Background

Diagnostic serology is a relatively new discipline within cetacean medicine. Only within the past few years have serologic assays for determining exposure of cetaceans to specific pathogens been developed. Similarly, serologic assays for quantifying concentrations of total circulating immunoglobulin G (IgG) have been lacking. Recently, a set of anti-IgG monoclonal antibodies (mAbs) for bottlenose dolphins was developed in our laboratory for determining the presence of antigen-specific antibodies in cetacean serum or plasma. The same reagents can also be used in a different format to quantify total circulating IgG in serum or plasma of dolphins. This could aid in the diagnosis of failure of passive transfer of antibody in neonate dolphins with unknown clinical histories, in the evaluation of the efficacy of colostrum supplementation therapies and, possibly, in the detection of humoral immuno-suppression and stimulation.

Here, we report on the development of a competitive enzyme-linked immunosorbent assay (ELISA) for the quantification of IgG in bottlenose dolphin (Tursiops truncatus) serum and the application of the assay in a clinical setting. First, preliminary normal ranges of IgG levels were established for various age groups of bottlenose dolphins, including neonates. Next, the assay was used to aid in the veterinary management of a neonate bottlenose dolphin not nursed by its dam upon birth.

Development of IgG Quantification Assay

A competitive ELISA format was selected for quantifying dolphin IgG. In this assay, dolphin sera with unknown IgG concentrations competed with a known amount of purified dolphin IgG for binding sites on a known amount of mAb. The amount of free binding sites on the mAb, which was inversely correlated with the IgG concentration of either the unknown dolphin samples or the known purified IgG samples, was then determined in an indirect ELISA format. Finally, IgG concentrations for the unknown serum samples were then determined from a standard curve (Figure 1) in which a known concentration of a mAb was reacted against varying amounts of purified dolphin IgG. All assay parameters were varied to optimize assay performance and inter-plate variability. Varying assay parameters allowed us to accurately quantify IgG concentration in samples with both low (< 1.5 mg/ml) and high (= 1.5 mg/ml) IgG levels. Overall, the measurable range of IgG concentrations for the assay was 0.050 mg/ml to 15.36 mg/ml.

Figure 1. Standard curve for quantifying bottlenose dolphin (Tursiops truncatus) immunoglobulin G (r2= 0.995). Measurable IgG range for this curve is 1.25 mg/ml to 15.36 mg/ml.

Establishing Normal Ranges for Serum IgG Levels

IgG levels were analyzed in a total of 34 normal serum samples collected during 06 August 1998 to 14 July 2005 from 19 clinically healthy bottlenose dolphins of the Navy Marine Mammal Program (NMMP). 'Normal' was defined as a routine serum sample with a total white blood cell count between 4,800 and 10,200 cells/µl, an erythrocyte sedimentation rate less than 19 mm/hr (modified Westergren method); and serum globulin concentrations between 2.1 and 3.6 g/dl. Normal CBC and serum chemistry reference ranges for NMMP dolphins were previously defined as the mean blood value +/- 1.5 times the standard deviation from 855 routine, non-hemolyzed serum samples collected from animals greater than 5 years old during years 1998 through 2002. Of the 34 samples analyzed, 17 (50%) were from females and the median age at time of sample collection was 13.8 years (range 1.3-44.6 years). The IgG reference range was defined as the mean IgG value +/- 1.5 times the standard deviation.

Given this definition, the normal IgG reference range for Tursiops truncatus aged greater than one-year-old at the NMMP is 2.9 +/- (0.75 * 1.5) = 1.8-4.0 mg/ml (Table 1). There were no statistically significant differences (significance defined as p < 0.05) when comparing mean IgG levels by age (continuous or categorical), sex, or individual animals (Table 1).

Assessment of Neonatal Immune Status

The assay was used to aid in the veterinary management of a neonate bottlenose dolphin not nursed by its dam upon birth (ref C.R. Smith et al. abstract). The neonate received a total of 180 cc fresh colostrum via stomach tube during hours 12 to 28 postpartum (12-33 cc administered per session, 7 sessions total), and it successfully nursed from an allomother; the IgG content of the colostrum was unknown. On day 1, a serum sample was collected prior to colostrum administration as well as after the calf had received 4 doses of colostrum, 105 cc total volume. IgG levels were determined in each sample using the newly developed ELISA.

Table 1. Comparison of mean IgG levels between age and gender categories and between individual bottlenose dolphins (Tursiops truncatus) aged = 1 year old. N/A: not applicable.

For comparison, a total of 12 serum samples from seven dolphins aged < 1 yr old were selected from the serum bank at NMMP and IgG levels were measured. Of these samples, 4 (33%) were from females; the median age at time of sample collection was 10 days (range 0-347 days). A summary of neonatal histories, outcomes, and IgG levels is provided in Table 2.

Summary

A preliminary normal reference range for serum IgG levels in Navy bottlenose dolphins aged greater than one-year-old has been established using the described assay (1.8-4.0 mg/ml). Among the normal study population, there were no significant differences in mean IgG levels when comparing age categories, sex, or individual animals.

In addition to assessing the immune status of dolphins aged greater than 1 year old, this assay may be useful for health assessment and outcome prediction of neonates; follow-up studies, including more data from successful calves during the first 14 days after parturition, are warranted to establish normal IgG reference ranges for this group.

Table 2. Serum IgG levels and outcomes in selected dolphin calves aged < 1 year old

*ND = Not detected; detection limit was 0.05 mg/ml

Acknowledgments

We would like to thank the Navy Marine Mammal Program training, management and veterinary staff for their support, especially Maj. Brad Blankenship, Dr. Mark Xitco, and Dr. Sam Ridgway; Randy Wells and Mike Walsh for providing the samples used in the development of this assay; and Paul Klein and Linda Green for their technical assistance.