Chapter 63 Longitudinal Monitoring of Immune System Parameters of Cetaceans and Application to Their Health Management
Infectious disease, trauma, and stress are all important contributors to morbidity and mortality in zoo and free-ranging mammals. Routine health assessment at the individual level largely relies on observation of abnormal (condition), appetite and/or behavior and population stability. Clinical evaluation of individual animals is based on laboratory analyses of accessible samples, including blood, urine, and feces. Ancillary diagnostic aids applied to such samples include hematology, serum biochemistry, microbe isolation, and serology; these all include relatively solid baseline data from which to draw tentative conclusions. From a hematologic perspective, the complete blood count (CBC) associated with varied chemical analyses continues to serve as the gold standard for diagnostic testing. Accurate identification of the causative insult may be difficult, especially in the early stages of clinical disease. This is significant in view of the fact that successful treatment and prevention of disease progression and/or development of chronic debilitating sequelae relies on the early accurate identification of the likely insult and specific disease processes. The administration of broad-spectrum antimicrobial agents, with its associated risks, is common in affected individuals in the absence of a specific diagnosis.
Development of programs to train zoo and marine mammals to present themselves voluntarily for blood collection has provided a much desired alternative to physical or chemical animal restraint techniques that often place the animals at risk for injury or death. Blood collection via voluntary presentation of extremities has permitted the initiation of routine hematologic analyses in valued terrestrial and aquatic mammalian species. Such programs have accelerated the establishment of baseline values for a given species and for individual animals. The ability to identify abnormal values, based on an animal’s own predetermined baseline, increases relative diagnostic sensitivity.
Establishment of routine bleeding programs in many marine parks has provided a window for advancing the science of clinical immunology in cetacean species. Successes enjoyed in human medicine, which have used analytic flow cytometry to identify perturbations in blood leukocytes, formulate prognoses, and evaluate the efficacy of treatment modalities, has logically inspired the initiation of similar approaches in veterinary medicine. Application of flow cytometry to zoo and free-ranging nondomestic species is limited in part by the paucity of monoclonal antibodies specific for leukocyte differentiation antigens; the great majority of antibodies developed for human, murine, and domestic mammals are species-specific and thus of limited use in comparative medicine. On a positive note, reagents developed for canine, feline, equine, and bovine species often cross-react with members in their greater families.
Toothed whales (suborder Odontoceti, order Cetacea), which are the primary focus of this chapter, have no close relatives in the world of domestic mammals, and thus limited immunologic reagents are available to assess perturbations in leukocyte phenotype. Given their monetary value, high visibility, and ongoing training programs directed at establishing voluntary fluke presentation for blood collection, efforts were successfully initiated to develop monoclonal antibodies specific for dolphin (Tursiops truncatus) and killer whale (Orcinus orca) leukocyte differentiation antigens,1–3 referred to in the literature as CD antigens; select monoclonal antibodies previously developed for equine8 and bovine5,11 species were also tested for cross-reactivity. A summary of these antibodies and their leukocyte differentiation antigen specificities, if known, are given in Table 63-1.
The antibodies shown in Table 63-1 have been applied to four killer whale populations over a period of 12 years. The antibody set was able to distinguish neutrophils (F6B+/ILA-24+, major histocompatibility complex [MHC] II−), monocytes (F6B+/ILA-24+/MHC II+), and lymphocytes (F6B+, MHC II+/ILA-24−). Lymphocytes could be divided into B cells (CD19+, CD21+) and T cells (CD2+), with T cells being subdivided further by differential density expression of CD2 and CD45R into naïve and memory T cell populations (CD2+/CD45R+/Hi and CD2+/CD45R+/Lo, respectively). The usefulness of using a longitudinal approach for identifying immunologic perturbations in killer whales was quickly revealed. As expected, all markers listed in Table 63-1 were determined to be susceptible to perturbation. Outliers could be identified through comparison to the baseline established for the total population. Sensitivity in identification of immunologic perturbations was increased for select animals—low variation in immune parameter values over time—when using their own individual baselines. Absolute numbers of T and B lymphocyte subpopulations, representing a subset of the data collected over a 3-year period from 25 animals, are illustrated in Figure 63-1. Such data may only be developed for the individual through longitudinal sampling. Figure 63-1 illustrates the obvious variability in T and B lymphocyte subpopulations among animals. Those animals with dramatic fluctuations may be experiencing multiple and/or recurring insults, some outwardly visible and others subclinical. On inspection of all leukocyte subpopulation data, total numbers of memory T, naïve T, and B lymphocyte numbers were sometimes abnormally elevated and sometimes depressed; this often occurred differentially, resulting in altered ratios of total T versus B lymphocytes and naïve versus memory T lymphocytes. The clinical significance of the various patterns is currently a matter of investigation. Diagnostically speaking, point in time perturbations and trends toward becoming abnormal may be more readily identified with comparative analyses using a combination of the species’ baseline and an animal’s own individual baseline. However, age-associated changes in leukocyte subpopulation numbers must be taken into account when establishing baseline values for a species.
Figure 63-1 Absolute numbers of T and B lymphocytes in peripheral blood samples derived from 25 killer whales over a period of 3 years. The means, associated percentiles (10th, 25th, 75th, and 90th), and outliers for each animal (male and female) are illustrated. Sample size and animal numbers ranged from 12 to 22.
As noted, the cell surface density of select immune parameter markers has provided a sensitive measure of immunologic perturbation. From a diagnostic perspective, the relative density of the putative cell adhesion molecule, CAM-D, appeared to be telling. As determined by flow cytometry, decreased fluorescence intensity on a variable percentage of polymorphonuclear leukocytes (PMNs) stained with fluorescein isothiocyanate (FITC)-labeled α–CAM-D was occasionally recorded. We have speculated that this could be the result of either a conformational change in the adhesion protein, resulting in reduced binding of the monoclonal antibody, or reduced cell surface density of the protein. Regardless, the appearance of a subpopulation of PMNs with low-density fluorescence was associated with clinical disease. Figure 63-2 illustrates a clinically ill animal with a subpopulation of PMNs expressing a low fluorescence as compared with those expressing a relatively high fluorescence following staining with α–CAM-D. Parallel samples obtained from a healthy companion are illustrated for comparative purposes. The first time point was obtained at the initiation of acute illness (top of the figure); the PMN profile slowly returned to a pattern similar to that of the control following treatment.
Figure 63-2 Alteration in the cell surface density of a putative cell adhesion molecule, CAM-D, on PMNs as a function of health. The clinical case is represented on the right and a time-matched healthy control on the left. Time points of blood collection for both animals are color-coded. Blood samples obtained from both the principal and control on the same date are illustrated in the same color. The first blood sample collected (at the time of clinical presentation) is at the top of the figure and subsequent samples are illustrated in a descending fashion. The unusual profile of high percentages of PMNs carrying a relative low density of CAM-D resolves with time.