Sauromalus obesus). Am. J. Physiol. 257
(Regulatory
Integrative
camp.
Physiol.
26)
R15GR155. Ortega C. E., Stranc D. S., Casal M. P., Hallman G. M. and Muchlinski A. E. (1991) A positive fever response in Agama agama and Sceloporus orcutri (Reptilia: Agamidae and Iguanidae). J. camp. Physiol. B. 161, 377-381. Rossetti Y. and Nagasaka T. (1988) Prostaglandin E,, prostaglandin E,, and endotoxin failure to produce fever in the Japanese fresh water snail Semisulcospira libertina. Jpn. J. Physiol. 38, 178-186.
Stitt J. T. (1991) Differential sensitivity in the sites of fever production by prostaglandin E, within the hypothalamus of the rat. J. Physiol. 432, 99-110. Stitt J. T., Shimada S. G. and Bemheim H. A. (1985) Comnarison of febrile responsiveness of rats and rabbits to enhogenous pyrogen. j. appl. Physiol. 59, 1121-1125. Vauahn L. K.. Bemheim H. A. and Kluaer M. J. (1974) F&er in the lizard Dipsosaurus dorsak. Nature 252, 413-474.
Zurovsky Y., Brain T., Labum H. and Mitchell D. (1987a) Pyrogens fail to produce fever in snakes Psammophis philipsii and Lamprophis fuliginosis. Comp. Biochem. Physiol. 87A, 911-914. Zurovsky Y., Mitchell D. and Labum H. (1987b) Pyrogens fail to produce fever in the leopard tortoise Geochelone pardalis.
Comp. Biochem. Physiol. WA, 467469.