© 2002 by The Society for Integrative and Comparative Biology
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Assessing Immunological Function in Toxicological Studies of Avian Wildlife1
1 Department of Biological Sciences, Wright State University, 3640 Colonel Glenn Highway, Dayton, Ohio 45435
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Laboratory and field studies have demonstrated that the immune system is sensitive to environmental contaminants. Testing protocols have been developed to screen for immunotoxic effects and elucidate mechanisms of toxicity in laboratory rodents. Similar methods have been applied to wildlife species in captivity and the wild. Several epizootics in wildlife have been associated with elevated exposure to contaminants. This paper discusses immunotoxicological techniques used in studies of avian wildlife. Measurements of immunological structure include peripheral white blood cell counts and the mass and cellularity of immune organs such as the thymus, spleen, and bursa of Fabricius. While contaminants can alter these measures of immunological structure, such measures do not directly assess how the immune system functions, i.e., responds to specific challenges. The two most commonly used in vivo immune function tests in birds are the phytohemagglutinin (PHA) skin response for T cell-mediated immunity and the sheep red blood cell (SRBC) hemagglutination assay for antibody-mediated immunity. In vitro tests of immune function in avian wildlife include proliferation of lymphocytes in response to various mitogens and phagocytosis of fluorescent particles by monocytes. While optimization of in vitro techniques for wildlife species is often time-consuming, these assays usually require only a single blood sample and can elucidate mechanisms of toxicity. In immunological studies of wildlife, investigators should consider factors that may influence immune responses, including age, body condition, date, developmental stage of the immune system, and time required for the progression of immune responses.
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Numerous laboratory studies have demonstrated that the immune system is sensitive to environmental contaminants (Wong et al., 1992
; Holladay and Luster, 1994). Exposure to contaminants may cause immunosuppression, increasing susceptibility to disease and (or) cancer. Alternatively, chemical-induced enhancement of immunological functions can lead to hypersensitivity, autoimmunity, and (or) decreased resources for other physiological responses. Immunotoxicity studies with rodents have investigated mechanisms of toxicity, including effects on various immunological functions and resistance to disease challenge. Furthermore, protocols for screening immunotoxic effects during product development have been developed for mice and rats (Luster et al., 1988; ICICIS Group, 1998).
One of the first studies in immunotoxicology was conducted by Friend and Trainer (1970), wildlife researchers who demonstrated that exposure to polychlorinated biphenyls (PCBs) increased the susceptibility of young mallards (Anas platyrhynchos) to duck hepatitis virus. A number of epizootics of infectious diseases in wildlife have been more severe in areas contaminated by environmental pollutants, demonstrating the possibility of population-level effects associated with contaminant-induced immunosuppression. Several disease outbreaks have occurred in marine mammals exposed to organochlorines, including California sea lions (Zalophus californianus) on San Miguel Island (Gilmartin et al., 1976), beluga whales (Delphinapterus leucas) in the St. Lawrence estuary (Martineau et al., 1988), common seals (Phoca vitulina) in Europe (McGourty, 1988), and bottlenose dolphins (Tursiops truncatus) in the Atlantic Ocean (Lahvis et al., 1995). In glaucous gulls (Larus hyperboreus), intestinal parasites were positively associated with organochlorine exposure (Sagerup et al., 2000). Several studies have suggested an interaction between high exposure to lead and infectious diseases in birds: Trichomonas gallinae, a potentially fatal protozoan parasite, in mourning doves (Zenaida macroura) (Locke and Bagley, 1967b); aspergillosis in Canada geese (Branta canadensis) and an Andean condor (Vultur gryphus) (Locke et al., 1969); and avian cholera, aspergillosis, and coccidiosis in waterfowl (Locke and Bagley, 1967a; Rocke and Samuel, 1991).
Many methods have been developed for investigating immunotoxic effects in laboratory animals, particularly rodents. Fewer methods have been developed for wildlife species, in part due to the lack of reagents such as species-specific antibodies against leukocyte receptors and immunoglobulins. Several reviews have described the immunotoxic effects of environmental contaminants in fish and wildlife (Wong et al., 1992; Luebke et al., 1997; Keller et al., 1999/2000; Fournier et al., 2000). The purpose of this paper is to discuss the immunological methods most commonly used in toxicological studies of wildlife species, with particular emphasis on free-living birds. However, laboratory studies of wildlife species are also discussed because methods employed in these studies often can be applied to wild animals. This paper reviews methods that assess the structure of the immune system (e.g., cell counts in immune organs or the blood). It then discusses in vivo and in vitro assays that test specific immunological functions. Recently, interest has grown in the area of immunoecology—the study of the physiological ecology of the immune system (Ros et al., 1997; Saino et al., 1995, 1997; Zuk and Johnsen, 1998). Some, but not all, of the assays employed in toxicological studies of wild birds have been applied in these ecological studies.
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Immune organs
In birds, the thymus and bursa of Fabricius are the sites of maturation of T and B lymphocytes, respectively. Mature T and B cells and macrophages interact in the spleen during immunological responses. Hence, the mass, cellularity, and histology of these organs can provide important general information on the maturation and structure of the immune system and have been assessed in several wildlife studies (Table 1). While not all chemicals cause atrophy and (or) histological changes in immune organs, when such alterations occur they are usually associated with altered immunological function, making these variables useful endpoints in studies where lethal sampling is employed.
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In laboratory animals, a characteristic effect of planar halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and coplanar polychlorinated biphenyls (PCBs) is the atrophy of thymus and bursa (Vos and Luster, 1989
; Fox and Grasman, 1999; Grasman and Whitacre, 2001), and similar effects have been observed in wild fish-eating birds. During 1991–92, we sampled 101 4 wk old herring gulls (Larus argentatus) from 9 Great Lakes colonies and 2 reference colonies (Lake Winnipeg and Bay of Fundy on the Atlantic coast) (Fox et al., 1998, 2002; Grasman et al., 2000b). Thymi were removed, fixed in formalin, and later trimmed of fat and re-weighed. Organochlorine contaminants were measured in pooled liver samples from each site, and liver ethoxyresorufin-O-deethylase (EROD) activity was measured as an index of exposure to dioxin-like chemicals. Although thymus mass (standardized to body mass) was not associated with liver concentrations of individual organochlorines, decreased thymus mass was associated with increased liver EROD, suggesting that complex mixtures of contaminants in the Great Lakes contribute to thymic atrophy in herring gulls (Fig. 1). In more recent studies, PCBs measured in the yolk sacs of pipping herring gull embryos were associated negatively with thymocyte numbers and viability, and DDE was associated negatively with numbers and viability of bursal lymphocytes (unpublished data, K.G.).
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In a study of nestling tree swallows in pesticide-sprayed orchards, some associations were seen between pesticide spraying and histological characteristics of the thymus and bursa (Bishop et al., 1998
). All nestlings were the same age when sampled, but sampling occurred over several weeks. There were associations between date of sampling and several immunological variables (thymic and bursal masses, thymic histology, hematology, and phagocytic response), indicating the influence of ecological and (or) phenological factors.
Leukocyte counts
White blood cells are the effector cells of immune responses and have been assessed in many immunotoxicological studies of avian wildlife (Table 1). Lymphocytes are important for T-cell mediated and antibody-mediated responses as well as nonspecific natural killer cell cytotoxicity. Monocytes are important for phagocytic responses. Neutrophils or heterophils provide an important first line of defense against bacterial infections, while eosinophils fight parasitic infestations. Blood cell counts are minimally invasive and relatively easy to conduct. Total and differential white blood cell counts are commonly used as clinical indicators of immunological and infection status. Decreased numbers of specific cell types may suggest reduced functions associated with these cells, and increased numbers may suggest a response to infection. In either case, altered white blood cell counts should be followed up by tests of immune function to better characterize potential immunological effects.
Heterophils and lymphocytes are the two most abundant circulating leukocytes in birds, so many studies have examined the ratio between these two cells. Adrenal stress responses consistently elevate the heterophil/lymphocyte ratio (Gross and Siegel, 1980, 1981), although other mechanisms also may affect these cells. Elevated heterophil to lymphocyte ratios have been observed in Japanese quail (Coturnix coturnix) exposed to paraquat and ethyl methanesulfonate (Clark et al., 1988), young American avocets (Recurvirostra americana) exposed in ovo to selenium, arsenic, and boron (Fairbrother et al., 1994), adult European captive starlings (Sturnus vulagaris) exposed to 7,12-dimethylbezn[a]anthracene (DMBA) (Trust et al., 1994), Japanese quail fed corn and exposed to lead (Grasman and Scanlon, 1995), and wild herring gull and Caspian tern (Sterna caspia) chicks exposed to dioxin-like chemicals, including PCBs (Grasman et al., 1996, 2000b). In many of these studies, these altered white blood cell numbers were associated with suppression of various immune functions (see below).
Plasma proteins
Assessment of plasma or serum protein concentrations can provide important data on health and physiological status, including immune and inflammation responses. Relative and total amounts of albumins, 
-, ß-, and 
-globulins are affected by infections, inflammation, and physiological status (see Grasman et al., 2000a for review). Antibody responses may increase -globulins. Inflammation may increase positive acute phase proteins (-globulins such as antitrypsin, 2-macroglobulin, and haptoglobin and ß-globulins such as fibrinogen, C3, C4, ferritin and amyloid A) and decrease prealbumin, albumin, and transferrin. In prefledgling herring gulls from the Great Lakes, ß2-globulins were positively associated with PCBs and DDE (Table 1) (Grasman et al., 2000a). In young Caspian terns, PCBs were negatively associated with -globulins and positively associated with ß1-globulins. The challenge in wildlife studies is identifying particular proteins in these fractions. In river otters (Lutra canadensis) exposed to the Exxon Valdez oil spill, alterations in albumins, haptoglobin, and ß2-globulins were observed, possibly as direct toxic effect of the oil and (or) a dietary effect of altered food supply (Duffy et al., 1993). Although analysis of plasma protein fractions and specific plasma proteins in wildlife immunotoxicology studies has been rare, these techniques, which fall into the expanding field of proteomics, hold promise in the future for elucidating the immunological and inflammation status of wild animals.
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T cell-mediated immunity
While measures of immunological structure assess the status of the immune system, they do not directly test immune function (i.e., how the immune system responds to specific challenges). The phytohemagglutinin (PHA) skin response test has been used as a measure of T lymphocyte function in laboratory experiments and human and veterinary medicine and is one of the most common assays in wildlife studies. Injection of PHA, a T cell mitogen, into the skin stimulates T cell proliferation, differentiation, and cytokine production (Stadecker et al., 1977
; Lochmiller et al., 1993), causing an influx of other leukocytes and fluid. Common sites of injection in birds include the wing web, wattle, dewlap, and interdigitary skin. Maximal inflammation occurs 12–24 hr after injection. Usually, phosphate buffered saline (PBS), the carrier for the PHA, is injected into an alternate site (e.g., other wing web or wattle). The response to PHA is calculated as the increase in skin thickness caused by PHA minus the increase caused by phosphate buffered saline (PBS) alone. While most investigators have used the PBS injection, several recent studies have simplified the procedure by eliminating the placebo (Zuk and Johnsen, 1998; Smits et al., 1999), citing a strong correlation between the PHA response and difference between the PHA and PBS responses (Smits et al., 1999). However, in our own studies of gulls and terns, the relationship between the PHA and PBS responses often varies between study groups, and the difference between the PHA and PBS responses often gives greater statistical power than the PHA response alone (unpublished data, K.G.). Hence, it seems wise for investigators to test whether the PBS placebo is appropriate for the species and age they are studying.
Elimination of T lymphocyte function by irradiation or immunosuppressive drugs decreases the PHA skin response 50–60% in birds in the laboratory (Edelman et al., 1986; Schrank et al., 1990; Grasman and Scanlon, 1995). In bobwhite quail, the PHA skin response was more sensitive to the immunosuppressive effects of a low protein diet as compared to in vitro proliferation of T lymphocytes stimulated by PHA (Lochmiller et al., 1993). The in vitro assay measures only events in T cell activation and proliferation. The skin test incorporates these events and differentiation and cytokine production. Overall, the PHA test is easy to adapt to different species and is an excellent indicator of T cell function. However, it can only be applied in studies where the test animals can be recaptured 24 hr after the initial measurements and injection.
Several studies have suggested that exposure to organochlorines, especially PCBs, is associated with suppressed T cell function as assessed by the PHA skin test. In a multiyear study during the early 1990s, the PHA skin response was evaluated in prefledgling herring gulls and Caspian terns from the Great Lakes (Grasman et al., 1996). Organochlorine contaminants were measured in pooled egg samples from each site, allowing the sites to be ranked by various contaminants. The PHA response was suppressed 30–45% in colonies in Lake Ontario and Saginaw Bay for both species and in western Lake Erie for herring gulls (Fig. 2). Co-correlation between PCB and DDE concentrations made it difficult to separate the relative importance of these contaminants, although PCBs were found in higher concentrations. This suppression of the PHA response continued in both species in the late 1990s (Grasman and Fox, 2001; unpublished data, K.G.). In Caspian terns, there was a strong negative association between the response and PCBs and, to a slightly lesser degree, DDE measured in the plasma of individual terns (Grasman and Fox, 2001).
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Immunosuppression was investigated in prefledgling black guillemots (Cepphus grylle) in Saglek Bay, Labrador, which was contaminated with PCBs during salvage operations at a military installation. No other organochlorines were present in significant concentrations. In guillemot chicks, increasing liver PCB concentrations measured in individuals were associated with suppressed skin responses to PHA (unpublished data, K.G.). The magnitude of suppression in the most contaminated birds was comparable to that found in Great Lakes gulls an terns at contaminated sites. Unlike the gulls and terns, age influenced the PHA response in guillemots, so statistical corrections for age were necessary to demonstrate the remaining effect of PCBs, emphasizing the importance of looking for relationships with age, body size, or condition when conducting immunological studies in wild birds.
While mitogen-induced responses stimulate large numbers of T cells, antigen-specific responses stimulate only those cells that can bind that particular antigen. Antigen-specific delayed type hypersensitivity (DTH) tests such as the tuberculin skin test require two injections to stimulate a response. The first immunization produces memory cells, which are stimulated by the second reaction. The advantage of the antigen-specific DTH test is that it incorporates antigen processing and presentation and memory cell functions, which are not part of the mitogen-induced PHA skin test. However, the requirement of two immunizations often is not possible in wildlife studies where repeated access to animals may be limited by mobility or rapid growth. Hence, PHA test has been more commonly used in field studies. While mitogen and antigen-specific skin tests often show similar results, the DTH test but not the PHA test was suppressed in mallards fed selenium, suggesting an effect on memory T cell function or antigen processing (Fairbrother and Fowles, 1990).
Antibody-mediated immunity
Like skin tests, measurement of antibody titers following immunization with an antigen integrates a large number of immunological functions and events. The tests are minimally invasive but do require recapture of test animals approximately one week after immunization. Enzyme-linked immunosorbant assays (ELISAs) are commonly used to assess immune function in humans, rodents, and domestic animals. However, ELISAs require antibodies that specifically bind to the antibodies produced by the test subjects. Such species-specific antibody reagents are not available for most wildlife species. Alternatively, the anti-sheep red blood cell (SRBC) hemagglutination test has been employed successfully in many species of wild birds. Blood plasma is collected from birds approximately 6 days after immunization with SRBC. Serial dilutions of the plasma are incubated with SRBC in microtiter plates. If sufficient antibody activity is present, the antibodies will agglutinate the SRBC, which causes a visible spreading of the SRBC on the bottom of the wells. If sufficient activity is not present, the SRBC will not spread and will form a distinct "button" at the bottom of the V-shaped well. The higher the antibody activity or titer, the more the plasma sample can be diluted before the agglutination stops. The agglutination activity is caused by both IgM and IgG. Preincubation of the plasma with 2-mercaptoethanol dissociates IgM, and any residual agglutinating activity is attributable to IgG, which is more prevalent in memory responses. An alternate form of the SRBC test, the Jerne plaque assay, has been shown to be one of the best immunotoxicity assays in laboratory rodents (Luster et al., 1992). In the Jerne plaque assay, spleen cells from SRBC-immunized animals are incubated in the presence of SRBC and complement. B cells that are secreting antibodies against SRBC can be counted under a microscope as the antibodies and complement lyse the SRBC around these lymphocytes.
In mallard ducks, ingestion of two #4 lead shot decreased the anti-SRBC PFC response (Rocke and Samuel, 1991) and ingestion of one #4 lead shot decreased anti-SRBC antibody titers (Trust et al., 1990). In captive red-tailed hawks, chronic lead ingestion caused a 5.7 fold decrease in antibody titers, but this was not statistically significant because of high variability and (or) low sample sizes (Redig et al., 1991). In Japanese quail fed corn, lead caused acute poisoning and reduced the primary antibody response against chuckar partridge (Alectoris graeca) red blood cells (Grasman and Scanlon, 1995). Lead did not cause acute toxicity or affect antibody titers in quail fed standard feed. Selenium did not affect PFC response or antibody titers in mallards (Fairbrother and Fowles, 1990). Cyclophosphamide, the positive control suppressed titers but not PFC numbers, suggesting that titers might be more sensitive indicators of immunotoxicity. In adult European starlings, subcutaneous but not oral exposure to DMBA decreased anti-SRBC antibody titers (Trust et al., 1994). In male mallards, crude oil (Rocke et al., 1984) and PCBs (Fowles et al., 1997) had no effect on anti-SRBC antibody titers.
In Great Lakes Caspian terns antibody responses were positively associated with PCBs and DDE measured in pooled egg samples and individual plasma samples (Grasman et al., 1996; Grasman and Fox, 2001). Although wildlife immunotoxicologists usually look for immunosuppression, chemical-induced enhancement can indicate disruption of regulatory pathways and can lead to hypersensitivity and (or) autoimmunity, which are important concerns in human immunotoxicology. In Great Lakes herring gulls, despite biologically significant intersite differences in antibody titers, there were no associations with organochlorines (Grasman et al., 1996).
The time of assessment of immune response, both with respect to the development of the immune and the time course of specific responses, is a crucial consideration in immunotoxicology studies. In a study of young zebra finches (Taeniopygia guttata) exposed to tailings water from oil sands, anti-SRBC titers were not detectable in control or treatment birds (Smits and Williams, 1999). Finches were immunized at 11 days of age and bled for antibody titers at 21 days. The authors hypothesized that altricial finches were unable to respond to immunization at 11 days because their immune system had not yet developed sufficiently. Antibody titers were also undetectable in three to four week old double-crested cormorants, another altricial species, from the Great Lakes and Lake Winnipeg (unpublished data, K.G.). The lack of a measurable response in the finches also could have been caused by the timing of blood sampling. In most birds, primary antibody responses peak 5–7 days following immunization and decline rapidly. Sampling at 10 days after immunization may have missed the peak titer.
Phagocytic defenses
Most assays of phagocytic function are conducted in vitro (see below), but the clearance of India ink carbon dye can be used as an in vivo measure of phagocytosis (Fairbrother and Fowles, 1990). In mallards, neither selenium ingestion nor cyclophosphamide (immunosuppressant for positive control) affected carbon clearance.
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Isolation and preservation of leukocytes in birds
In vitro assays for immune function using peripheral white blood cells hold much promise because they incorporate the rapidly expanding tools of molecular biology. Some in vitro assays provide information about specific immunological mechanisms, while others integrate a series of mechanisms involved in the response. Because these assays are frequently used in laboratory studies on immunotoxicity, use of similar assays in field studies facilitates comparisons between laboratory and the wild. Blood sampling is minimally invasive. Most in vitro tests require that an animal be captured only once, although some require pre-immunization. These techniques are easier in large bodied species where blood samples of several milliliters can be obtained, although several investigators have applied these techniques to passerines.
Development of in vitro tests of immune function for wild species is often time-consuming because culture and assay conditions must be optimized for each species. However, in studies of wild birds, two other potential problems must be confronted before beginning cell culture: the isolation and preservation of leukocytes. Cell isolation is not necessary if whole blood assays are employed (Fairbrother and Fowles, 1990; Redig et al., 1991; Fairbrother et al., 1994; Trust et al., 1994; Fowles et al., 1997). However, whole blood assays are standardized to consistent blood volumes but not cell numbers. Hence, the response of each blood sample can be influenced by the activity per cell and the cell number.
In mammals, mononuclear cells (lymphocytes and monocytes) are isolated by centrifugation of blood through a density gradient (e.g., Ficol or Histopaque). Mononuclear cells remain on top of the gradient, while other cells move through it. These techniques have been used successfully in some avian studies (Trust et al., 1994; Fowles et al., 1997; Bishop et al., 1998). However, avian thrombocytes often remain in the mononuclear layer in large numbers (70% or more). Avian thrombocytes can be difficult to differentiate from lymphocytes using a viability dye such as Trypan blue, so other stain stains such as Natt and Herricks diluent must be used (Gross, 1984). Thrombocytes can interfere with lymphoproliferation assays (inaccurate lymphocyte counts) and phagocytosis assays (phagocytic activity of thrombocytes vs. monocytes). Several strategies have been used to reduce thrombocyte contamination: dilution of blood with saline or tissue culture medium before centrifugation over the density gradient, and slow-spinning of blood without a density gradient. The slow-spin technique can reduce thrombocyte contamination to approximately 5–20% of the preparation (Gogal et al., 1997; unpublished data, K.G.).
In many instances tissue culture facilities are not available close to field study sites. In several studies blood samples have been rapidly shipped to the laboratory (Fairbrother et al., 1994; Lahvis et al., 1995). Cryopreservation of cells in the field allows for longer storage time and for cells from multiple sites to be tested at the same time.
Lymphoproliferation tests for T and B cell-mediated immunity
Mitogen-induced proliferation or blastogenesis tests have been employed in numerous avian immunotoxicology studies. PHA and concanavalin-A (Con-A) stimulate T cell proliferation. Bacterial LPS stimulates B cell proliferation. Pokeweed mitogen stimulates both T and B cells. High chronic exposure to lead in captive red-tailed hawks suppressed proliferation responses to PHA and Con-A. Exposure to DMBA decreased proliferation responses to Con-A in young and adult starlings (Trust et al., 1994). Pesticide exposure in orchards was associated with suppressed proliferation in response to PWM, which was surprising because responses to T cell-specific mitogens (PHA and Con-A) and a B cell-specific mitogen (LPS) were unaffected (Bishop et al., 1998). Recent investigations on cryopreserved lymphocytes from chickens, herring gulls, and black-crowned night herons (Nycticorax nycticorax) have shown good mitogen-induced proliferation (unpublished data, K.G.).
Phagocytosis
In vitro assays for phagocytosis have been used frequently in immunotoxicology studies of fish and marine mammals, which have been recently reviewed (Fournier et al., 2000). In avocets exposed to selenium, arsenic, and boron, phagocytosis of fluorescent yeast was decreased (Fairbrother et al., 1994). In starlings, DMBA reduced phagocytosis of fluorescent yeast in nestlings but not adults (Trust et al., 1994). Pesticide spraying in orchards did not affect phagocytosis of fluorescent beads by monocytes from tree swallows (Bishop et al., 1998). However, phagocytosis was correlated with collection date despite the fact that all birds were 16 days old at time of sampling.
Beyond ingestion of foreign particles, other mechanisms are related to phagocytosis. Following phagocytosis, cells often release reactive oxygen species to kill and (or) degrade the ingested particles. Pesticide exposure in orchards did not affect the respiratory burst of monocytes in young tree swallows (Bishop et al., 1998).
Cell-mediated cytotoxicity
Natural killer cell cytotoxicity is an important nonspecific defense against viral infections. Peripheral lymphocytes are incubated with target cells for a short period of time (e.g., 4 hr), and the extent of target cell lysis is measured. In a modification of this test, a T cell mitogen such as Con-A can be added to activate T lymphocytes. The amount of target cell lysis is then a measure of the combined activity of natural killer cells and cytotoxic T lymphocytes. Subchronic exposure to PCBs did not affect either of these responses in mallards (Fowles et al., 1997).
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The most obvious consequence of contaminant-induced immunosuppression is increased susceptibility to infectious diseases, which could have impacts on the individual and population levels. Immune function tests and disease prevalence rates rarely have been monitored in same individuals or populations in the wild. However, as described previously (see Introduction), there are numerous examples of increased rates of infections in wild birds and marine mammals exposed to elevated concentrations of contaminants. Following observations of higher mortality associated with phocine distemper virus in common seals from polluted areas of the Baltic Sea (McGourty, 1988
), young captive seals were fed organochlorine-contaminated fish from the Baltic, and a variety of immune responses were suppressed compared to seals fed uncontaminated fish (Ross et al., 1996). Suppression of T cell-mediated immunity has been associated with PCB exposure in prefledgling Caspian terns in Saginaw Bay, L. Huron (Grasman et al., 1996; Grasman and Fox, 2001), and this immunosuppression could be contributing to the low recruitment rate observed at this colony (Mora et al., 1993). Associations between suppressed immune function assays and increased morbidity and (or) mortality due to challenge infections have well documented in laboratory studies, and future studies should examine these associations in wild animals.
Avian ecologists have investigated relationships among immunocompetence and various physiological and behavioral parameters, including growth and development (Fair et al., 1999); testosterone, body mass, display behavior, and secondary sex characteristics (Saino et al., 1995; Ros et al., 1997); mate choice (Zuk and Johnsen, 1998); and feeding behavior (Saino et al., 1997). Contaminant-induced alterations in immunological development and (or) function are likely to influence or be influenced by these physiological and behavioral responses. In particular, the immune and endocrine systems communicate via many mechanisms, and chemicals that act primarily as endocrine disruptors or immunosuppressants may also influence the other system (Ahmed, 2000).
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Over the last two decades, a wide variety of techniques has been developed for the assessment of immune function in wildlife species. These methods can be applied to toxicological, physiological, and ecological studies. Measurements of immunological structure often suggest important effects on the immune system, but measures of immunological function generally provide more insightful data on the significance and mechanisms of these alterations. Some methods require lethal sampling (immune organ masses and cellularity, culture of cells from immune organs), while others are nonlethal (blood sampling for cells or plasma, in vivo skin and antibody testing). Some methods require that an animal be handled only one time (cell culture, plasma protein fractions). Tests that require an in vivo stimulation prior to the measurement of the response (PHA skin test, DTH skin test, SRBC hemagglutination test) require recapture. Measures of immunological structure (immune organ masses and cellularity, white blood cell counts) and in vivo tests of immune function (PHA skin test and SRBC hemagglutination test) have proven to be relatively easy to adapt to wildlife species. In vitro assays for immune function require more experimentation to optimize protocols for each species. Despite their difficulty, these in vitro assays have several advantages: they require only a blood sample, they often reveal more about mechanisms of toxicity, and they take advantage of recent advances in cell biology and immunology. In the future, application of molecular techniques such as measuring expression of immunological genes may provide additional assays for immunotoxic effects in wildlife.
Disease defenses are important for survival and fitness, as shown by recent studies on the physiological ecology of the immune system. Both laboratory and field studies have demonstrated the sensitivity of the immune system to environmental contaminants, especially during the perinatal period. Several epizootics have been associated with elevated contaminant exposure. One important avenue for future investigations in wildlife immunotoxicology is the increased utilization of state of the art immunological techniques, including biochemical, cell culture, and gene expression assays. These methods are increasingly adaptable to species other than humans and rodents, and they can supply important functional and mechanistic information about immunotoxicity in wild animals. Further work is needed to link contaminant-induced suppression of immune function to increased morbidity or mortality of infectious diseases in wild animals. Finally, other energetic, physiological, and behavioral costs of contaminant-induced immunosuppression should be investigated. This last area is a potentially fruitful field of collaboration between environmental toxicologists and physiological ecologists.
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FOOTNOTES |
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1 From the symposium Taking Physiology to the Field: Advances in Investigating Physiological Function in Free-living Vertebrates presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 3–7 January 2001, at Chicago, Illinois.
2 E-mail: keith.grasman{at}wright.edu
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