Immunology, 1970, 19, 621.

Antibody Responses of Goldfish to Bovine Serum Albumin

PRIMARY AND SECONDARY RESPONSES

G. N. TRUMP* AND W. H. HILDEMANN

Department of Medical Microbiology and Immunology, University of California,
Los Angeles, California 90024

(Received 19th January 1970)

Summary. Serum antibody responses of a teleost fish (Carassius auratus) to a

soluble immunogen (bovine serum albumin) were investigated as a function of time
and environmental temperature. Temperature variable experiments were used to
accentuate time differences between major events of the immune responses. Three
groups of adult goldfish were maintained and tested separately at 200, 250 or 300.
Sensitive monitoring of the anti-BSA responses was achieved by passive haemag-
glutination test. Comparisons among successive stages of antibody production at
the three disparate temperatures indicated their temperature sensitivity: all major
events occurred more rapidly at higher temperatures. The exponential rise period
appeared to be the most temperature dependent. All phases of the secondary
immune response occurred at increased rates at all temperatures. Observations
of two distinct anti-BSA populations of whole antiserums indicated that they
were induced sequentially by BSA during the primary response. The time difference
between their respective inductions was shortened with increasing temperature.
Both populations arose at the same time following secondary stimulation.
Differences in the qualitative and quantitative characteristics of the anti-BSA
response are taken as evidence for immunologic memory.

INTRODUCTION
The purpose of this study was to investigate the effect of temperature on the primary
and secondary antibody response of goldfish to a soluble immunogen. An evaluation of the
temporal relationships between two populations of antibodies directed against bovine
serum albumin (BSA) at different temperatures permitted primary and secondary res-
ponsiveness of goldfish to be further considered in the framework of immunological
phylogeny.
A close temperature dependence of the immune response in ectothermic vertebrates is
well documented (cf. Hildemann and Cooper, 1963). Immunologic memory involving
circulating antibody production has been demonstrated in lampreys (Finstad and Good,
1966), sharks (Sigel and Clem, 1966), marine teleosts (Clem and Sigel, 1966), and
* Present address: Department of Genetics, School of Medicine, University of Hawaii, Honolulu; Hawaii 96822,
U.S.A.
621
622 G. N. Trump and W. H. Hildemann
amphibians (Evans, Kent, Bryant and Moyer, 1966). The antibody response of many
lower vertebrates involves a single antibody class. This limited response may be more
amenable to study than the complex immunological memory of certain mammalian species
involving the synthesis of first one and then another class of immunoglobulin. However,
many representatives do produce fast and slow sedimenting or mercaptoethanol sensitive
and insensitive populations of antibodies (cf. Smith, Miescher and Good, 1966). The
temporal relationships between different types of antibodies in primary as compared to
secondary response should provide additional criteria for assessing the phylogenetic
development of immunological memory. Such an approach may contribute to under-
standing of the more complex regulatory patterns of higher vertebrate systems.

MATERIALS AND METHODS

Adult goldfish, Carassius auratus, were obtained commercially. These fish ranged from
17 to 20 cm in length and 200-400 g in weight, and were estimated to be young adults
more than 2 years old. Purina Trout Chow, a well balanced food high in protein content,
was provided six times a week. Fish were maintained within + 10 of the desired experi-
mental temperature at a population density of one fish per 12-20 gallons of water. Indi-
viduals were identified by numbered tags inserted in the caudal peduncle.
Purified bovine serum albumin (Pentex Corporation, Kankakee, Illinois) was dissolved
in phosphate buffered saline, pH 7-4.
All fish were acclimatized at their experimental temperatures for 2 weeks or more
before immunization. Fish were immunized, bled and maintained at either 20, 25 or 300.
Each fish was immunized with 300 pg BSA/kg body weight on days 0, 2 and 4 by intra-
cardiac, intraperitoneal and intramuscular routes, respectively. Bleedings were performed
at 3-4-day intervals until peak antibody levels were observed, and then at more extended
intervals for 2-3 months.
All fish were given an intraperitoneal injection of 500 jug of BSA as a secondary immuni-
zation at times indicated in the text.
Antibody was assayed by passive haemagglutination of bis-diazotized sheep red blood
cells coated with BSA (Hyslop and Roeder, 1966). Amounts ofBSA and bis-diazobenzidine
were adjusted so that conjugated cells and a standard antiserum produced identical titres
from batch to batch with very abrupt transitions from negative to positive settling
patterns.
Separation of antiserums into two antigenically and electrophoretically distinct anti-
body populations (Trump, 1970) was accomplished by a micro-adaptation of a diethyla-
minoethyl (DEAE) method (Baumstark, Laffin and Bardawil, 1964). DEAE-Sephadex
was prepared according to suggestions by the manufacturer (Pharmacia Fine Chemicals,
Piscataway, New Jersey) and suspended in 0-01 M potassium phosphate, pH 6-5. 0-2 ml
of whole serum was added to 0X6 g moist resin. After incubation for 1 hour, the unadsorbed
proteins were removed by centrifugation and the gel was washed twice with 0X5-ml
amounts of starting buffer. The pH ofthe combined fluids was adjusted to 7f4 with NaOH.
1 ml of 0-01 M potassium phosphate-0-3 M NaCl was added to the gel, mixed, incubated,
centrifuged through; and the gel was washed with 0 5 ml eluting buffer. The pH was ad-
justed to neutrality. Both samples were adjusted to 2 ml with 0 01 M Tris-0'14 M NaCl, pH
7 5, then concentrated separately to 0-2 ml with dry Sephadex G-25.
 Goldfish Primary and Secondary Response to BSA 623
RESULTS
PRIMARY RESPONSE AT 200

No antibody was detected before the 17th day after cardiac immunization in fish
maintained at 200. After that time anti-BSA was observed in an increasing number of
fish. By the 38th day, 80 per cent of the fish had responded. At this temperature a mean
peak titre of 5 (i.e. 1: 32 or 25) was reached at 50 days (Fig. 1).

6_ = -

2-

00 l0 20 30 40 50 60 70
Days
FIG. 1. The mean anti-BSA primary responses at 200 (A), 250 (0) and 300 (h). The responses of
goldfish maintained at three different temperatures to weight-adjusted doses of BSA are indicated as
haemagglutination titres. The different groups of fish are indicated by the temperature of their
environment.

PRIMARY RESPONSE AT 250

All fish produced anti-BSA by day 10 at 250. However, no antibody was detected at 7
days. Peak titres of 3 to 7 were achieved in most animals by 20 days. Antibody production
was demonstrated to persist for as long as 245 days after immunization (Fig. 1).

PRIMARY RESPONSE AT 30°

Anti-BSA production at 300 was markedly different in many respects. Although the
induction period appeared to take 7 days, peak titres were reached by 13 days. However,
the mean highest titre was only 3 and antibody production was sustained for a significantly
shorter period of time at this temperature than at the other temperatures (Fig. 1).
Table 1 summarizes the kinetic data presented in Fig. 1 for the primary response at 20,
25 and 300.

SECONDARY RESPONSE AT 200

The mean induction period at 200 following secondary stimulation occupied 7 days.
Although most peak titres were reached by 30 days the rate of secondary antibody pro-
duction at this temperature was still notably slower than the rates of primary responses
at the higher temperatures. The titres for individual fish never exceeded those of the pri-
mary response at this temperature. In contrast to the primary response, antibody pro-
duction remained undiminished by 60 days after secondary stimulation (Fig. 2).
624 G. N. Trump and W. H. Hildemann

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 Goldfish Primary and Secondary Response to BSA 625
SECONDARY RESPONSE AT 250
Accelerated production of anti-BSA at 250 began 3 days after secondary immunization.
The rate was one and a half times faster than during the primary response. Peak titres at
this temperature exceeded primary responses at any temperature. However, antibody
production began to decline some 20 days following its peak although antibody remained
detectable for more than 90 days (Fig. 2).
8I

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2-

IoD 10 20 30 40 50 60 89
Days
FIG. 2. The mean anti-BSA secondary responses at 200 (A), 250 (0) and 300 (Lii). The responses of
goldfish maintained at three different temperatures to a secondary dose of BSA are indicated as haemag-
glutination titres. The different groups of fish are indicated by the temperature of their environment.

SECONDARY RESPONSE AT 300

Anti-BSA production at the highest temperature was noteworthy for its generally
feeble nature. While only half the time was required to induce secondary responses, the
rate and magnitude of antibody production was less than during the primary response at
this temperature. It appeared from an extrapolation of the secondary response at this
temperature that no antibody would be detectable by 75 days (Fig. 2).
Table 1 summarizes the kinetic data presented in Fig. 2 for the secondary responses at
20, 25 and 300.

ANTIBODIES OF THE PRIMARY AND SECONDARY ANTI-BSA RESPONSES

Two antigenically and electrophoretically distinguishable antibodies (termed 'Popula-

tion 1' and 'Population 2'), participate in the goldfish response to BSA (Trump, 1970). A
simple one-step procedure was employed to determine the relative amounts of both
populations of goldfish anti-BSA. Table 2 expresses the mean day of appearance of the two
populations of antibodies in several individual fish at 20, 25 and 300. The number of days
between the detection of the two populations or delta value (A) at each temperature is
revealing. Note especially the change from a 17- to 0-day interval in the primary-versus-
secondary responses of the 200 group.
The anti-BSA response at all temperatures appeared to be the product of at least two
electrophoretically and antigenically distinguishable populations of antibodies. Only one
626 G. N. Trump and W. H. Hildemann
TABLE 2
A COMPARATIVE SUMMARY OF THE DAY (EXTRAPOLATED TO ZERO TITRE) WHEN ANTI-BSA POPULATION 1 AND 2 BEGAN
TO APPEAR IN THE SERUM IN THE PRIMARY AND SECONDARY RESPONSE

Primary Secondary
Ab population Ab population
Temperature
(0C) No. 1 No. 2 A No. 1 No. 2 A
200 21 38 17 20 20 0
250 13 18 5 10 12 2
300 11 11 0 11 11 0
The delta value (A) is the difference in number of days between disappearance of Population 1 and 2.

of the two populations could be demonstrated during the early phases of antibody pro-
duction in the primary response at 20° or 250. Table 1 shows the time difference between
appearance of both antibodies to be inversely related to the temperature. Although 17
days separated the appearance of the two populations at 20°, only 5 days separated their
induction at 250. No detectable time differences were noted at 300. There appeared to be
no appreciable differences in the detection of the two antibody populations during the
secondary responses at any temperature. However, the titres of Population 1 generally
exceeded those of Population 2. There appeared to be no dramatic switch-over from one
antibody to the other.

DISCUSSION
Previous experiments have shown a correlation between the absolute rates of immune
responses and temperature in diverse ectothermic vertebrates. The present work compares
phases of antibody production at different temperatures. 50 temperature differences
were found to change the temporal parameters of the immune response in goldfish. They
suggest that all stages of the primary anti-BSA response involve temperature-dependent
reactions. However, the exponential rise periods appear to be the most temperature
sensitive stages. While induction periods at 200 and 250 differed by a factor of 2 and the
length of time at peak titre by 3, the titre rise periods revealed a full four-fold difference.
The striking difference at 300 was the abrupt end of the immune response. In this con-
nection, the period of titre rise was also shortened as was the time of peak antibody
production. Recent work suggests that immunogenic stimulation leading to antibody pro-
duction involves multiple cell interaction (cf. Groves, Lever and Makinodan, 1969).
Rates of such interactions may be thermally dependent. It has also been suggested that
slower elevation of titres at lower temperatures reflects a lowered rate of antibody
secretion (Elek, Rees, and Gowing, 1962).
The magnitudes of goldfish anti-BSA responses following secondary immunization
ranked in decreasing order at temperatures of 250, 200 and 300. This may have reflected
the number of memory cells (Coons, 1965) available for stimulation. At all temperatures,
the so-called latent period of the secondary responses was shorter than during the primary
responses. Except for the 300 group, rates of antibody appearance increased following
secondary stimulation. Consequently peak titres were reached in a comparatively shorter
time. In a kinetic study of allograft rejection in goldfish temperature was found to affect
secondary responses much less than primary response (Hildemann and Cooper, 1963).
 Goldfish Przmary and Secondary Response to BSA 627
There were two distinctive populations of antibodies to BSA separable by chromato-
graphy. Antibodies of Population 1 apeared before those of Population 2 in the primary
responses at 200 and 250. The decreasing delta values (Table 2) with increasing temperature
indicate that temporal differences were not a product of disparate agglutinating efficiency
of the two antibodies. That no such temporal differences were noted at any temperature
during the secondary response indicates a qualitative difference between the develop-
mental stages of the cell populations in primary and secondary responses. This effect could
be mimicked at the highest temperature during a primary response. This may suggest that a
series of closely spaced immunizations has a priming effect at higher temperatures.
The present study reveals that goldfish possess a capacity for secondary responsiveness
which is both qualitatively and quantitatively different from the primary response. Thus,
these teleosts may represent a phylogenetic level where mechanisms for anamnesis and
memory cell formation are present even though only a limited repertoire of immuno-
globulins may be elicited.

ACKNOWLEDGMENTS
This work was supported by United States National Institutes of Health Grant Al-
07970.
G.N.T. was supported by United States Public Health Fellowship 5FI-GM-22,767.

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