Heterophil cytokine mRNA profiles from genetically distinct lines of chickens with differential heterophil-mediated innate immune responses

Abstract

Previously we demonstrated that increased in-vitro heterophil function translates to increased in-vivo resistance to Salmonella enteritidis infections in broilers (line A > B). Heterophils produce cytokines and modulate acute protection against Salmonella in neonatal poultry. We hypothesized that heterophils from S. enteritidis-resistant chickens produce an up-regulated pro-inflammatory cytokine/chemokine response compared with S. enteritidis-susceptible chickens. In this study, heterophils were isolated 1, 14, and 28 days post-hatch, treated with RPMI or phagocytic agonists, and the cytokine/chemokine mRNA expression assessed using quantitative real-time reverse transcriptase-polymerase chain reaction. At all time-points, heterophils from S. enteritidis-resistant chickens (line A) had higher levels of pro-inflammatory cytokine/chemokine mRNA expression upon stimulation compared with heterophils from S. enteritidis-susceptible chickens (line B). Furthermore, heterophils from line A chickens had decreased mRNA expression of transforming growth factor-β4, an anti-inflammatory cytokine, compared with line B. These data indicate a relationship between cytokine/chemokine mRNA expression by heterophils and determining overall immune competence. Therefore, heterophil functional efficiency, accompanied by evaluating cytokine/chemokines produced by heterophils, may be useful biomarkers for breeders to consider when developing new immunocompetent lines.

Profils des ARNm des cytokines d'hétérophiles de lignées de poulet génétiquement différentes, présentant des réponses immunitaires à médiation hétérophile différentes dès l’éclosion

Lors d'études préalables nous avons montré qu'une fonction hétérophile accrue in vitro entraîne une résistance accrue in vivo aux infections par Salmonella enteritidis (SE) chez les poulets de chair (lignée A > B). Les hétérophiles produisent des cytokines et modulent la protection contre les salmonelles chez le poussin de 1 jour. Nous avons émis l'hypothèse que les hétérophiles des poulets résistants à SE présentent une sur-expression des cytokines/chemokines pro-inflammatoires comparée à celle des poulets sensibles à SE. Dans cette étude, les hétérophiles ont été isolés à 1, 14, et 28 jours après éclosion, traités avec du RPMI ou des agonistes phagocytaires, et l'expression de l'ARNm des cytokines/chemokines a été évaluée en utilisant la RT-PCR quantitative en temps réel. A toutes les dates d'analyse les hétérophiles des poulets résistants à SE (A) avaient des niveaux plus élevés d'expression de l'ARNm des cytokines/chemokines pro-inflammatoires lors de stimulations, comparés à ceux des hétérophiles des poulets sensibles à SE (B). De plus les hétérophiles des poulets de la lignée A présentaient une diminution de l'expression de l'ARNm du TGF-4, une cytokine anti-inflammatoire, comparée à la lignée B. Ces données indiquent une relation entre l'expression des ARNm des cytokines/chemokines par les hétérophiles et la détermination de la compétence immunitaire globale. Par conséquent, l'efficacité de la fonction hétérophile ainsi que l'évaluation des cytokines/chemokines produites par les hétérophiles, peuvent être des biomarqueurs utiles à prendre en considération par les sélectionneurs lors du développement de lignées immunocompétentes.

mRNS-Profile von heterophilen Zytokinen aus genetisch unterschiedlichen Hühnerlinien mit verschiedenen angeborenen von Heterophilen vermittelten Immunantworten

Vor Kurzem konnten wir nachweisen, dass eine gesteigerte in vitro-Funktion der Heterophilen in eine erhöhte Resistenz gegenüber Salmonella enteritidis (SE)-Infektionen bei Broilern (Linie A > B) übertragen wurde. Heterophile produzieren Zytokine und modulieren akute Schutzmaßnahmen gegen Salmonellen in frisch geschlüpften Geflügelküken. Wir stellten die Hypothese auf, dass Heterophile von SE-resistenten Hühnern im Vergleich zu SE-empfänglichen Hühnern die pro-inflammatorische Zytokin/Chemokin-Antwort hochregulieren. In dieser Studie wurden Heterophile 1, 14 und 28 Tage nach dem Schlupf isoliert, mit RPMI oder phagozystischen Antagonisten behandelt und die Exprimierung der Zytokin/Chemokin-mRNS wurde mittels Real Time RT-PCR gemessen. Zu allen Zeitpunkten zeigten die Heterophilen aus den SE-resistenten Hühnern (A) nach der Stimulation eine höhere Exprimierung der pro-inflammatorischen Zytokin/Chemokin-mRNS als die Heterophilen aus den SE-empfänglichen Hühnerküken (B). Außerdem wiesen die Heterophilen aus der Hühnerlinie A im Vergleich zu denen aus der Linie B eine erniedrigte Exprimierung des transformierenden Wachstumsfaktors-4, eines anti-inflammatorischen Zytokins, auf. Diese Ergebnisse weisen auf eine Beziehung zwischen der Zytokin/Chemokin-mRNS-Exprimierung und der bestimmenden Gesamtimmunkometenz hin. Aus diesem Grund kann die Bestimmung der Effizienz der Heterophilenfunktion zusammen mit der Zytokin/Chemokin-Produktion der Heterophilen ein nützlicher Biomarker für Züchter sein bei der Entwicklung neuer immunkompetenter Linien.

Perfiles de mRNA de citoquinas de heterófilos procedentes de distintas líneas genéticas de pollos con respuestas inmunes innatas diferenciadas mediadas por heterófilos

Anteriormente demostramos que funciones heterofílicas incrementadas in vitro se traducen en un incremento in vivo de la resistencia a infecciones por Salmonella enteritidis (SE) en pollos de engorde (línea A > B). Los heterófilos producen citoquinas y modulan la protección aguda frente a Salmonella en aves neonatales. Hipotetizamos que los heterófilos procedentes de pollos SE-resistentes producían una respuesta pro-inflamatoria de citoquinas/quimioquinas sobreregulada en comparación con pollos SE-susceptibles. En este estudio, se aislaron heterófilos a 1-, 14-, y 28-días post eclosión, se trataron con RPMI o agonistas fagocíticos, y se evaluó la expresión de mRNA de citoquinas/quimioquinas mediante RT-PCR a tiempo real. En todos los tiempos, los heterófilos procedentes de pollos SE-resistentes (A) tenían niveles más altos de expresión de mRNA de citoquinas/quimioquinas pro-inflamatorias bajo estimulación en comparación con los heterófilos de pollos SE-susceptibles (B). Además, los heterófilos de la línea de pollos A tenían la expresión de mRNA del factor de crecimiento de transformación 4, una citoquina anti-inflamatoria, disminuída en comparación con la línea B. Estos resultados indican una relación entre la expresión de mRNA de citoquina/quimioquina por heterófilos y una competencia inmune media determinante. Por lo tanto, la eficiencia funcional de los heterófilos, podría ser un biomarcador útil en el desarrollo de nuevas líneas inmunocompetentes de aves reproductoras.

Introduction

Studies over the past decade indicate that innate immunity, in addition to the first line of defence, provides instruction for acquired immunity (Medzhitov & Janeway, 2002; Hoebe et al., 2004). This cascade of events begins with specific receptors (pattern recognition receptors) on the host's innate immune response cells recognizing conserved molecular motifs unique to invading organisms (pathogen-associated molecular patterns) (Akira, 2001; Janeway & Medzhitov, 2002). The end product of microbial recognition by the innate system is activation of intracellular signalling pathways that initiate cellular processes, such as activation of microbicidal killing mechanisms, production of pro-inflammatory and/or anti-inflammatory cytokines/chemokines, and production of co-stimulatory molecules required for antigen presentation to the acquired immune system (Medzhitov & Janeway, 1997; Imler et al., 2001).

As the first cells to migrate to the infection site, polymorphonuclear leukocytes (PMN) are critical components of innate immunity and subsequent inflammatory response (Hachicha et al., 1998; Kobayashi et al., 2002). Heterophils, the primary PMN in chickens, are the avian counterpart to mammalian neutrophils (Burton & Harrison, 1969; Maxwell & Robertson, 1998). Functionally, heterophils modulate the acute innate host response through phagocytosis of invading microbes and foreign particles, production of oxygen intermediates, and releasing proteolytic enzymes (Desmidt et al., 1996; Kogut et al., 2001; Swaggerty et al., 2003b). Until recently, heterophils were thought to be terminally differentiated cells, deficient of transcriptional activity, and with little protein synthesis. However, using quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) measuring specific mRNA expression of cytokines and chemokines, we showed that heterophils isolated from 1-day-old chickens have a distinct pro-inflammatory or anti-inflammatory response dependent on the stimulant (Kogut et al., 2003b). We have demonstrated that increased in-vitro heterophil functional efficiency translates to increased in-vivo resistance to Salmonella enteritidis and vancomycin-resistant Enterococcus gallinarum infections utilizing a pair of parental broiler lines (line A is more resistant than line B) (Swaggerty et al., 2003b 2005a b; Ferro et al., 2004). Additionally, heterophils isolated from 1-day-old line A and B chickens have a differential constitutive and agonist-stimulated cytokine profile (Swaggerty et al., 2004).

Cytokines/chemokines are essential effector molecules of innate and acquired immunity that initiate and coordinate responses aimed at eradicating pathogens. Detecting avian cytokines is limited by the lack of specific antibodies and reliable bioassays. However, recent cloning of chicken cytokines has enabled development of a more comprehensive array of reagents for investigating innate and acquired immune responses at cellular and molecular levels. Chicken orthologues of T1 cytokines interferon-γ (Digby & Lowenthal, 1995), interleukin (IL)-18 (Schneider et al., 2000), and IL-12 (Balu & Kaiser, 2003; Degen et al., 2004), pro-inflammatory cytokines IL-1β (Weining et al., 1998) and IL-6 (Schneider et al., 2001), chemokine CXCLi2 (formerly IL-8) (Bedard et al., 1987; Sugano et al., 1987), T2-specific cytokines, IL-4, IL-13, and IL-3 (Avery et al., 2004), and anti-inflammatory cytokines transforming growth factor-β4 (TGF-β4) (Jakowlew et al., 1988) and IL-10 (Rothwell et al., 2004) have been cloned and sequenced (Kaiser et al., 2005). This makes it possible to design probes and primers to quantify cytokine mRNA expression using qRT-PCR in a specific population of cells, such as heterophils. Measuring cytokine mRNA expression by qRT-PCR does not equate to production of bioactive protein. However, recent publications show qRT-PCR is the most highly sensitive method available to reliably quantify a broad spectrum of avian cytokines, particularly in the absence of effective bioassays (Kaiser et al., 2000 2002 2003; Beal et al., 2004; Swaggerty et al., 2004; Withanage et al., 2004; Smith et al., 2005).

Economic pressure on the poultry industry has directed genetic selection to develop birds that grow rapidly while consuming less feed. However, selection based on growth characteristics alone can adversely affect immune competence (Han & Smyth, 1972; Bayyari et al., 1997; Beaumont et al., 1999). Assessment of innate immune indices, specifically cytokine mRNA expression by heterophils, may be a useful selection tool that will indicate the overall immunological responsiveness of a particular line of commercial poultry. The objectives of the present study were to further characterize the cytokine/chemokine profile of heterophils isolated from chickens from lines A and B and to determine whether the differences observed 1-day post-hatch are maintained over time. Specifically, we used qRT-PCR to measure mRNA expression levels of IL-6, CXCLi2, IL-18, and TGF-β4 following treatment with phagocytic agonists 1, 14, and 28 days post-hatch.

Materials and Methods

Chickens

Chickens were obtained from a commercial breeder and designated line A and line B. Fertilized eggs were incubated and hatched under standard conditions (Stromberg, 1975). At hatch, chickens were placed in floor pens containing wood shavings, provided supplemental heat, water, and unmedicated corn and soybean meal-based chick starter diet ad libitum until day 14, then transitioned to grower diet. Both feeds met or exceeded standards established by the National Research Council (1994).

Bacteria

A poultry isolate of S. enteritidis (#97-11771) was obtained from the National Veterinary Services Laboratory (Ames, IA, USA) and approved by the United States Department of Agriculture. S. enteritidis was cultured in tryptic soy broth (Difco Laboratories, Becton Dickinson Co., Sparks, Maryland, USA) overnight at 41°C. Stock S. enteritidis (1×10⁹ colony forming units/ml) was prepared as previously described (Swaggerty et al., 2003b). S. enteritidis were opsonized with normal chicken serum (NCS-OpSE) or hyper-immune serum from adult chickens immunized against S. enteritidis (IgY-OpSE) as previously described (Kogut et al., 2001).

Heterophil isolation

Heterophils were isolated from peripheral blood of 50 chickens per line on day 1, 12 chickens per line on day 14 and eight chickens per line on day 28. Blood from 1-day-old chickens was collected in vacutainer tubes containing disodium ethylenediamine tetraacetic acid (EDTA) (BD vacutainer, Franklin Lakes, New Jersey, USA) and mixed thoroughly. Blood was collected into 50 ml tubes containing 5 ml K₃EDTA (0.15 M) on days 14 and 28. Following blood collection, heterophils were isolated as previously described (Swaggerty et al., 2003b). Tissue culture reagents obtained from Sigma Chemical Company (St Louis, Missouri, USA).

RNA isolation

Heterophils (1×10⁷) were treated with 300 µl RPMI, SE, NCS-OpSE, or IgY-OpSE for 30 min at 39°C on a rotary shaker. Treated heterophils were pelleted and washed with RPMI (485×g for 15 min at 4°C), the supernatant discarded, and the cells re-suspended in lysis buffer (Qiagen RNeasy mini RNA extraction kit; Qiagen Inc., Valencia, California, USA), and frozen. Total RNA was extracted from homogenized (QIAshredder columns) lysates according to the manufacturer's instructions.

Quantitative real-time reverse transcriptase-polymerase chain reaction

Cytokine/chemokine mRNA expression in control and agonist-treated heterophils were quantitated using a method described by Kaiser et al. (2000) and Moody et al. (2000). Primers and probes have been described (Kaiser et al., 2000; Kogut et al., 2003b; Swaggerty et al., 2004). qRT-PCR was performed using the Reverse Transcriptase qPCR Master Mix RT-PCR kit (Eurogentec, San Diego, California, USA). Amplification and detection of products were performed using the ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems). Results are expressed as the fold change from controls.

Statistical analyses

Anti-coagulated blood from 50, 12, and eight chickens per line (1, 14, and 28 days post-hatch, respectively) were pooled and heterophils were isolated. Each blood collection and heterophil isolation was conducted on three separate days (heterophils pooled from 50, 12, and eight chickens for each experiment; i.e. 150, 36, and 24 total chickens were used per line as cell donors on days 1, 14, and 28, respectively). The mean and standard error of the mean were calculated from pooled data. Statistical analyses (Student's t test) were performed using Microsoft® Excel 2000 (P≤0.05).

Results and Discussion

We recently showed that heterophils express increased levels of cytokine mRNA upon receptor-mediated phagocytosis (Kogut et al., 2003b), indicating that heterophils are capable of transcriptional activity and protein synthesis. Previously, we quantified basal and agonist-stimulated cytokine mRNA expression in heterophils from 1-day-old line A and B chickens (Swaggerty et al., 2004). The ability to initiate effective inflammatory and T1 responses results in efficient stimulation and direction of acquired immunity (Fearon & Locksley, 1996; Medzhitov & Janeway, 1997). In-vivo challenges using line A and B chickens showed line A chickens are more resistant to S. enteritidis-induced mortality (Swaggerty et al., 2005a) and extra-intestinal organ invasion (Ferro et al., 2004) and vancomycin-resistant E. gallinarum organ invasion (Swaggerty et al., 2005b).

Herein, heterophils were isolated from line A and B chickens and the mRNA expression of cytokines and chemokines quantified through 28 days post-hatch. Data indicate heterophils from genetically distinct chickens have a differential cytokine/chemokine profile and responsiveness to phagocytic agonists. The differences in mRNA expression are maintained to 28-days post-hatch and therefore may be potential selection parameters to identify poultry resistant to diverse pathogens.

There were differences (P≤0.05) between IL-6 mRNA expression of heterophils isolated from the two lines regardless of the phagocytic agonist (SE, NCS-OpSE or IgY-OpSE) (Figure 1). Heterophils isolated from line A chickens expressed significantly more IL-6 mRNA (Figure 1a) compared with heterophils from line B chickens (Figure 1b). The increase in IL-6 expression was maintained from 1, 14, and 28 days post-hatch. The fold change levels ranged from 7.88 to 10.39 in heterophils from line A chickens compared with 1.4 to 4.67 in heterophils from line B chickens.

Figure 1.  Quantitation of IL-6 mRNA expression in heterophils isolated from (1a) line A chickens and (1b) line B chickens on days 1, 14, and 28 post-hatch. Data are expressed as the fold change in IL-6 mRNA levels when treated samples (S. enteritidis [SE], NCS-OpSE, and IgY-OpSE) were compared with control heterophils treated with RPMI. Error bars show the standard error of the mean from triplicate experiments.

Pro-inflammatory and Th1 cytokines are important in immunological responses. In mammals, IL-6 stimulates neutrophils to degranulate (Borish et al., 1989). It is possible that the increased degranulation observed between lines A and B (Swaggerty et al., 2003b) is attributed to increased IL-6 expression, thus creating an environment where heterophils respond more efficiently and eliminate pathogens. However, it is also likely that IL-6 alone is not sufficient to confer resistance against Salmonella, as recombinant chicken IL-6 does not affect in-vitro heterophil function (Ferro et al., 2005). In mammals, IL-6 accompanied with other cytokines may act collaboratively (Keller et al., 1996). It is likely that additional cytokines and/or other factors influence resistance of chickens to S. enteritidis. Heterophils migrate to the liver and intestinal villi of newly hatched chickens infected with Salmonella typhimurium, accompanied by elevated levels of pro-inflammatory cytokine in the tissues (Withanage et al., 2004), further indicating a potential role of the heterophil and the acute inflammatory response in neonatal chickens. Taken together, these data indicate previously observed increased resistance against bacterial infections by line A chickens compared with line B chickens is influenced, in part, by the presence of a strong pro-inflammatory cytokine response, including IL-6, elicited by heterophils.

Expression of the pro-inflammatory chemokine CXCLi2 (formerly IL-8) was up-regulated in heterophils from both lines of chickens following treatment and was maintained throughout the experiment (Figure 2). Recent analysis of the chicken genome sequence revealed that the previous nomenclature for chicken inflammatory chemokines was incorrect; therefore, a new nomenclature has been proposed (Kaiser et al., 2005). As with IL-6, expression of CXCLi2 mRNA was higher (P≤0.05) in heterophils from line A chickens (Figure 2a) compared with line B (Figure 2b). The fold changes for heterophils from line A chickens ranged from 9.83 to 11.51 over the course of the experiment compared with 2.72 to 4.3 for heterophils from line B chickens.

Figure 2.  Quantitation of CXCLi2 mRNA expression in heterophils isolated from (2a) line A chickens and (2b) line B chickens on days 1, 14, and 28 post-hatch. Data are expressed as the fold change in CXCLi2 mRNA levels when treated samples (S. enteritidis [SE], NCS-OpSE, and IgY-OpSE) were compared with control heterophils treated with RPMI. Error bars show the standard error of the mean from triplicate experiments.

In addition to an effective pro-inflammatory cytokine response, a strong chemotactic response may also influence susceptibility of chickens to S. enteritidis. Chemokines are small, structurally related chemoattractant molecules that regulate movement of various leukocytes (Zlotnick & Yoshie, 2000). In mammals, IL-8 is a pro-inflammatory chemokine involved in recruitment of PMN to the site of infection (Rot, 1991), and an IL-8-like (CXCLi2) chemokine is involved in heterophil recruitment to the site of infection in S. enteritidis-infected neonatal chickens (Kogut, 2002). One-day-old chickens infected with S. typhimurium had elevated mRNA expression of pro-inflammatory cytokines/chemokines in the liver and small intestine (Withanage et al., 2004). Additionally, there is increased infiltration of heterophils into tissues of S. typhimurium-infected chickens. Based on studies from our laboratory, increased numbers of heterophils in tissues may contribute to up-regulation of pro-inflammatory cytokine mRNA. We have shown a differential IL-8 (CXCLi2) response in heterophils from S. enteritidis-challenged chickens and observed a significant up-regulation in line A chickens compared with line B (Ferro et al., 2004). Similarly, IL-8 (CXCLi2) mRNA expression was up-regulated in other Salmonella-resistant chickens (Sadeyen et al., 2004). Since heterophils from line A chickens are functionally more efficient in vitro (Swaggerty et al., 2003a b) it is likely that the recruited heterophils are more efficient at eliminating an infection. In fact, we have shown that chickens from line A are more resistant to bacterial infections than chickens from line B (Ferro et al., 2004; Swaggerty et al., 2005a b).

IL-18 mRNA expression was evaluated in heterophils from line A and B chickens at 1, 14, and 28-days post-hatch (Figure 3). Again, heterophils from line A chickens had higher (P≤0.05) IL-18 mRNA expression than heterophils from line B chickens. Line A fold changes ranged between 2.85 and 5.12 compared with −0.94 to 1.72 for line B.

Figure 3.  Quantitation of IL-18 mRNA expression in heterophils isolated from (3a) line A chickens and (3b) line B chickens on days 1, 14, and 28 post-hatch. Data are expressed as the fold change in IL-18 mRNA levels when treated samples (S. enteritidis [SE], NCS-OpSE, and IgY-OpSE) were compared with control heterophils treated with RPMI. Error bars show the standard error of the mean from triplicate experiments.

IL-18 is critical in initiating an inflammatory response, is associated with a T1 cytokine response, and dramatically enhances innate and acquired immunity, thereby affecting host resistance to bacterial infections (Okamura et al., 1998; Garcia et al., 1999; Barreiros et al., 2000). In humans, increased IL-18 plays a protective role against systemic Salmonella infections (Mizuno et al., 2003). In the present study, heterophils from chickens more resistant to S. enteritidis produced higher mRNA expression levels of IL-18. In mammals, IL-18 activates neutrophils by promoting cell migration, cytokine production, generation of a respiratory burst, and degranulation (Leung et al., 2001) mediated by p38 mitogen-associated protein kinase (Wyman et al., 2002). Avian heterophils also undergo degranulation mediated by p38 mitogen-associated protein kinase (Kogut et al., 2003a). Since PMN are activated by IL-18, increased mRNA expression of IL-18 may result in a population of cells primed and subsequently more able to respond.

As would be expected, mRNA expression of the anti-inflammatory cytokine TGF-β4 is different from the pro-inflammatory profile. At 1, 14, and 28 days post-hatch, TGF-β4 mRNA levels in line A heterophils were slightly down-regulated or changed very little from control values following treatment (−2.05 to 1.35) (Figure 4a). In contrast, expression levels in line B heterophils were higher (P≤0.05) regardless of the time post-hatch or the agonist (1.97 to 6.78) (Figure 4b).

Figure 4.  Quantitation of TGF-β4 mRNA expression in heterophils isolated from (4a) line A chickens and (4b) line B chickens on days 1, 14, and 28 post-hatch. Data are expressed as the fold change in TGF-β4 mRNA levels when treated samples (S. enteritidis [SE], NCS-OpSE, and IgY-OpSE) were compared with control heterophils treated with RPMI. Error bars show the standard error of the mean from triplicate experiments.

In the current study, heterophils from resistant line A chickens had up-regulated mRNA expression of pro-inflammatory (IL-6), T1 (IL-18), and chemotactic (CXCLi2) cytokines and chemokines, while mRNA expression of the anti-inflammatory cytokine, TGF-β4, was down-regulated compared with the response observed in heterophils from line B chickens. These data provide evidence that down-regulation of TGF-β4 in heterophils from line A chickens enables these chickens to quickly and efficiently initiate an acute pro-inflammatory cytokine/chemokine response, resulting in increased resistance to bacterial infections. In mammals, high levels of TGF-β are associated with increased mortality in Plasmodium infections (Omer et al., 2003). A separate study showed TGF-β4 mRNA expression was up-regulated in the spleen from S. typhimurium-infected 6-week-old Leghorn chickens (Beal et al., 2004), which is similar to those observed in our Salmonella-susceptible chickens (line B). It is likely that S. typhimurium and S. enteritidis produce a differential cytokine response or that the differences observed are attributed to the type of chickens evaluated. Age is probably not the key factor in the differences, as the current study evaluated 4-week-old chickens compared with 6-week-old chickens (Beal et al., 2004). We postulate that there is a direct relationship between the up-regulation of TGF-β4, decreased heterophil functional efficiency, and increased susceptibility to extra-intestinal S. enteritidis infections in poultry.

Based on data from the present study accompanied by our previous findings utilizing these parental lines of chickens (Swaggerty et al., 2003a b 2005a b; Ferro et al., 2004), we propose isolating heterophils and evaluating cytokine mRNA expression as a novel method to identify poultry with an efficient immune response while maintaining the desired growth parameters required by industry.

Acknowledgments

The authors thank Virginia Lowry, Pamela Ferro, and Zane Brandenberger for technical assistance. Experiments were conducted according to regulations established by the United States Department of Agriculture animal care committee. Mention of commercial products provides information, not recommendation/endorsement.