general measures of avian immune system

These measures are applied to provide a general indication of the strength of the immune system in birds. However, they do not provide a direct assessment of the functionality of the immune system against antigens and infectious diseases. Measurement of the general status of the avian immune system includes assessment of weights and cellularity of immune organs such as the thymus, spleen and bursa of Fabricius, peripheral leukocyte count, and assessment of plasma proteins concentration.

assessment of weights and cellularity of immune organs

The main immune organs in poultry are the thymus, spleen and bursa of Fabricius. Upon immunological response, mature lymphocytes and other immune cells interact with antigens in these immune tissues. Consequently, the mass and cellular status of these organs can generally indicate an immune status (Smith and Hunt, Reference SMITH and HUNT2004; Grasman, Reference GRASMAN2002; Swamy et al., Reference SWAMY, SMITH, KARROW and BOERMANS2004).

peripheral leukocyte count

Circulating blood leukocytes represent a pivotal measure of the immune system status. They possess an effective influence on foreign particles in immune responses. Consequently, they are considered to reflect immune strength. Thus, counting the total and differential numbers of leukocytes is a general parameter of the health status. Abnormal numbers of the specific types of leukocytes may indicate immunosuppression or immunocompetence in response to an immunomodulator or an infectious challenge (Brennan et al., Reference BRENNAN, HENDRICKS III, EL-SHEIKH and MASHALY2002; Eeva et al., Reference EEVA, HASSELQUIST, LANGEFORS, TUMMELEHT, NIKINMAA and ILMONENND2005; Swamy et al., Reference SWAMY, SMITH, KARROW and BOERMANS2004; Mashaly et al., Reference MASHALY, HENDRICKS III, KALAMA, GEHAD, ABBAS and PATTERSON2004).

assessment of plasma protein concentrations

Immunological and health status in terms of infection and inflammation is clearly characterised by the production and release of specific plasma or serum proteins. Examples of such proteins are the complement system proteins and the acute phase proteins and their concentration in the plasma is strongly associated with an inflammatory response. Some proteins such as antitrypsin, haptoglobin, fibrinogen, and ferritin are increased in response to inflammation. On the other hand, the concentrations of other proteins such as pre-albumin, albumin, and transferrin are decreased during inflammatory responses. Moreover, antibody responses are commonly associated with the production of γ- globulins. Accordingly, monitoring the level of proteins that are associated with infection and inflammation reflects the health status of birds (Grasman, Reference GRASMAN2002; Takahashi et al., Reference TAKAHASHI, KAWAMATA, AKIBA and OKADA2002; Al-Mayah, Reference AL-MAYAH2006; Bunchasak et al., Reference BUNCHASAK, POOSUWAN, NUKRAEW, MARKVICHITR and CHOOTHESA2005).

markers of functional avian immune responses

The various measures of avian immune response give general information about the health status of birds. Consequently, these measures are unreliable in decision making regarding vaccinations or analysing mechanisms with which immunomodulators affect responses. However, these general measures are useful in monitoring the health status and can be applied as a first step before confirming using other parameters or markers that are more reliable in studying the avian immune functions and responses. The latter measures give more information about the mechanisms with which the immune system responds to a specific challenge. These functional measures can be applied in vivo or in vitro and include numerous immune parameters as follows.

measures of antibody-mediated immunity

Immunisation of birds with antigens induces the production of specific antibodies. The pattern of antibody production reflects numerous immunological functions in response to different antigenic stimulants. Primary and secondary immunisation measures are often applied against specific antigens. This is because when a bird is challenged with an antigen, a low magnitude and short duration primary antibody will be produced. A secondary immunisation with the same antigen produces a secondary antibody response that lasts longer and is greater in magnitude (Poli et al., Reference POLI, ZANELLA, DALL'ARA and BONIZZI2000; Goldsby et al., Reference GOLDSBY, KINDT, OSBORNE and KUBY2003; Sijben et al., Reference SIJBEN, SCHRAMA, NIEUWLAND and PARMENTIER2000b; Fritsche and Cassity, Reference FRITSCHE and CASSITY1992; Bedecarrats and Leeson, Reference BEDECARRATS and LEESON2006; Puthpongsiriporn and Scheideler, Reference PUTHPONGSIRIPORN and SCHEIDELER2005).

There are numerous techniques that are used to measure antibody production in response to a specific antigen in vitro or in vivo. Paramount of all is the use of the enzyme-linked immunosorbent assay (ELISA) technique that utilises species-specific antibodies which bind to the antibodies of the species in question (Elsafty et al., Reference ELSAFTY, ALI and FATHI2006; Kondo et al., Reference KONDO, GOTO and ABE2004). This technique makes it possible to measure the circulating concentrations of total and subclasses of Ig, specific Ig in response to antigenic challenge, concentration of immunoglobulin A (IgA) in secretions such as mucosa, saliva and tears, as well as the circulating concentrations of cytokines and cytokine receptors (Verhulst et al., Reference VERHULST, RIEDSTRA and WIERSMA2005; Friedman et al., Reference FRIEDMAN, BARTOV and SKLAN1998; Abdukalykova and Ruiz-Feria, Reference ABDUKALYKOVA and RUIZ-FERIA2006; Christe, Reference CHRISTE2002). Interestingly, the humoral immune response can be induced by a wide range of immunomodulators. Sheep red blood cells (SRBC), Newcastle disease virus (NDV), Keyhole limpet haemocyanin (KLH), 2,4-Dinitrophenol, a cellular metabolic poison (DNP), M. butyricum and Escherichia coli (E. coli) are commonly used in avian immunology studies to induce antibody production (Wang et al., Reference WANG, FIELD and SIM2000; Fritsche et al., Reference FRITSCHE, CASSITY and HUANG1991; Praharaj et al., Reference PRAHARAJ, DUNNINGTON, GROSS and SIEGEL1997; Guo et al., Reference GUO, KWAKKEL, WILLIAMS, PARMENTIER, LI, YANG and VERSTEGEN2004; Bartlett and Smith, Reference BARTLETT and SMITH2003; Muhammad et al., Reference MUHAMMAD, HAMAYUN, SAJID UR and MUHAMMAD2006; Velhner et al., Reference VELHNER, KAPETANOV, KAPETANOV, MITEVSKI and ORLIC2001; Hangalapura et al., Reference HANGALAPURA, NIEUWLAND, DE VRIES REILINGH, HEETKAMP, VAN DEN BRAND, KEMP and PARMENTIER2003; Sijben et al., Reference SIJBEN, GROOT, NIEUWLAND, SCHRAMA and PARMENTIER2000a; Parmentier et al., Reference PARMENTIER, NIEUWLAND, BARWEGEN, KWAKKEL and SCHRAMA1997; Huang et al., Reference HUANG, DENG, YANG, YIN, XIE, WU, LI, LI, TANG, KANG, HOU, DENG, XIANG, KONG and GUO2007). However, the limited availability of species-specific antibodies for poultry makes the use of ELISA techniques limited. In these cases, other alternatives such as SRBC haemagglutinatin test and Jerne plaque assays are often used successfully. SRBC haemagglutinatin test involves immunisation of birds with SRBC and blood is collected after a period of time, usually 1-3 weeks, and serial dilutions of the plasma are incubated with SRBC. Sufficient antibody response is reflected by agglutination of SRBC by antibodies (Grasman, Reference GRASMAN2002; Takahashi et al., Reference TAKAHASHI, ONODERA and AKIBA1999). In the Jerne plaque test, birds are first immunised with SRBC and their spleen cells are then incubated with SRBC and complement. Splenic B-lymphocytes will lyse SRBC in its vicinity in the presence of complement system (Luster et al., Reference LUSTER, PORTIER, PAIT, WHITE, GENNINGS, MUNSON and ROSENTHAL1992) .

phytohemagglutinin skin-testing technique (PHA skin test)

PHA skin test is commonly used to measure T- cell mediated immunity because PHA is considered a T-lymphocytes mitogen. It is a carbohydrate binding lectin extracted from red kidney beans called Phaseolus vulgaris (Kennedy and Nager, Reference KENNEDY and NAGER2006; Grasman, Reference GRASMAN2002; Calder, Reference CALDER2007; Abdukalykova and Ruiz-Feria, Reference ABDUKALYKOVA and RUIZ-FERIA2006).

The PHA-skin swelling technique is a widely used quantitative technique to measure the acquired immunity in terms of T-cell activity in birds. In this technique, birds are injected in their wing web, wattle, dewlap, or interdigitary skin with PHA. A control saline solution such as phosphate buffered saline (PBS) is injected into an alternate site. PHA response is then calculated 12-24 or 48 hours after injection as the difference in skin thickness increase between the two cases of PHA and PBS injections using a micrometer (Smits et al., Reference SMITS, BORTOLOTTI and TELLA1999; Grasman, Reference GRASMAN2002; Verhulst et al., Reference VERHULST, RIEDSTRA and WIERSMA2005; Martinez-Padilla, Reference MARTINEZ-PADILLA2006; Moore and Siopes, Reference MOORE and SIOPES2002; Kliger et al., Reference KLIGER, GEHAD, HULET, ROUSH, LILLEHOJ and MASHALY2000; Cheema et al., Reference CHEEMA, QURESHI and HAVENSTEIN2003; Sijben et al., Reference SIJBEN, NIEUWLAND, KEMP, PARMENTIER and SCHRAMA2001a; Bedecarrats and Leeson, Reference BEDECARRATS and LEESON2006; Babu et al., Reference BABU, WIESENFELD, RAYBOURNE, MYERS and GAINES2005; Korver and Klasing, Reference KORVER and KLASING1997; Hudson et al., Reference HUDSON, DOZIER III, WILSON, SANDER and WARD2004; Fritts et al., Reference FRITTS, ERF, BERSI and WALDROUP2004).

It was concluded by Martin et al. (Reference MARTIN, HAN, LEWITTES, KUHLMAN, KLASING and WIKELSKI2006) that PHA as a foreign protein induces both innate and acquired branches of the avian immune system. Directly after PHA injection, an influx of heterophils and thrombocytes occurs, followed by basophils and macrophages. This innate response is associated with inflammation response and skin trauma. After 24 hours post-injection, infiltration of lymphocytes, especially T-lymphocytes peaks at the site of injection. The pivotal involvement of T-lymphocytes in the PHA response assay was further confirmed by Edelman et al. (Reference EDELMAN, SANCHEZ, ROBINSON, HOCHWALD and THORBECKE1986), and Schrank et al.(1990). These authors concluded that immuno-suppression of avian T-lymphocytes by irradiation or chemical drugs significantly decreased PHA skin response up to 50-60%. Similarly, the PHA response was suppressed by 30-45% as a result of organochlorines exposure, these chemicals are known to suppress T-lymphocytes function in birds (Grasman and Fox, Reference GRASMAN and FOX2001). Interestingly, some authors studied the primary and secondary PHA injection in birds and have shown that the secondary response induced greater swelling than the primary response, indicating that the immunity involved is characterised by a memory response (Johnsen et al., Reference JOHNSEN, ANDERSEN, SUNDING and LIFJELD2000; Kondo et al., Reference KONDO, GOTO and ABE2004). On the contrary, Reid et al. (Reference REID, ARCESE and KELLER2003) showed no significant difference between the primary and secondary PHA responses.

antigen-specific delayed type hypersensitivity test (DTH)

Unlike the PHA-skin test that induces large numbers of T-lymphocytes that are not specific to PHA, antigen-specific DTH such as the tuberculin skin test induces specific lymphocytes for a particular antigen. Primary injection induces memory and antigen presenting cells that are activated in a secondary response. Interestingly, both PHA and antigen-specific DTH skin tests give the same results (Fairbrother and Fowles, Reference FAIRBROTHER and FOWLES1990; Grasman, Reference GRASMAN2002; Wang et al., Reference WANG, AJUYAH, SUNWOO, CHERIAN and SIM2002).

Another modification of DTH skin tests that does not cause skin trauma is the use of skin patches of contact-sensitising agents such as oxazolone or dinitrofluorobenzene. These agents stimulate T-lymphocytes and natural killer cells upon primary and secondary applications. The resulting response can be measured in the same way as PHA and DTH skin tests (O'Leary et al., Reference O'LEARY, GOODARZI, DRAYTON and VON ANDRIAN2006).

phagocytosis assay

In vitro assays require the isolation of white blood cells of interest in order to use them in functional immunological assays. One of the most common methods of isolation of avian mononuclear cells (i.e. lymphocytes and monocytes) is by centrifugation of blood samples to create a density gradient. This separates more than 70% of the lymphocytes and monocytes at the top of the gradient (Fowles et al., Reference FOWLES, FAIRBROTHER, TRUST and KERKVLIET1997; Trust et al., Reference TRUST, FAIRBROTHER and HOOPER1994). Nevertheless, contamination of monocytes with quite large numbers of thrombocytes characterises this method. Gogal et al. (Reference GOGAL, AHMAD and LARSEN1997) have reported that thrombocyte contamination can be reduced by 5-20% by a slow-spin technique without a density gradient. Moreover, it was shown that diluting the blood samples with saline or culture medium with application of the density gradient may significantly reduce thrombocyte contamination (Grasman, Reference GRASMAN2002).

In addition the purified cell preparations, whole blood samples can be used to assess in vitro or ex vivo functional assays of the immune system. The latter has the advantage of retaining all the components of the in vivo situation in the same ratios. Nevertheless, purified cell cultures give information about the number of cells in the assay and the cells can be stored in a liquid nitrogen compartment for future use (Calder, Reference CALDER2007; Fairbrother and Fowles, Reference FAIRBROTHER and FOWLES1990; Fowles et al., Reference FOWLES, FAIRBROTHER, TRUST and KERKVLIET1997).

The phagocytic capability of avian heterophils and mononuclear cells can be measured by the phagocytosis assay that quantitatively measures the percentage of phagocytes and their enzymatic activity (i.e. the number of ingested antigens per phagocytic cell). Bacteria such as Escherichia coli, SRBC, and yeast cells are commonly used as substrates for phagocytosis. Opsonisation of these substrates by complements and/or antibodies is usually applied to facilitate phagocytosis, although unopsonised substrates may be used (Thies et al., Reference THIES, MILES, NEBE-VON-CARON, POWELL, HURST, NEWSHOLME and CALDER2001; Arunachalam et al., Reference ARUNACHALAM, GILL and CHANDRA2000). Opsonisation degree, temperature, incubation time and the ratio of substrates to phagocytes are the main factors to be considered when conducting phagocytosis assays. Labelling the substrates for phagocytosis with fluorescent stains enables detection and counting of these substrates by techniques such as flow cytometry or fluorescence microscopy (Janaczyk et al., Reference JANACZYK, PLISZCZAK-KROL, GRACZYK, HOUSZKA and ROUIBAH2006).

oxidative burst

Measuring the phagocytic activity of leukocytes is commonly associated with the oxidative burst assay that involves production of anti-microbial and cytotoxic substances in the form of reactive oxygen and nitrogen intermediates such as hydroxyl radicals, hydrogen peroxide and nitric oxide (Farnell et al., Reference FARNELL, CRIPPEN, HE, SWAGGERTY and KOGUT2003a;Farnell et al., Reference FARNELL, HE, GENOVESE and KOGUT2003b;Kogut et al., Reference KOGUT, GENOVESE and LOWRY2001;Salgar et al., Reference SALGAR, PAAPE, ALSTON-MILLS and MILLER1991). The oxidative burst assay, commonly referred to as phago-burst or burst test, involves the use of unlabelled opsonised bacteria or any other antigen and a stimulus for the reaction that is usually a protein kinase C ligand phorbol, as well as a chemotactic factor such as N-formyl-MetLeuPhe. A flourogenic substrate is used to enable quantitative determination of the reaction by the use of flow cytometry or fluorescence microscopy techniques (Farnell et al., Reference FARNELL, HE, GENOVESE and KOGUT2003b; Chen et al., Reference CHEN, LI, CHANG, GONG, DAI and YI2003a; Reference CHEN, LI, CHANG, GONG, PIAO, YI and ZHANG2003b; Fritts et al., Reference FRITTS, ERF, BERSI and WALDROUP2004; Kew et al., Reference KEW, BANERJEE, MINIHANE, FINNEGAN, MUGGLI, ALBERS, WILLIAMS and CALDER2003a).

bactericidal killing by whole blood and plasma

In addition to phagocytosis by cellular components, bacteria can be killed by soluble immune elements in whole blood and plasma. For example, the complement system can initiate an enzymatic cascade capable of lysing targeted bacterial cells (Nordahl et al., Reference NORDAHL, RYDENGARD, NYBERG, NITSCHE, MORGELIN, MALMSTEN and BJORCK2004). In addition, plasma lysozymes and acute phase proteins such as mannose-binding proteins exhibit bactericidal competence by attacking bacterial cell walls (Zheng et al., Reference ZHENG, YE, BAI, WU, LAO and LUO2005; Fernie-King et al., Reference FERNIE-KING, SEILLY, DAVIES and LACHMANN2002). This bacteria-killing ability contributes to the innate immune response, and can be easily measured in vitro by using whole blood or isolated plasma. One advantage of this assay is that it does not require species-specific reagents (Matson et al., Reference MATSON, TIELEMAN and KLASING2006).

chemotactic response

Chemotaxis is the movement of immune cells toward the site of infection under the influence of certain stimuli (Harmon, Reference HARMON1998; Qureshi, Reference QURESHI2003; Qureshi et al., Reference QURESHI, PETITTE, LASTER and DIETERT1993). Chemotactic response of heterophils and monocytes can be studied by using different stimuli such as leukotriene B4, antigenic peptides of bacterial cell walls, some interleukins, and autologous serum (Walrand et al., Reference WALRAND, MOREAU, CALDEFIE, TRIDON, CHASSAGNE, PORTEFAIX, CYNOBER, BEAUFRERE, VASSON and BOIRIE2001; Calder, Reference CALDER2007).

natural killer cell (NK) and cytotoxic t-lymphocytes activity

These cytotoxic activities are usually combined with each other in in vitro assays. The target cells are incubated with NK cells and cytotoxic T-cells for specific periods of time and the assay is described by measuring the lytic ratio between the cytotoxic and the target cells (Calder, Reference CALDER2007; Calder et al., Reference CALDER, YAQOOB, THIES, WALLACE and MILES2002; Fowles et al., Reference FOWLES, FAIRBROTHER, TRUST and KERKVLIET1997). Classical responses mediated by NK cells, monocytes and eosinophils can also be measured (Fritsche and Cassity, Reference FRITSCHE and CASSITY1992; Fritts et al., Reference FRITTS, ERF, BERSI and WALDROUP2004). Antigen-coated chicken red blood cells are widely used to conduct the cytotoxicity assay (Boyd and Wick, Reference BOYD and WICK1980; Maccubbin and Schierman, Reference MACCUBBIN and SCHIERMAN1986).

mitogen-stimulated proliferation of T- and B- lymphocytes

T- and B- lymphocytes are well known to proliferate in vitro by increasing in number and by synthesizing DNA during blastogenic transformation in response to certain stimuli such as mitogens, antigens, antibodies, or even vaccines. For example, concanavalin A (Con A) and PHA mitogens induce proliferation of T-lymphocytes, while bacterial lipopolysaccharides (LPS) and Staphylococcus aureus stimulate proliferation of B-lymphocytes. Pokeweed mitogens stimulate proliferation of both T- and B- lymphocytes (Meydani et al., Reference MEYDANI, ENDRES, WOODS, GOLDIN, SOO, MORRILL-LABRODE, DINARELLO and GORBACH1991; Fritsche et al., Reference FRITSCHE, CASSITY and HUANG1991; Calder, Reference CALDER2007; Sijben et al., Reference SIJBEN, GROOT, NIEUWLAND, SCHRAMA and PARMENTIER2000a; Barber et al., Reference BARBER, FEARON and ROSS2005; Lee, Reference LEE1978).

Lymphocyte proliferation is traditionally measured by radioactivity of incorporated thymidine into the DNA of mitotic lymphocytes. Incorporation of bromodeoxyuridine into the deoxyribonucleic acid (DNA) of lymphocytes may also be used as a measure of lymphocyte proliferation (Wang et al., Reference WANG, FIELD and SIM2000; Calder, Reference CALDER2007; Takahashi et al., Reference TAKAHASHI, ONODERA and AKIBA1999; Hangalapura et al., Reference HANGALAPURA, NIEUWLAND, DE VRIES REILINGH, HEETKAMP, VAN DEN BRAND, KEMP and PARMENTIER2003). A flow cytometry technique was recently used to measure cell proliferation by incorporation of carboxyfluoroscein succinimidyl ester (CFSE) stain (Lyons, Reference LYONS2000; Lyons and Parish, Reference LYONS and PARISH1994; Angulo and Fulcher, Reference ANGULO and FULCHER1998; Bird et al., Reference BIRD, BROWN, MULLEN, MOSKOWITZ, MAHOWALD, SIDER, GAJEWSKI, WANG and REINER1998; Hodgkin et al., Reference HODGKIN, CHIN, BARTELL, MAMCHAK, DOHERTY, LYONS and HASBOLD1997).

production of cytokines

Cytokines are known to be produced upon stimulation of lymphocytes and monocytes as regulators of the immune response. Cytokines can be measured in cultures using ELISA techniques in which monoclonal antibodies are directed to them. Flow cytometry techniques are used to measure cytokines and to phenotype the cells producing them. In addition, intracellular cytokines can be measured by flow cytometry by using specific stains after treatment of cell membranes (Meydani et al., Reference MEYDANI, ENDRES, WOODS, GOLDIN, SOO, MORRILL-LABRODE, DINARELLO and GORBACH1991; Thies et al., Reference THIES, MILES, NEBE-VON-CARON, POWELL, HURST, NEWSHOLME and CALDER2001; Tricon et al., Reference TRICON, BURDGE, KEW, BANERJEE, RUSSELL, GRIMBLE, WILLIAMS, CALDER and YAQOOB2004; Yang and Yuming, Reference YANG and YUMING2006; Kromer et al., Reference KROMER, SCHAUENSTEIN and WICK1984). Up-regulation or down-regulation of different types of cytokines can be used as an indication of the immune status. For example, immuno-suppression in chickens is commonly evaluated as a decrease in IFN- α and IFN-γ m-RNA that can be detected by using nucleic acid hybridisation micro-titre assays (Kew et al., Reference KEW, BANERJEE, MINIHANE, FINNEGAN, WILLIAMS and CALDER2003b; Novak and Scheideler, Reference NOVAK and SCHEIDELER2001; Chen et al., Reference CHEN, LI, CHANG, GONG, DAI and YI2003a; Reference CHEN, LI, CHANG, GONG, PIAO, YI and ZHANG2003b; Sijben et al., Reference SIJBEN, SCHRAMA, PARMENTIER, VAN DER POEL and KLASING2001b; Meydani et al., Reference MEYDANI, ENDRES, WOODS, GOLDIN, SOO, MORRILL-LABRODE, DINARELLO and GORBACH1991).

expression of cell membrane molecules

Stimulation of different types of immune cells induces the expression of specific molecules on their surfaces. Consequently, determination of different immune cells can be achieved by identifying molecules expressed on their cell membranes. This requires the use of specific stains or markers for these surface molecules. Examples of cell surface molecules include toll-like receptors, major histocompatibility complex (MHC) subtypes, clusters of differentiation (CD) subtypes and cytokine receptors. Determination of the profiles of different cell types is then achieved by the use of the flow cytometry technique (Farnell et al., Reference FARNELL, CRIPPEN, HE, SWAGGERTY and KOGUT2003a; Calder, Reference CALDER2001; Reference CALDER2007). In addition, other immuno-fluorescence techniques such as fluorescence microscopy can be used to detect reactions with surface antigens (Swamy et al., Reference SWAMY, SMITH, KARROW and BOERMANS2004). Some antibodies have been developed to chicken antigens such as to chicken CD3, CD4, CD8, and to cytokines (Bacon, Reference BACON1992; Chan et al., Reference CHAN, CHEN and COOPER1988). This approach enables identification of the morphological characteristics of lymphocytes during their differentiation and developmental stages. For example, Chen et al. (Reference CHEN, CIHAK, LOSCH and COOPER1988) showed that two receptors TcR1 and TcR2 are expressed on separate T cell sub-lineages during their early development in the thymus and then in the periphery. This technique of labelling immune cells with monoclonal antibodies directed against surface antigens is called immuno-phenotyping.