Are we enlightened about the immunocompetence of a severely inbred population of New Zealand robins? Challenges inherent in studies using immunological endpoints.

The relatively new field of immunotoxicology developed as an offshoot of traditional, medically based immunology, to study the effects of environmental toxicants on immune function (Fairbrother et al., 2004). A parallel application of methods used in immunotoxicology has been to investigate the evolutionary significance of a robust immune system to such diverse topics as sexual signalling, reproductive effort and population persistence. In a broad sense, Hale & Briskie's (2007) work confirms that there is a high level of interest in evaluating the health of wildlife using non-lethal methods, and that tests of immune function may function as sensitive bioindicators of well-being in wild birds. Scientists, as well as concerned citizens, exercise huge efforts to ensure survival of threatened species, although reduced genetic diversity is expected to result in constraints on the adaptability, disease resistance and overall fitness of the individuals in those populations. If we accept that challenges to the immune response include many types of stressors, severe inbreeding in Motuara robins, described by Hale & Briskie, would qualify as such a stressor. The authors cite numerous publications in which inbred populations have suffered disproportionate losses from environmental stressors, including infectious disease, presumably because of suboptimal immune function. They identify a gap in experimental research linking low genetic diversity with compromised immune status, and, with the current study, set out to address this deficiency. The authors study two island populations of New Zealand robins, one of which is the original, while the other has 600 birds that grew from five individuals translocated from the ancestral population 32 years ago. The bottlenecked population has recently shown poor reproductive performance, speculated to be due to inbreeding depression. The authors compare parasitism and several features of the immune systems in these populations. The testing of immunocompetence is a sound and valuable tool for field scientists but we must be cautious about how to proceed. Too often, tests are selected without sufficient attention to their relevance or application. With a system as robust and redundant as the immune system, it is most meaningful to compare birds during periods of stress when it is particularly costly for them to compensate for additional challenges such as infectious or parasitic diseases. Therefore, studies of immune function are most enlightening if they are carried out during socially or physiologically demanding times such as the breeding or winter seasons, rather than the benign pre- and post-breeding seasons that the authors selected. The following comments are based upon examples taken from Hale & Briskie's study, but the shortcomings are not unique to these authors. One of the two ectoparasites the authors selected to reflect immunocompetence was a feather mite (Astigmata spp.). There is substantial evidence (Blanco et al., 2001; Pap, Tokolyi & Szep, 2005) that feather mites are commensal organisms with no pathological costs to the host. Thus, mites would be highly unlikely to stimulate any immunological response. Regarding the other costly, blood-sucking hippoboscid flies (Ornithomya spp. and Ornithoica spp.) that they examined, if the birds had been dusted with insecticidal powder, the researchers would have had definitive counts of the flies as well as any other ectoparasites. Instead, we have only estimates of these parasites based on how many were seen on, or flying off the birds. Hippoboscid flies would exert an immunological cost. Proteins present in the saliva of haematophagous ectoparasites are foreign antigens that elicit potent localized immune responses (Wikel & Alarcon-Chaidez, 2001). In the immunologically competent host, the reaction to being bitten results in an inflammatory response triggering a series of events that impairs parasite engorgement and provokes its detachment from the host (Pruett, 1999). Thus, immunocompromised birds should be detectable because they would suffer higher ecoparasitism. The most widely used test of avian immune function is the phytohaemagglutinin (PHA) skin test, so other researchers are familiar with what an increased or decreased PHA response should mean, but this only holds true if the test is conducted consistently. Hale & Briskie changed the standard protocol they cited by Smits et al. (1999), reducing the time given the birds to mount a T cell response from the normal 24 to 6 h. Many biologists may not appreciate the significance of this action, for it invalidates conclusions about the T cell response. Immunological responses are complex, and as leucocytes have inherent temporal behavior, responses require time to occur. In the first 60 min of the inflammatory response in birds, venules dilate and leucocytes involved in the innate, or non-specific immune response (heterophils, monocytes and basophils), emigrate from the blood into the surrounding tissue. Then, the heterophils degenerate, monocytes become macrophages and from about 12 h onwards, lymphoid cells (including the T cells of interest in the PHA response) begin to appear, continuing to increase over the next 12 to 24 h (Barnes, 1996 and references therein). In the PHA response, variations to this sequence have historically (Stadecker et al., 1977; Goto et al., 1978) and recently (Martin et al., 2006) been described, but heterophils are always the early responders, whereas lymphocytes including T cells are late recruits to the challenged site. With their 6-h PHA test, Hale & Briskie were measuring the nonspecific heterophil and monocyte, rather than the specific T cell-mediated response in their robins. The biological or immunological interpretation of the PHA test can be ambiguous under the best of circumstances, and people using it need to be aware of its usefulness, but also of its limitations. In all species, one of the fundamental indices of immune status is a total white blood cell (WBC) count plus a differential count that distinguished the different types of WBC. The heterophil to lymphocyte ratio (H:L) determined from a blood smear is broadly heralded as a meaningful indicator of stress when the H:L ratio increases. But, without knowing the total WBC count, it is impossible to know whether the heterophils have increased (a stress response) or whether the lymphocytes have decreased while heterophil numbers have remained constant (no stress response). In birds, nucleated RBCs preclude automated counts, making time-consuming, manual WBC counts necessary. There are three methods of determining WBC counts that I present in order of accuracy. The direct method involves diluting whole blood, staining it and counting the leucocytes on a haemacytometer (Campbell, 1988). Then, the total WBC per microliters is calculated from a formula. The indirect method uses the eosinophil Unopette system, in which diluted, stained blood is placed onto a haemacytometer, all eosinophils and heterophils are counted, and then from the blood smear, a WBC differential is performed, the percentages of heterophils and eosinophils are determined and the total leucocyte count is calculated. The third method used by the authors provides an estimate only, which, by definition, is the least accurate. Because it is also dependent upon the quality of the blood smear, this crude estimation is best used when quantitative counts are unavailable. To add to the complexity of interpreting haematology in wild species, WBC counts pose another challenge because they are rarely biologically meaningful unless one knows the health status of the individuals tested. Hale & Briskie correctly quote that a ‘high lymphocyte count is correlated with a marked immune stimulation’, but what constitutes ‘high’ or ‘low’ is different for each species, and the range of normal is very broad. As healthy birds have a wide reference range of WBC counts, even with statistical differences between the populations, such data are unlikely to be enlightening about immunocompetence. Overall, Hale & Briskie designed a sound approach to study immunocompetence in a genetically bottlenecked population, by looking at parasite resistance, a true reflection of integrated immunocompetence, as well as measures of other aspects of the immune response. The environmental similarity of the habitats of their two populations strengthens the authors' conclusions that genetic diversity was driving the differences observed in immunological endpoints. Their observations of a muted cell-mediated immune response support similar findings by others who have looked at populations lacking genetic diversity. Testing the immune response provides a valuable, sensitive and minimally invasive approach to learning about physiological, developmental and toxicological impacts, as well as genetic constraints in wild populations. Challenges in using tests of immune function in wild birds exist but can mostly be overcome by ensuring a holistic and well-considered approach. For example, the PHA test is valuable if one recognizes it as a single test of immune function, rather than being representative of overall immunological competence with implications about disease resistance. Generally, the aim of a study using immunology testing is to reach a deeper understanding of the immunological competence (i.e. the ability to resist infectious and neoplastic diseases) of a population. To reach this point, multiple tests of immune function that include both innate (non-specific) and adaptive (specific, antibody and T cell mediated) immunity, as well as the interaction between these two arms of the immune system, must be studied in the same individuals. Researchers must strive to identify and use the most meaningful and practical suite of tests available that accurately reflect immunological competence and are linked to relevant biological effects. This will ensure and enhance the value of studies of immune function associated with conservation efforts into the future.

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