Cellular and humoral aspects of the primary immune response of the toad, Bufo marinus

The primary immune response of the toad, Bufo marinus, to Salmonella adelaide flagella has been analysed in terms of three immunological parameters: (a) the kinetics of appearance and morphology of cells producing specific antibodies; (b) antigen retention in one of the lymphoid organs, the jugular bodies; and (c) the nature of the immunoglobulins produced. It was found that toads responded to the bacterial antigen by producing antibody-forming cells and serum antibody in quantities comparable to those observed in mammals. In the early phase of antibody production, small and medium lymphocytes played an important role in antibody synthesis. In the later stages of the response, large cells which resembled immature plasma cells were the predominant producers of antibody. Studies on the incorporation of tritiated thymidine suggested that the majority of antibody-forming cells observed during the logarithmic phase of the response arose by division. Electron microscopic studies showed that the localization of radioactively-labelled antigen in the jugular bodies was similar to the follicular localization in mammalian lymph nodes, i.e. the antigen was associated with the surfaces of cells, rather than with intracellular sites. Although the toad was found to possess distinct classes of 18S and 7S immunoglobulins, antibody activity to this antigen was present only in the 18S molecules.These results are consistent with the hypothesis that the major patterns of differentiation and proliferation of immunologically competent cells, antigen retention and immunoglobulin structure have emerged by the phylogenetic level of anuran amphibians.     

Cyclicity and memory in the humoral immune response of the marine toad Bufo marinus

Bufo marinus toads immunized with a single dose of 100 μg of polymerized flagellin (POL) produce a longlasting cyclic humoral response at 22 °C in which the serum antibody titers rise and fall within periods of 2–3 weeks. This seems to be regulated by the catabolism of immunoglobulins, since passive ¹²⁵I-labeled IgM in the serum has a half-life of 17 days, corresponding to the cyclicity of the titers of active antibodies. In toads injected with between 10 μg and 10 pg antigen the serum antibody titer peaks between 4 and 7 weeks, depending on the antigen dose, and declines by the 9th week. At this time a second stimulation with an equal dose of POL induces a secondary response during which higher titers of antibodies rise faster than in the primary response. A dose of 10 μg POL stimulates an optimal primary response and a second equal dose, given when the serum antibodies are disappearing, induces a secondary response enhanced in time but not in antibody titers.     

Salt sensitivity and hydration behavior of the toad, Bufo marinus

Toads, Bufo marinus, were placed on laboratory tissue saturated with water or with hyperosmotic (250 or 500 mM NaCl or KCl) solutions, and their behavior was observed for 5 min. Toads placed on water initially allowed their ventral skin to touch the surface without abducting the hind limbs. During this "seat patch down" (SPD) behavior toads appeared to be evaluating the suitability of a hydration source prior to initiating "water absorption response" (WR) behavior with the hind limbs fully abducted and the ventral skin pressed to the moist surface. Toads dehydrated by more than 10% showed significantly shorter periods of SPD behavior and initiated WR behavior more frequently than did hydrated toads. Dehydrated toads placed on 250 mM NaCl initiated WR behavior in only 18% of the trials, but spent significantly more time showing SPD behavior than they did on water, indicating that this concentration is marginally acceptable to them. Recordings from spinal nerve #6 showed an increase in activity when 250 mM NaCl or KCl solutions were perfused over the outer surface of the ventral skin. The response to KCl was significantly greater than NaCl. The addition of 10 microM amiloride to 250 mM NaCl resulted in a higher frequency of WR behavior and reversibly inhibited the neural response to 250 mM NaCl. These results suggest that epithelial Na+ channels in the skin serve a sensory function in this species. Neither the hydrated nor dehydrated toads initiated WR behavior on 250 or 500 mM KCl solutions, indicating that toads have a lower tolerance of K+ than of Na+ salts.     

Vagal stimulation and somatostatin potentiate cardiac vagal action in the toad, Bufo marinus.

Cholinergic postganglionic neurones of the cardiac vagus in the toad, Bufo marinus, have been shown to contain the peptide somatostatin (SOM), which causes direct negative inotropic and chronotropic effects on the heart. In anaesthetised toads, high frequency stimulation (10 Hz) of cardiac vagus nerves results in prolonged cardiac slowing and potentiation of the cardiac slowing measured in response to a train of vagal stimuli at low frequency. Intravenous administration of the tetradecapeptide form of SOM also results in prolonged cardiac slowing and potentiation of cardiac vagal action. Effects on heart rate of small bolus doses of acetylcholine (ACh) were unaltered by administration of SOM, at the same time as cardiac vagal slowing was enhanced. It is suggested that SOM is released from vagal nerve endings by high frequency stimulation and enhances cardiac vagal action by a presynaptic mechanism.     

Spontaneous chromomycosis in the marine toad (Bufo marinus).

Post-mortem examinations were performed on two marine toads, one animal showing neurological disorders and the other multifocal dermatitis. In one case, lesions consisted of a severe granulomatous encephalomyelitis and in the other of multiple granulomas in the nasal cavity, lungs, heart, bone marrow, ovaries and skin. Histologically, the lesions revealed varying amounts of dark brown fungal elements, predominantly sclerotic bodies indicative of a mycotic infection due to a pigmented fungus.     

A primary immune response of Bufo marinus spleen cells in vitro

Single-cell suspensions of Bufo marinus spleen have been induced to produce a primary immune response to a soluble purified protein. Using polymerized flagellin from Salmonella adelaide as antigen and culture conditions commonly available in most laboratories but new for amphibian cells, it has been found that in vitro at 37°C, toad spleen cells produce an antibody-forming cell response optimal at day 6. The response depends on the number of cultured cells and dose of antigen, and parallels that obtained in vivo. The optimal immune response is preceded at day 4 by the peak uptake of tritiated thymidine. The antibody-forming cell response is suppressed in the presence of allogeneic serum.     

Baroreceptor control of heart rate in the conscious toad Bufo marinus

Perivascular balloons were used to elicit heart rate (HR) responses to imposed changes in mean arterial pressure (Pa) in conscious unrestrained toads (Bufo marinus). It is clear that functional baroreceptors lie in the pulmocutaneous arteries of B. marinus, and that these can elicit compensatory heart rate responses to perturbations of blood pressure. Functional baroreceptors were not present in the carotid circulation and are probably absent from the lateral aortae and conus arteriosus as well. Normalized gain of the baroreceptor-heart rate reflex (delta HR%/delta Pa) in five toads was 13%/kPa, which is considerably less than that found in other vertebrates to date.     

Galanin-like immunoreactivity in sympathetic and parasympathetic neurons of the toad Bufo marinus

Immunoreactivity (IR) to galanin (GAL) was detected in a wide range of peripheral autonomic neurons in the toad Bufo marinus. Forty percent of adrenergic nerve cell bodies in paravertebral sympathetic ganglia had GAL-IR in addition to neuropeptide Y (NPY)-IR. Some of these neurons projected to systemic arteries. GAL-IR was localized in parasympathetic neurons supplying the heart, lung, pulmonary artery, bladder, rectum and tongue. Eighty-two percent of intracardiac vagal nerve cell bodies had both GAL-IR and somatostatin (SOM)-IR. GAL-IR and SOM-IR were also co-localized in cholinergic post-ganglionic vagal neurons supplying the lung musculature and the pulmonary artery, and in neurons intrinsic to the bladder. Many postganglionic glossopharyngeal neurons in the tongue contained both GAL-IR and vasoactive intestinal peptide (VIP)-IR. Therefore, in Bufo marinus, a GAL-like peptide, in combination with other peptides or with adrenaline or acetylcholine, may be involved in neurotransmission in several different functional classes of autonomic neurons.     

Primary and secondary immune responses of the marine toad (Bufo marinus) to bacterophage f2

The primary and secondary immune responses of toads (Bufo marinus) were studied using the bacteriophage f2 as antigen. Antibodies were identified in the serum 2 weeks after primary immunization and peak antibody levels were reached at 6 weeks. Although both IgM and IgG antibodies can be found in toad serum, most of the antibody activity in our animals persisted in the IgM fraction until 8 weeks after immunization. When a second injection of antigen was given 4 weeks after primary immunization, there was a marked increase in the total serum antibody activity and IgG antibodies were found as early as 4 weeks after the second injection of antigen. These observations suggest that the toad, B. marinus, is capable of a true secondary response to the bacteriophage f2.     

Thermal regulation of the immune response in South American toads (Bufo marinus)

Thermal regulation of the immune response was studied in toads following single injections of bacteriophage f2. The immune response was markedly inhibited in animals kept at 15° as compared to the controls (25°). The appearance of serum antibodies was delayed in animals kept at 15° for the first post-immunization week but their peak antibody levels were similar to those in toads maintained at 25° throughout. Transfer of animals from 25° to 15° 2 weeks after immunization only temporarily depressed the serum antibody levels but caused a marked delay in conversion from heavy to light antibodies.

Our results are in keeping with the hypothesis that lowered environmental temperatures inhibit a variety of metabolic processes which may be concerned with the utilization of antigen and/or the synthesis of antibodies.

     

Z and W sex chromosomes in the cane toad (Bufo marinus)

The cane toad (Bufo marinus) is one of the most notorious animal pests encountered in Australia. Members of the genus Bufo historically have been regarded as having genotypic sex determination with male homogamety/female heterogamety. Nevertheless, as with many toads, karyotypic analyses of the cane toad have so far failed to identify heteromorphics sex chromosomes. In this study, we used comparative genomic hybridization, reverse fluorescence staining, C-banding, and morphometric analyses of chromosomes to characterize sex chromosome dimorphism in B. marinus. We found that females consistently had a length dimorphism associated with a nucleolus organizer region (NOR) on one of the chromosome 7 pair. A strong signal over the longer NOR in females, and the absence of a signal in males indicated sex-specific DNA sequences. All females were heterozygous and all males homozygous, indicating a ZZ/ZW sex chromosomal system. Our study confirms the existence of sex chromosomes in this species. The ability to reliably identify genotypic sex of cane toads will be of value in monitoring and control efforts in Australia and abroad.     

Phylogenetic studies on the immune response: II. The immune response of the Australian echidna Tachyglossus aculeatus*

The immunological response of the primitive mammal Tachyglossus aculeatus has been studied. We have shown that this animal responds to flagella from Salmonella adelaide with production of mercaptoethanol-resistant antibodies during both primary and secondary responses. Although an anamnestic secondary response could be demonstrated, it was of much smaller magnitude than that of phylogenetically higher placental mammals. Serological investigations demonstrated the presence of a mercaptoethanol-sensitive antibody of high molecular weight and an antibody of lower molecular weight which was mercaptoethanol resistant. These two antibodies seem to be analogous to IgM and IgG antibodies of higher mammals.     

Active transport of calcium across the urinary bladder and colon of the toad Bufo marinus

Unidirectional fluxes of calcium were measured (in vitro) across the urinary bladder and colon of the toad Bufo marinus in the absence of electrochemical gradients. A net calcium flux was observed in each tissue, but the polarity differed; it occurred from the mucosal (luminal) to the serosal side of the colon and from the serosal to mucosal (urinary) side of the bladder. The active transport in each tissue appeared to involve different mechanisms; that across the colon exhibited a sodium dependence, possibly involving a sodium-calcium exchange, but this was not seen in the urinary bladder. The net flux in the latter was, however, abolished by metabolic inhibitors, possibly reflecting a role of a calcium-adenosine triphosphatase mechanism. The results are discussed in relation to the calcium metabolism of this amphibian.