Antibodies to larval Taenia crassiceps in hibernating woodchucks, Marmota monax

EXPERIMENTAL PARASITOLOGY 24, 42-46

Antibodies

(1969)

to Larval

Taenia

Woodchucks, Naval

Medical

North Carolina

crassiceps

Marmota

monaxl

Richard L. Beaudoin Research Institute, Bethesda, David E. Davis Stale University, Raleigh,

in Hibernating

Maryland

North

Carolina

20014

27607

and Naval

Medical

(Submitted

K. D. Murrell Research Unit 2, Taipei, for publication,

22 July

Taiwan 1968)

BEAUDOIN, RICHARD L., DAVIS, DAVID E., AND MURRELL, K. D. 1969 Antibodies Marmota monax. Erperito Larval Taenia crassiceps in Hibernating Woodchucks, mental Parasitology 24, 42-46. An experiment was designed to study the effects of hibernation on the production and induction of antibody response to cysticerci of Taenia crassiceps in woodchucks (Marmota monax). Eight woodchucks were placed at 6OC (hibernators) and eight at 20°C (nonhibernators). The food was removed from all woodchucks to induce hibernation or lethargy. Four of each group were infected before hibernation and four after. Cysticerci were injected intraperitoneally. The response was judged by hemagglutination titration. The results showed no difference in antibody level among the four groups. Thus, it is concluded that antibodies to at least some helminths are produced during the period of hibernation. Whether or not production occurs only during the brief periods of arousal was not determined. INDEX DESCRIPTORS: Taenia crassiceps Hibernation; Antibody; Antibody induction; glutination; Microtiter.

larvae; Marmota Antibody

monax; Woodchucks; production; Torpor; Hemag-

ceived less attention. In one investigation Chute (1961) showed that lowered temperatures had a protective effect in golden hamsters infected with Trichinella spiralis. However, it is not clear whether this protection was a direct effect on the worms or an alteration of the host’s resistance. Previous studies with the hibernating animal indicate that production of humoral antibody is suppressed (Andjus and Matic, 1959; Cahill et al., 1967; Jaroslow and Smith, 1964). However, as Schmidt ( 1967) emphasizes, a direct correlation between antibody titer and resistance may not always exist. Woodchucks (1Marmota monax) in Pennsvlvania harbor considerable numbers of

The role of hibernation in epidemiology of viral and bacterial disease has been the subject for numerous investigations (see review of Schmidt, 1967). Its effect on helminth host-parasite relationships has re1 This work was supported in part by National Institutes of Health grant HE 062662 and Bureau of Medicine and Surgery, Navy Department Research Tasks MR005.09.0003 and MR005.20.0098. The experiments reported herein were conducted according to the principles enunciated in “Guide for Laboratory Animal Facilities and Care,” prepared by the Committee on the Guide for Laboratory Animal Resources, NAS-NRC. The opinions and assertions contained herein are those of the authors and are not to be construed as official or reflecting the views of the Navy Department or the Naval service at large. 42

ANTIBODIES IN HIBERNATING WOODCHUCKS

ruenia crassiceps cysticerci on emergence in the spring. A program was planned to study the effects of hibernation on the humoral antibody response of woodchucks to one of its natural parasites, larval T. crusiceps. MATERIALS AND METHODS

The woodchucks were trapped on the Letterkenny Ordnance Depot near Chambersburg, Pennsylvania, where numerous studies of population dynamics have determined the annual sequence of events (e.g. Davis et aZ., 1964; Bailey, 1965; Davis, 1967a,b). In brief, the woodchucks emerge from hibernation in February, breed, raise their young, and then enter hibernation in October. The young are born in the first half of April, leave the maternal burrow in June, and disperse in July and August. The woodchucks were captured in box traps (Snyder et al., 1960) and transported to the Animal Behavior Laboratory at Pennsylvania State University. They were kept individually in cages that measured 50 x 80 X 50 cm and were fed pellets of rat chow. Day length was kept at 16 hours. Woodchucks destined to hibernate were kept in a walk-in cooler at 6°C the normal burrow temperature ( Davis, 1967a). The controls were kept at 20°C. Removal of food (as occurs in nature) resulted in hibernation. Since it was not possible to obtain woodchucks in sufficient numbers to ensure uniformity in age, sex, and weight, an attempt was made to randomize animals among the experimental groups. In considering problems of host resistance in hibernators, it is important to remember that the woodchucks, during the hibernation period, arouse for a couple of days at normal body temperature (37’C) and then return to torpor for a variable period (4-12 days) at low temperature (8-10°C). Thus during the 2 months of hibernation, the woodchuck

43

has normal temperature intermittently for a total of about 10 days. Failure to recognize this sequence has prevented interpretation of manv research reports. EXPERJ~~ENTAL PROCEDURE

The experiment was designed to contrast induction and production of antibody before and during hibernation. Since woodchucks will become torpid normally when food is absent, food was withdrawn at the appropriate time. The woodchucks in the cooler at 6°C maintained the torpor-arousal routine, permitting their bodv temperature to drop to 8-10°C (Bailey, I965). In contrast, woodchucks in a room at 20°C became lethargic when food was withdrawn and had a body temperature of 3337°C but never entered true torpor. The woodchucks in the cooler are called experimentals and those in the room are called controls because their temperature dropped only to 33°C. Eight woodchucks, four experimental animals with their four corresponding controls, were infected on November 21, before hibernation. Similarly, another group of eight woodchucks, four experimentals and four controls, were infected on January 9 after the onset of torpor. If torpor affected the production of antibodies, the four woodchucks infected before entrance into torpor and kept at 6’C should have a low titer. Likewise, if torpor affects induction of antibodies, the four woodchucks infected after entrance into torpor should have low titers. Unfortunately, it is not possible to examine woodchucks for parasites before the experiment. However, hemagglutination titrations were accomplished on all animals before each was experimentally infected to determine whether they had had prior experience from a natural infection, and only animals without demonstrable titers were used (Table I-Nov. reading). On termination of all experiments, each animal was

44

BEAUDOIN, DAVIS, AND MURRELL

examined thoroughly for the presence of larval T. crassiceps. When the four experimental woodchucks that were infected prior to hibernation aroused in April, they were challenged while alive by injecting 10” larvae into the groin. Infection The Taenia crassiceps cysticerci were originally obtained from an infected woodchuck captured at Letterkenny Ordnance Depot in 1966. Larvae were maintained in laboratory mice by syringe passage, following the method of Freeman (1964). Woodchucks were infected by intraperitoneal injection of 10 larvae. Serum samples were collected on the day of infection, and at monthly intervals following onset of torpor. Antigen Preparation Lyophilized larvae from mice were powdered in a Ten-Broeck tissue grinder. Two hundred milligrams were suspended in 10 ml of cold phosphate-buffered saline (PBS) (pH 7.2). The homogenate was diluted with PBS to 100 ml, and sonicated in an ice bath for four e-minute intervals, followed by stirring with a magnetic stirrer for 12 hours at 4°C. The suspension was centrifuged at 5,OOOgfor 30 minutes at 4°C and the clear supernate was lyophilized until used. The total protein concentration was determined by the Biuret method to be 450 ugm/ml. Carbohydrate content was measured by the Molisch method and determined as 594 ugm/ml. Hemagglutination The formolized tanned sheep cell procedure described by Stein and Desowitz ( 1963) was used. The cells were sensitized with a 1:30 dilution of the antigen. The tests were performed in plexiglass microplates. Antibody was titrated by twofold serial dilutions using microdiluters. (Cooke Engineering Company)

One animal belonging to the experimental group infected before hibernation died ai the time of the February bleeding, presumably from the anaesthetic. Similarly, one animal from the experimental group infected after hibernation died following administration of anaesthesia at the time challenge was attempted in April. The three surviving woodchucks of this group were challenged and kept for an additional 6 weeks and then necropsied. The others were necropsied in April. RESULTS

Postmortem examination failed to reveal in the peritoneal cavity any of the worms used to immunize. Possibly this small number of larvae became entangled and obscured within the very large amounts of fat customarily deposited prior to hibernation. However, some larvae were recovered in all challenged animals. Although the site of challenge was the groin, larvae were recovered from the abdominal muscles and the pleural cavity. There was no evidence of increase in numbers though the search for larvae was not exhaustive. Recovered larvae were viable and active, several promptlv evaginating after being in saline for only a few minutes. One experimental animal which did not develop a very high titer was shown to have numerous large growths in the liver suggestive of advanced tumors and probably suffered from impaired liver function. Serological 0hervation.s The torpid state had no detectable effect on the antibody response in the woodchuck against larval T. crassiceps as determined by a specific hemagglutination test (Table I). The results were the same whether infection occurred before or after onset of hibernation. Differences in median titers between hibernating and non-hibernating group are not considered significant since

ANTIBODIES

Antibody

IN

HIBERNATING

45

WOODCHUCKS

TABLE I Response to Taenia crassiceps in Woodchucks

Titer of antibodies to Taenia crassiceps in woodchucks kept in a cooler at 6°C (experimentals) r room at 20°C (controls). One group was infected (Nov. 21) before hibernation occurred cember, another after (Jan. 9).

or in in De-

TITER Nov. Hibernators (6°C) When infected Before Before Before Before After After After After

a Challenged

March

Age

Sex

Wt

(Experimentals) < < < <

1:lO 1:lO 1:lO 1:lO

1: 1280 1:640 1:640 1: 1280

1:5120a 1:2560 1: 1280a -

Ad. Juv. Juv. Ygl.

F F M F

5700 4075 5025 4000

< < < <

1:lO 1:lO 1:lO 1:lO

1:640 1: 1280 1:640 1: 2560

1: 1280a 1: 1280 1:640 1: 1280

Ad.

JUV.

F M M F

4500 5725 4950 6475

1:640 I:2560 1: 1280 1:640

1:640 1: 1280 1:2560 1:640

Ad. Ad.

F F M F

7200 5250 3050 3600

1:640 I:2560 1:2560 1:1280

1:640 1: 1280 1: 1280 I:2560

Ad. Ad. Juv. Juv.

F F M F

6500 5100 4700 4550

Nonhibernators (20°C ) ( Controls) When infected < 1:lO Before < 1:lO Before < 1:lO Before < 1:lO Before < < < <

After After After After

Feb.

1:lO 1:lO 1:lO 1:lO

Ad. Ad.

ft

:

at arousal in April.

they represent single dilution.

in most instances

only

a

DISCUSSION

The apparent antibody synthesis demonstrated in these experiments was unexpected in view of the findings of other investigators. Andjus and Matic (1959) reported that hemagglutinin production was suppressed during the dormant period in ground squirrels; ahhough these animals demonstrated higher titers after arousal than those observed in controls. Jaroslow and Smith ( 1964) could detect no evidence of antibody synthesis by hibernating ground squirrels. Their findings agree with the concept that a decrease in metabolism during hypothermia results in a corresponding decrease in synthetic activity. Foord (1918) concluded that the chilling of rabbits during

the immunization period resulted in an increase in anti-typhoid agglutinin. However, very few reports of antibody response to parasitic infections have appeared. Certain phases of the antibody-forming processes have been reported to occur even during hibernation, Jaroslow and Smith (1964) suggest that induction may occur during hibernation in ground squirrels as judged by the range of latent periods that occurred after arousal. They also determined that antigen disappearance was much slower in hibernating animals. In our study, the presumed presence of the living larvae throughout the hibernation period provides a constant, antigenic stimulus for antibody synthesis. However, it may not have occurred during the actual torpor since it could occur during spontaneous arousals. The brevity of these arousal

46

BEAUDOIN,

DAVIS,

periods argue strongly against this alternative. These periods (totaling about 10 days) to have a significant effect would have to result in extremely rapid synthesis to account for the antibody levels observed, although the titers generated would probably remain somewhat stable. Andjus et al. (1964) showed that hibernation slowed the decay rate of hemagglutinins in ground squirrels. If the torpid state significantly affected either initiation of antibody response or antibody formation, then a difference in antibody levels should be obtained between the experimental groups infected before and after onset of torpor. But, no significant difference was found between any of the groups. In weighing the facts, therefore, we interpret the data to indicate that the titers produced in these hibernating woodchucks are too high to be the result of production only during brief arousal periods. In any case, the results indicate that the hibernation period does not affect either induction or production of antibody of woodchucks against larval T. crassiceps. Challenged animals exhibited peculiar behavior patterns at the time they were inoculated with the challenge larvae. This behavior consisted of a shrill whistle followed abruptly by curling up or sitting up and rubbing and licking the area of the groin receiving the challenge dose, suggestting possibly a reaction to a localized itching or allergic response. It did not especially suggest acute pain since the reaction was somewhat delayed and the movements slow. REFERENCES ANDJUS, P. K. AND MATIC, 0. 1959. Hibernation and immunobiological reactivity. Pflueger’s archiv fiir die gesamte physiologic des menschen und der tiere 270, 55-56. ANDJUS, P. K., MATIC, O., PETROVIC, U., AND RAJEVSKI, V. 1964. Influence of hibernation and of intermittent hypothermia on the formation of immunohemagglutinins in the

AND

MUBBELL

ground squirrel. Annales academiae scientarun Fennicae series A:III:Biol:71-2. BAILEY, E. D. 1965. Seasonal changes in metabolic activity of nonhibernating woodchucks. Cana. dian Journal of Zoology 43, 905-909. CAHILL, J. R., LEWERT, R. M., AND JAROSLOW, B N. 1967. Effect of hibernation on course of infection and immune response in Citellus tridecemilineatus infected with Nippostrongy lus brasiliensis. Jozrrnal of Parasitology 53, 110-115. CHUTE, R. M. 1960. Over wintering of helminths in hibernating animals. Journal of Parasitology 46, 539. CHUTE, R. M. 1961. Infections of Trichinella spiralis in hibernating hamsters. Journal of Parasitology 47, 25-29. CHUTE, R. M. 1964. Hibernation and parasitism: Recent developments and some theoretical consideration. Annales academiae scientarum Fennicae series A:IV:71-7. DAVIS, D. E., CHRISTIAN, J. J., AND BRONSON, F. 1964. Effect of exploitation on birth, mortality, and movement rates in a woodchuck population. Journal of Wildlife Management 28, 1-9. DAVIS, D. E. 1967a. The role of environmental factors in hibernation of woodchucks (Marmota monar). Ecology 48, 683-689. DAVIS, D. E. 1967b. The annual rhythm of fat deposition in woodchucks ( Marmota monax) . Physiological Zoology 40, 391-402. FOORD, A. G. 1918. The effect of exposure to cold on antibody production. Journal of Infectious Diseases 23, 159-168. FREE&IAN, R. S. 1962. Studies on the biology of Taenia crassiceps (Zeder, 1800) Rudolphi, 1810 (Cestoda). Canadian JournaE of Zoology 40, 969-990. JAROSLOW, B. N., AND SMITH, D. R. 1964. Effect of hibernation on the latent period in normal and X-irradiated ground squirrels. Journal of Immunology 93, 649-655. SCHMIDT, J. P. 1967. Response of hibernating mammals to physical, parasitic, and infectious agents. In “Mammalian Hibernation,” (K. C. Fisher, ed.), Vol. III, pp. 421-438. Oliver & Boyd, Edinburgh & London. SNMER, R. L., AND CHRISTIAN, J. J. 1960. Reproductive cycle and litter size of the woodchuck. Ecology 41(4 ), 647-656. STEIN, B., AND DESOWITZ, R. S. 1964. The measurement of antibody in human malaria by a formolized tanned sheep cell hemagglutination test. Bulletin of World Health Organization 30, 45-49.