Introduction

The Maintenance of Marine mammals in captivity requires that these animals undergo occasional transportations and to major environmental changes. Both events frequently result in reduction or suppression of the animal's immune status, rendering them vulnerable to invasion by infectious microorganisms. Pathogenic bacteria and viruses which predominate in certain geographic regions represent a specific health threat. To date we know little of the extent of natural exposure to pathogens in the wild, nor of the ability and type at immunological response these animals can produce.

The health evaluation program described here includes investigations into the basis immune functions of dolphins. Tursiops truncatus. Characterization of the numeral component, IgG, was performed allowing for the development of field use implementation of radial-immunodiffusion plate (RID) diagnostic procedures. Animals from captive and wild environments were utilized for these studies.

Methods

Immunoglobulin IgG was isolated from a 40% saturated ammonium sulfate (SAS) precipitate of pooled Tursiops truncatus sera. The IgG was isolated on DEAE (Affigel Blue BioRad). Elution profiles were determined on each Ig by testing pooled fractions against rabbit anti-dolphin whole serum by immunoelectrophoresis.

Single radial immunoelectrophoresis. (RID) plates were prepared using polyclonal rabbit or guinea pig antisera to dolphin IgG. Preliminary Studies indicate a standard of incubation of 24 hrs for IgG produced satisfactory and reproducible values. Pre-prepared plates, containing built-in mm scale for direct measurements (Miles Lab.), plus standard curve reference sheets were provided for on-site use. Six (a) mcl of serum from each test dolphin was placed into a prepared well of a RID plate and the zone of precipitation stained with Light Yellow Fast stain and measured using a millimeter scale.

Results

1.  Ig Standards

a.  The isolated IgG of wild dolphin 71J was checked for purity against rabbit anti-whole 71J sera by immuno-electrophoresis. The results indicated that the IgG migrated in a single band. The preparation was then further purified by Isolab Systems II chromatographic columns. Further immuno-electrophoresis of these products indicated the IgG to consist of subclasses IgG, IgG, and IgG.

b.  Rabbit antisera to the above IgG was standardized in the RID plate at dilutions of 1:5. 1:105 1:20. The Ig6 concentrations were 12.0 and 27.5 mgms.

c.  Three concentrations of IgG are provided with each plate and a standard curve constructed for each set of tests (see Figure 1). The optimum dilution of the current rabbit antiglobulin was found to be at 1:20 dilution.

d.  Sera from fifteen "wild" dolphins from a study population in Sarasota bay, Florida, were selected for determining the base-line levels of IgG. The levels of immunoglobulins are shown in Table 1. As noted, the range for IgG was 22 mg/ml to 27.5 mg/ml with an average of 24.53mg/ml.

2.  Test of RID Plates
Sera from clinically normal dolphins were selected from a pool of frozen specimens for use in the field testing of the RID plates (see table 2 for results). The data here exhibits an IgG range of 21.0 mg/ml to 27 mg/ml with an average of 25.20 mg/ml, results very similar to those from table 1 as expected. All procedures were performed by a technician previously unfamiliar with the RID plate analysis.

3.  Clinical cases
Sera from selected clinical cases were retained from dolphins undergoing clinically suspicious circumstances and run on available RID plates (see table 0 for results). Of the cases presented, relevant findings include; 1) Sundance, an 9/5/84, exhibited a marked rise in IgG over 4/11/80, at a time just before the initiation of a rising WBC, and on 9/19 the manifestation of clinical symptoms a+ what was later determined to be lung inflammation: 2) newly caught dolphin J86F3. showing a rising IQ from (a) time of capture to (b) time of transport to holding pool, to (c) onset of concern over apparent maladaptation to (d) outright maladaptation and rising WBC, compared to J86F2 of the same group who adapted normally; 3) Aletta, representing a captive-born yearling female who was undergoing a period of undiagnosed regression in appetite and behavior; 4) Misty, at 6/19/86 showing a very high IgG immediately following a period of elevated WBC, responsive anemia and unresponsive behavior (a condition which responded to antibiotic therapy during which time the test sample was obtained); 4) the two aged animals Zippi and Echo who were free of clinical problems other than their 30 plus years of age.

4.  Retrospective Studies
Sera from six cases resulting in mortality were selected from frozen specimens and run for IgG on RID plates (see Table 4 for results). Where possible, multiple samples were run for each case representing time periods associated with normal, early and late stages of the fatal diseases. Of these, relevant comments include: 1) Angel, from normal at 10/13/79 to early chronic active hepatitis on 9/23/81, to severe active hepatitis an 6/19/82 with a significantly lower IgG. to partial recovery clinically and with a rising IgG on 7/11/81. This animal eventually died of the disease in July, 1984; 2) Merlin, from subclinical chronic-active hepatitis on 5/15/34 to a drooping IgG on 8/6/84 three weeks before onset of clinical symptoms of an acute infection on 9/19/84. At this time. the IgG was precipitously low and marked the beginning day of a marked leukocytosis which progressed through 9/24/84 to the animal's death an 9/27/84; 3) Leilani, who was normal on 11/28/83 and who died shortly after 6/27/85 of an acute apparent viral infection (as evidence by severe leukopenia and lymphopenia) with a rising IgG: 4) Billy-Bon, an aged male Tursiols gilli, featured mere with his apparent normal IgG on 2/10/82 (a very high normal level. similar to Zippi, also an aged male), an identical level an 4/26/80 at the beginning of a rising WBC associated with clinical regression and a necrotizing dermatitis, and finally a rising IgG shortly before death with a marked leukocytosis; 5) the dolphin Sal, representing the classic post-transport stress syndrome here with an already low IgG on 4/6/85, two weeks after arrival, followed by a progressive and precipitous drop in IgG as the animal went into renal failure resulting from inappetance and dehydration, with death ensuing on 4/24/25; and 6) Captain, who died on 6/22/85 approximately one year post-capture with an encephalitis, who exhibited a high IgG six weeks before death, but at a time when clinical symptoms were at their onset.

Discussion

The specimens designated for baseline IgG levels represented blood serum obtained from wild Tursiops truncatus in captured in Sarasota bay, Florida. Each animal utilized was evaluated by physical examination and considered to be in good health. Specimens from a total number of 15 animals were utilized to establish a reliable norm for this species. The clinical normal group was drawn from a group of clinically normal dolphins housed at Marineland at Palos Verdes, California. The study investigating responses in ten clinical cases include animals undergoing either interesting clinical states (Sundance, Aletta, Misty. Echo, Zippy) or post capture and transport episodes (J86F2 and J86F3) for which we might anticipate alterations in immune competence. The six animals utilized in the retrospective study represent interesting cases from a variety of sources which terminated in mortality.

The retrospective study data offers the best opportunity to observe the relationship between IgG level alterations and disease. The dolphins Sal, Angel and Merlin show classic reduction in immune competance leading to clinical regression. In the case of Merlin and Angel, a transient rise in IgG occurred in an overwhelming infection in one and a clinical response in the other. In Captain, Billy-bon and Leilani, there appeared to be an elevation in IgG in response to an ongoing disease state. This responsive condition was also observed in the dolphin J86F3 who underwent a disease episode but whose condition resolved and who continues to remain healthy to this day. As indicated here, the RID plates can indeed be used to reference the animal's immune state.

This research funded by the Naval Ocean Systems Center, San Diego, CA (Contract No. 35-M-6495), Dr. J. F. Schroeder, project sponsor under the Palos Verdes Animal Care Foundation. Rancho Palos Verdes, CA.

Figure 1

Editors Note: Tables 1 through 5 have been omitted as they were not readable in the original provided pdf.