A hemolysis-hemagglutination assay for characterizing constitutive innate humoral immunity in wild and domestic birds

Department of Biology, University of Missouri, 8001 Natural Bridge Road, St Louis, MO 63121-4499, USA.
Developmental & Comparative Immunology (Impact Factor: 2.82). 02/2005; 29(3):275-86. DOI: 10.1016/j.dci.2004.07.006
Source: PubMed

ABSTRACT Methods to assess immunocompetence requiring only a single sample are useful in comparative studies where practical considerations prevent holding or recapturing individuals. The assay for natural antibody-mediated complement activation and red blood cell agglutination described here, requiring approximately 100 microl of blood, is highly repeatable. The effects of complement deactivation, 2-mercaptoethanol (2-ME), age, and lipopolysaccharide (LPS)-induced sickness response were examined to validate comparisons among diverse avian species. Complement deactivation by heating significantly reduces lysis and treatment with 2-ME reduces both lysis and agglutination. Lysis and agglutination both increase with age in chickens; LPS treatment does not influence these variables in 11-week-old chickens. In a comparison of 11 species, both lysis (0.0-5.3 titers) and agglutination (1.8-8.0 titers) vary significantly among species. Accordingly, this assay can be used to compare constitutive innate humoral immunity among species and with respect to age, sex, and experimental treatments within populations.

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Available from: Kevin D Matson, Sep 25, 2015
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    • "Half scores were recorded when SRBC precipitated partially but not to the extent of control wells. Lysis of erythrocytes was not scored since this process has been shown to be subjective (Matson et al., 2005). "
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    ABSTRACT: Stress is typically characterized as "acute" (lasting from minutes to hours) or "chronic" (lasting from days to months). These terms are of limited use as they are inconsistently used and only encompass one aspect of the stressor (duration). Short and long duration stress are generally thought to produce specific outcomes (e.g. acute stress enhances while chronic stress suppresses immune function). We propose that aspects of stress other than duration, such as frequency and intensity, are important in determining its outcome. We experimentally manipulated duration, frequency, and intensity of application of exogenous corticosterone, CORT, in Sceloporus undulatus (Eastern fence lizards) and measured the immune outcomes. Our findings reveal that immune outcomes of stress are not easily predicted from the average amount or duration of CORT elevation, but that intensity plays an important role. Although three of our treatments received the same average amount of CORT, they produced different effects on immune outcomes (hemagglutination). As predicted by the literature, short-duration exposure to low-dose CORT enhanced hemagglutination; however, short-duration exposure to high-dose CORT suppressed hemagglutination, suggesting that stressor intensity affects immune outcomes of stress. While both are traditionally termed "acute' based on duration, these treatments produced different immune outcomes. Long-duration ("chronic") exposure to CORT did not produce the expected suppression of hemagglutination. Frequency of CORT application did not alter immune outcomes at low intensities. These results highlight the need to quantify more than just the duration of a stressor if we are to understand and manage the ecological consequences of stress. Specifically, we should consider stressor frequency and intensity, as well as duration, for a more complete characterization and understanding of stress. Copyright © 2015 Elsevier Inc. All rights reserved.
    General and Comparative Endocrinology 07/2015; DOI:10.1016/j.ygcen.2015.07.008 · 2.47 Impact Factor
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    • "To assess the levels of circulating natural antibodies and the activity of the complement system, we used the hemolysis–hemagglutination assay as developed by Matson et al. (2005), with several minor alterations . This assay is based on the interaction of avian plasma samples and rabbit red blood cells, which results in agglutination (HA) and lysis (HL) of the cells. "
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    ABSTRACT: Abstract Excessive deposition of metals in the environment is a well-known example of pollution worldwide. Chronic exposure of organisms to metals can have a detrimental effect on reproduction, behavior, health and survival, due to the negative effects on components of the immune system. However, little is known about the effects of chronic sublethal metal exposure on immunity, especially for wildlife. In our study, we examined the constitutive innate immunity of great tit (Parus major) nestlings (N = 234) living in four populations along a metal pollution gradient. For each nestling, we determined the individual metal concentrations (lead, cadmium, arsenic) present in the red blood cells and measured four different innate immune parameters (agglutination, lysis, haptoglobin concentrations and nitric oxide concentrations) to investigate the relationship between metal exposure and immunological condition. While we found significant differences in endogenous metal concentrations among populations with the highest concentrations closest to the pollution source, we did not observe corresponding patterns in our immune measures. However, when evaluating relationships between metal concentrations and immune parameters at the individual level, we found negative effects of lead and, to a lesser extent, arsenic and cadmium on lysis. In addition, high arsenic concentrations appear to elicit inflammation, as reflected by elevated haptoglobin concentrations. Thus despite the lack of a geographic association between pollution and immunity, this type of association was present at the individual level at a very early life stage. The high variation in metal concentrations and immune measures observed within populations indicates a high level of heterogeneity along an existing pollution gradient. Interestingly, we also found substantial within nest variation, for which the sources remain unclear, and which highlights the need of an individual-based approach.
    Science of The Total Environment 03/2015; 508:297 - 306. DOI:10.1016/j.scitotenv.2014.11.095 · 4.10 Impact Factor
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    • "Although limited in number, there are reports of the successful use of this assay to investigate complement activity in two species of opossum (Croix et al. 1989; Koppenheffer et al. 1998; Wirtz and Westfall 1967), but our study is the first to report the use of this assay for dasyurid marsupials. Using two well-established functional assays based on complement-mediated lysis of RBC (Matson et al. 2005; Morgan 2000), initial standard testing of haemolytic activity in RTP serum in male and female RTPs indicates the presence of a functional alternative complement pathway (Fig. 1) and classical complement pathway in RTPs (Figs. 4, 5 and 6). This response is similar to marsupials (Koppenheffer et al. 1998; Wirtz and Westfall 1967) and eutherian mammals (Klerx et al. 1983), where there is an increase in SE and RbE lysis as serum concentration increases. "
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    ABSTRACT: Very few assays that are used to assess the status of mammalian immunity have proved useful for assessment of marsupial health and/or diagnosis of disease. This is largely due to the lack of species cross-reactive reagents that underpin such experiments. To begin to address this deficit, we describe the activation of classical and alternative complement pathways of red-tailed phascogales (RTP; Phascogale calura). Using standard haemolytic assays, the existence of both complement pathways were established in RTP serum based on its ability to lyse unsensitised rabbit erythrocytes (RbE) and sensitised sheep erythrocytes (SE), respectively. The alternative complement pathway assays were conducted using pooled serum of male and female RTPs, and the remaining RTP sera were opportunistically used to test the presence of a functional classical complement system in individual animals, a first in non-eutherian animals. Observations from this study suggest that the activation of these two complement pathways in RTPs are comparable to that seen in other mammals. Since this assay was able to be used on very small samples of blood, it could serve as a useful tool to gather data for comparative immunological studies and to further our knowledge of the mechanisms of immunity available to marsupial young.
    Comparative Clinical Pathology 01/2015; DOI:10.1007/s00580-015-2111-2 · 0.37 Impact Factor
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